The free-energy cost of interaction between DNA loops.

Science.gov (United States)

Huang, Lifang; Liu, Peijiang; Yuan, Zhanjiang; Zhou, Tianshou; Yu, Jianshe

2017-10-03

From the viewpoint of thermodynamics, the formation of DNA loops and the interaction between them, which are all non-equilibrium processes, result in the change of free energy, affecting gene expression and further cell-to-cell variability as observed experimentally. However, how these processes dissipate free energy remains largely unclear. Here, by analyzing a mechanic model that maps three fundamental topologies of two interacting DNA loops into a 4-state model of gene transcription, we first show that a longer DNA loop needs more mean free energy consumption. Then, independent of the type of interacting two DNA loops (nested, side-by-side or alternating), the promotion between them always consumes less mean free energy whereas the suppression dissipates more mean free energy. More interestingly, we find that in contrast to the mechanism of direct looping between promoter and enhancer, the facilitated-tracking mechanism dissipates less mean free energy but enhances the mean mRNA expression, justifying the facilitated-tracking hypothesis, a long-standing debate in biology. Based on minimal energy principle, we thus speculate that organisms would utilize the mechanisms of loop-loop promotion and facilitated tracking to survive in complex environments. Our studies provide insights into the understanding of gene expression regulation mechanism from the view of energy consumption.

Sub-Ensemble Monitoring of DNA Strand Displacement Using Multiparameter Single-Molecule FRET.

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Baltierra-Jasso, Laura E; Morten, Michael J; Magennis, Steven W

2018-03-05

Non-enzymatic DNA strand displacement is an important mechanism in dynamic DNA nanotechnology. Here, we show that the large parameter space that is accessible by single-molecule FRET is ideal for the simultaneous monitoring of multiple reactants and products of DNA strand exchange reactions. We monitored the strand displacement from double-stranded DNA (dsDNA) by single-stranded DNA (ssDNA) at 37 °C; the data were modelled as a second-order reaction approaching equilibrium, with a rate constant of 10 m -1  s -1 . We also followed the displacement from a DNA three-way junction (3WJ) by ssDNA. The presence of three internal mismatched bases in the middle of the invading strand did not prevent displacement from the 3WJ, but reduced the second-order rate constant by about 50 %. We attribute strand exchange in the dsDNA and 3WJ to a zero-toehold pathway from the blunt-ended duplex arms. The single-molecule approach demonstrated here will be useful for studying complex DNA networks. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Studying DNA Looping by Single-Molecule FRET

OpenAIRE

Le, Tung T.; Kim, Harold D.

2014-01-01

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can als...

Integrating DNA strand-displacement circuitry with DNA tile self-assembly

Science.gov (United States)

Zhang, David Yu; Hariadi, Rizal F.; Choi, Harry M.T.; Winfree, Erik

2013-01-01

DNA nanotechnology has emerged as a reliable and programmable way of controlling matter at the nanoscale through the specificity of Watson–Crick base pairing, allowing both complex self-assembled structures with nanometer precision and complex reaction networks implementing digital and analog behaviors. Here we show how two well-developed frameworks, DNA tile self-assembly and DNA strand-displacement circuits, can be systematically integrated to provide programmable kinetic control of self-assembly. We demonstrate the triggered and catalytic isothermal self-assembly of DNA nanotubes over 10 μm long from precursor DNA double-crossover tiles activated by an upstream DNA catalyst network. Integrating more sophisticated control circuits and tile systems could enable precise spatial and temporal organization of dynamic molecular structures. PMID:23756381

Sub-ensemble monitoring of DNA strand displacement using multiparameter single-molecule FRET

OpenAIRE

Baltierra Jasso, Laura; Morten, Michael; Magennis, Steven William

2018-01-01

Non-enzymatic DNA strand displacement is an important mechanism in dynamic DNA nanotechnology. Here we show that the large parameter space that is accessible by single-molecule FRET is ideal for the simultaneous monitoring of multiple reactants and products of DNA strand exchange reactions. We monitored the strand displacement from double-stranded DNA (dsDNA) by single-stranded DNA (ssDNA) at 37 °C; the data were modelled as a second-order reaction approaching equilibrium, with a rate constan...

Connecting localized DNA strand displacement reactions

Science.gov (United States)

Mullor Ruiz, Ismael; Arbona, Jean-Michel; Lad, Amitkumar; Mendoza, Oscar; Aimé, Jean-Pierre; Elezgaray, Juan

2015-07-01

Logic circuits based on DNA strand displacement reactions have been shown to be versatile enough to compute the square root of four-bit numbers. The implementation of these circuits as a set of bulk reactions faces difficulties which include leaky reactions and intrinsically slow, diffusion-limited reaction rates. In this paper, we consider simple examples of these circuits when they are attached to platforms (DNA origamis). As expected, constraining distances between DNA strands leads to faster reaction rates. However, it also induces side-effects that are not detectable in the solution-phase version of this circuitry. Appropriate design of the system, including protection and asymmetry between input and fuel strands, leads to a reproducible behaviour, at least one order of magnitude faster than the one observed under bulk conditions.Logic circuits based on DNA strand displacement reactions have been shown to be versatile enough to compute the square root of four-bit numbers. The implementation of these circuits as a set of bulk reactions faces difficulties which include leaky reactions and intrinsically slow, diffusion-limited reaction rates. In this paper, we consider simple examples of these circuits when they are attached to platforms (DNA origamis). As expected, constraining distances between DNA strands leads to faster reaction rates. However, it also induces side-effects that are not detectable in the solution-phase version of this circuitry. Appropriate design of the system, including protection and asymmetry between input and fuel strands, leads to a reproducible behaviour, at least one order of magnitude faster than the one observed under bulk conditions. Electronic supplementary information (ESI) available. See DOI: 10.1039/C5NR02434J

Simplified Real-Time Multiplex Detection of Loop-Mediated Isothermal Amplification Using Novel Mediator Displacement Probes with Universal Reporters.

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Becherer, Lisa; Bakheit, Mohammed; Frischmann, Sieghard; Stinco, Silvina; Borst, Nadine; Zengerle, Roland; von Stetten, Felix

2018-04-03

A variety of real-time detection techniques for loop-mediated isothermal amplification (LAMP) based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However, these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization. Here, we present the first universal real-time detection technique for multiplex LAMP. The novel approach allows simple assay design and is easy to implement for various targets. The innovation features a mediator displacement probe and a universal reporter. During amplification of target DNA the mediator is displaced from the mediator displacement probe. Then it hybridizes to the reporter generating a fluorescence signal. The novel mediator displacement (MD) detection was validated against state-of-the-art molecular beacon (MB) detection by means of a HIV-1 RT-LAMP: MD surpassed MB detection by accelerated probe design (MD: 10 min, MB: 3-4 h), shorter times to positive (MD 4.1 ± 0.1 min shorter than MB, n = 36), improved signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, MB: 2.7 ± 0.4; n = 15), and showed equally good or better analytical performance parameters. The usability of one universal mediator-reporter set in different multiplex assays was successfully demonstrated for a biplex RT-LAMP of HIV-1 and HTLV-1 and a biplex LAMP of Haemophilus ducreyi and Treponema pallidum, both showing good correlation between target concentration and time to positive. Due to its simple implementation it is suggested to extend the use of the universal mediator-reporter sets to the detection of various other diagnostic panels.

Protected DNA strand displacement for enhanced single nucleotide discrimination in double-stranded DNA.

Science.gov (United States)

Khodakov, Dmitriy A; Khodakova, Anastasia S; Huang, David M; Linacre, Adrian; Ellis, Amanda V

2015-03-04

Single nucleotide polymorphisms (SNPs) are a prime source of genetic diversity. Discriminating between different SNPs provides an enormous leap towards the better understanding of the uniqueness of biological systems. Here we report on a new approach for SNP discrimination using toehold-mediated DNA strand displacement. The distinctiveness of the approach is based on the combination of both 3- and 4-way branch migration mechanisms, which allows for reliable discrimination of SNPs within double-stranded DNA generated from real-life human mitochondrial DNA samples. Aside from the potential diagnostic value, the current study represents an additional way to control the strand displacement reaction rate without altering other reaction parameters and provides new insights into the influence of single nucleotide substitutions on 3- and 4-way branch migration efficiency and kinetics.

DNA loop domain organization in nucleoids from cells of different types.

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Afanasieva, Katerina; Chopei, Marianna; Lozovik, Alexandra; Semenova, Anastasia; Lukash, Lyubov; Sivolob, Andrei

2017-01-29

The loop domain organization of chromatin plays an important role in transcription regulation and thus may be assumed to vary in cells of different types. We investigated the kinetics of DNA loop migration during single cell gel electrophoresis (the comet assay) for nucleoids obtained from human lymphocytes, lymphoblasts and glioblastoma T98G cells. The results confirm our previous observation that there are three parts of DNA in nucleoids: DNA on the nucleoid surface, loops up to ∼150 kb inside the nucleoid, and larger loops that cannot migrate. However, the relative amounts of the three parts were found to be very different for different cell types. The distributions of the loop length up to 150 kb were shown to be exponential, with the distribution parameter, the loop density, to be dependent on the cell type. Copyright © 2016 Elsevier Inc. All rights reserved.

DNA looping by FokI: the impact of synapse geometry on loop topology at varied site orientations

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Rusling, David A.; Laurens, Niels; Pernstich, Christian; Wuite, Gijs J. L.; Halford, Stephen E.

2012-01-01

Most restriction endonucleases, including FokI, interact with two copies of their recognition sequence before cutting DNA. On DNA with two sites they act in cis looping out the intervening DNA. While many restriction enzymes operate symmetrically at palindromic sites, FokI acts asymmetrically at a non-palindromic site. The directionality of its sequence means that two FokI sites can be bridged in either parallel or anti-parallel alignments. Here we show by biochemical and single-molecule biophysical methods that FokI aligns two recognition sites on separate DNA molecules in parallel and that the parallel arrangement holds for sites in the same DNA regardless of whether they are in inverted or repeated orientations. The parallel arrangement dictates the topology of the loop trapped between sites in cis: the loop from inverted sites has a simple 180° bend, while that with repeated sites has a convoluted 360° turn. The ability of FokI to act at asymmetric sites thus enabled us to identify the synapse geometry for sites in trans and in cis, which in turn revealed the relationship between synapse geometry and loop topology. PMID:22362745

Single nucleotide polymorphisms in the D-loop region of mitochondrial DNA is associated with the kidney survival time in chronic kidney disease patients.

Science.gov (United States)

Xu, Jinsheng; Guo, Zhanjun; Bai, Yaling; Zhang, Junxia; Cui, Liwen; Zhang, Huiran; Zhang, Shenglei; Ai, Xiaolu

2015-02-01

The mitochondrial displacement loop (D-loop) is known to accumulate mutations and SNPs at a higher frequency than other regions of mitochondrial DNA (mtDNA). We had identified chronic kidney disease (CKD) risk-associated SNPs in the D-loop of CKD patients previously. In this study, we investigated the association of SNPs in the D-loop of mtDNA with the kidney survival of CKD. The D-loop region of mtDNA was sequenced for 119 CKD patients from the inpatient of the Fourth Hospital of Hebei Medical University. The Kaplan-Meier method was used to identify disease outcome-associated SNPs in the D-loop of CKD patients. The Cox proportional hazards model was used to identify risk factors for the kidney survival of CKD. In the present study, we identified 20 SNPs with a frequency higher than 5% and assessed the relationship of these SNPs with kidney survival time in CKD patients, a SNP of 146 was identified by log-rank test for statistically significant prediction of the kidney survival time. In an overall multivariate analysis, allele 146 was identified as an independent predictor of kidney survival time in CKD patients. The survival time of kidney in the CKD patients with 146C was significantly shorter than that of kidney in CKD patients with 146T (relative risk, 2.336; 95% CI, 1.319-3.923; p = 0.001). SNPs in the D-loop can predict the kidney survival of CKD patients. Analysis of genetic polymorphisms in the mitochondrial D-loop can help to identify CKD patient subgroup at high risk of a poor disease outcome.

Transcription-Replication Conflict Orientation Modulates R-Loop Levels and Activates Distinct DNA Damage Responses.

Science.gov (United States)

Hamperl, Stephan; Bocek, Michael J; Saldivar, Joshua C; Swigut, Tomek; Cimprich, Karlene A

2017-08-10

Conflicts between transcription and replication are a potent source of DNA damage. Co-transcriptional R-loops could aggravate such conflicts by creating an additional barrier to replication fork progression. Here, we use a defined episomal system to investigate how conflict orientation and R-loop formation influence genome stability in human cells. R-loops, but not normal transcription complexes, induce DNA breaks and orientation-specific DNA damage responses during conflicts with replication forks. Unexpectedly, the replisome acts as an orientation-dependent regulator of R-loop levels, reducing R-loops in the co-directional (CD) orientation but promoting their formation in the head-on (HO) orientation. Replication stress and deregulated origin firing increase the number of HO collisions leading to genome-destabilizing R-loops. Our findings connect DNA replication to R-loop homeostasis and suggest a mechanistic basis for genome instability resulting from deregulated DNA replication, observed in cancer and other disease states. Copyright © 2017 Elsevier Inc. All rights reserved.

Exonuclease of human DNA polymerase gamma disengages its strand displacement function.

Science.gov (United States)

He, Quan; Shumate, Christie K; White, Mark A; Molineux, Ian J; Yin, Y Whitney

2013-11-01

Pol γ, the only DNA polymerase found in human mitochondria, functions in both mtDNA repair and replication. During mtDNA base-excision repair, gaps are created after damaged base excision. Here we show that Pol γ efficiently gap-fills except when the gap is only a single nucleotide. Although wild-type Pol γ has very limited ability for strand displacement DNA synthesis, exo(-) (3'-5' exonuclease-deficient) Pol γ has significantly high activity and rapidly unwinds downstream DNA, synthesizing DNA at a rate comparable to that of the wild-type enzyme on a primer-template. The catalytic subunit Pol γA alone, even when exo(-), is unable to synthesize by strand displacement, making this the only known reaction of Pol γ holoenzyme that has an absolute requirement for the accessory subunit Pol γB. © 2013. Published by Elsevier B.V.

MRI-guided percutaneous cryoablation of renal tumors: Use of external manual displacement of adjacent bowel loops

International Nuclear Information System (INIS)

Tuncali, Kemal; Morrison, Paul R.; Tatli, Servet; Silverman, Stuart G.

2006-01-01

Purpose: We sought to investigate retrospectively the safety and effectiveness of using external hand compression to displace adjacent bowel loops during MRI-guided percutaneous cryoablation of renal tumors. Materials and methods: Fourteen patients (six women, eight men; mean age: 72 years) with 15 renal tumors (mean diameter: 2.4 cm; range: 1.4-4.6 cm) adjacent to bowel were treated with MRI-guided percutaneous cryoablation during which bowel was displaced manually. Bowel loop of concern was ascending colon (n 5), descending colon (n = 8), descending colon and small bowel (n = 1), ascending colon and small bowel (n = 1). To analyze effectiveness of the maneuver, mean distance between tumor margin and bowel before and after the maneuver were compared and analyzed using paired Student's t-test. Minimum distance between iceball edge and adjacent bowel with external manual displacement during freezing was also measured. Safety was assessed by analyzing post-procedural MR imaging for adjacent bowel wall thickening and focal fluid collections as well as patients' clinical and imaging follow-up. Results: Mean distance between tumor margin and closest adjacent bowel increased from 0.8 cm (range: 0-2 cm) before external manual compression to 2.6 cm (range: 1.6-4.1 cm) with manual displacement (p < 0.01). Mean minimum distance between iceball edge and closest adjacent bowel during the procedures was 1.6 cm (range: 0.5-3.5 cm). No evidence of bowel injury was encountered. Twelve of 15 tumors had follow-up (mean: 10 months) that showed no tumor recurrence. Conclusion: MRI-guided percutaneous cryoablation of renal tumors adjacent to bowel can be done safely and effectively using external hand compression to displace bowel loops

RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes.

Science.gov (United States)

NandyMazumdar, Monali; Nedialkov, Yuri; Svetlov, Dmitri; Sevostyanova, Anastasia; Belogurov, Georgiy A; Artsimovitch, Irina

2016-12-27

Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β' clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.

DNA fork displacement rates in human cells

International Nuclear Information System (INIS)

Kapp, L.N.; Painter, R.B.

1981-01-01

DNA fork displacement rates were measured in 20 human cell lines by a bromodeoxyuridine-313 nm photolysis technique. Cell lines included representatives of normal diploid, Fanconi's anemia, ataxia telangiectasia, xeroderma pigmentosum, trisomy-21 and several transformed lines. The average value for all the cell lines was 0.53 +- 0.08 μm/min. The average value for individual cell lines, however, displayed a 30% variation. Less than 10% of variation in the fork displacement rate appears to be due to the experimental technique; the remainder is probably due to true variation among the cell types and to culture conditions. (Auth.)

DNA fork displacement rates in human cells

Energy Technology Data Exchange (ETDEWEB)

Kapp, L.N.; Painter, R.B. (California Univ., San Francisco (USA). Lab. of Radiobiology)

1981-11-27

DNA fork displacement rates were measured in 20 human cell lines by a bromodeoxyuridine-313 nm photolysis technique. Cell lines included representatives of normal diploid, Fanconi's anemia, ataxia telangiectasia, xeroderma pigmentosum, trisomy-21 and several transformed lines. The average value for all the cell lines was 0.53 +- 0.08 ..mu..m/min. The average value for individual cell lines, however, displayed a 30% variation. Less than 10% of variation in the fork displacement rate appears to be due to the experimental technique; the remainder is probably due to true variation among the cell types and to culture conditions.

Toehold-Mediated Displacement of an Adenosine-Binding Aptamer from a DNA Duplex by its Ligand.

Science.gov (United States)

Monserud, Jon H; Macri, Katherine M; Schwartz, Daniel K

2016-10-24

DNA is increasingly used to engineer dynamic nanoscale circuits, structures, and motors, many of which rely on DNA strand-displacement reactions. The use of functional DNA sequences (e.g., aptamers, which bind to a wide range of ligands) in these reactions would potentially confer responsiveness on such devices, and integrate DNA computation with highly varied molecular stimuli. By using high-throughput single-molecule FRET methods, we compared the kinetics of a putative aptamer-ligand and aptamer-complement strand-displacement reaction. We found that the ligands actively disrupted the DNA duplex in the presence of a DNA toehold in a similar manner to complementary DNA, with kinetic details specific to the aptamer structure, thus suggesting that the DNA strand-displacement concept can be extended to functional DNA-ligand systems. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dynamic protein assembly by programmable DNA strand displacement

Science.gov (United States)

Chen, Rebecca P.; Blackstock, Daniel; Sun, Qing; Chen, Wilfred

2018-03-01

Inspired by the remarkable ability of natural protein switches to sense and respond to a wide range of environmental queues, here we report a strategy to engineer synthetic protein switches by using DNA strand displacement to dynamically organize proteins with highly diverse and complex logic gate architectures. We show that DNA strand displacement can be used to dynamically control the spatial proximity and the corresponding fluorescence resonance energy transfer between two fluorescent proteins. Performing Boolean logic operations enabled the explicit control of protein proximity using multi-input, reversible and amplification architectures. We further demonstrate the power of this technology beyond sensing by achieving dynamic control of an enzyme cascade. Finally, we establish the utility of the approach as a synthetic computing platform that drives the dynamic reconstitution of a split enzyme for targeted prodrug activation based on the sensing of cancer-specific miRNAs.

Design and Characterization of DNA Strand-Displacement Circuits in Serum-Supplemented Cell Medium.

Science.gov (United States)

Fern, Joshua; Schulman, Rebecca

2017-09-15

The functional stability and lifetimes of synthetic molecular circuits in biological environments are important for long-term, stable sensors or controllers of cell or tissue behavior. DNA-based molecular circuits, in particular DNA strand-displacement circuits, provide simple and effective biocompatible control mechanisms and sensors, but are vulnerable to digestion by nucleases present in living tissues and serum-supplemented cell culture. The stability of double-stranded and single-stranded DNA circuit components in serum-supplemented cell medium and the corresponding effect of nuclease-mediated degradation on circuit performance were characterized to determine the major routes of degradation and DNA strand-displacement circuit failure. Simple circuit design choices, such as the use of 5' toeholds within the DNA complexes used as reactants in the strand-displacement reactions and the termination of single-stranded components with DNA hairpin domains at the 3' termini, significantly increase the functional lifetime of the circuit components in the presence of nucleases. Simulations of multireaction circuits, guided by the experimentally measured operation of single-reaction circuits, enable predictive realization of multilayer and competitive-reaction circuit behavior. Together, these results provide a basic route to increased DNA circuit stability in cell culture environments.

Solving probability reasoning based on DNA strand displacement and probability modules.

Science.gov (United States)

Zhang, Qiang; Wang, Xiaobiao; Wang, Xiaojun; Zhou, Changjun

2017-12-01

In computation biology, DNA strand displacement technology is used to simulate the computation process and has shown strong computing ability. Most researchers use it to solve logic problems, but it is only rarely used in probabilistic reasoning. To process probabilistic reasoning, a conditional probability derivation model and total probability model based on DNA strand displacement were established in this paper. The models were assessed through the game "read your mind." It has been shown to enable the application of probabilistic reasoning in genetic diagnosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

A specific subdomain in φ29 DNA polymerase confers both processivity and strand-displacement capacity

Science.gov (United States)

Rodríguez, Irene; Lázaro, José M.; Blanco, Luis; Kamtekar, Satwik; Berman, Andrea J.; Wang, Jimin; Steitz, Thomas A.; Salas, Margarita; de Vega, Miguel

2005-01-01

Recent crystallographic studies of φ29 DNA polymerase have provided structural insights into its strand displacement and processivity. A specific insertion named terminal protein region 2 (TPR2), present only in protein-primed DNA polymerases, together with the exonuclease, thumb, and palm subdomains, forms two tori capable of interacting with DNA. To analyze the functional role of this insertion, we constructed a φ29 DNA polymerase deletion mutant lacking TPR2 amino acid residues Asp-398 to Glu-420. Biochemical analysis of the mutant DNA polymerase indicates that its DNA-binding capacity is diminished, drastically decreasing its processivity. In addition, removal of the TPR2 insertion abolishes the intrinsic capacity of φ29 DNA polymerase to perform strand displacement coupled to DNA synthesis. Therefore, the biochemical results described here directly demonstrate that TPR2 plays a critical role in strand displacement and processivity. PMID:15845765

On the biophysics and kinetics of toehold-mediated DNA strand displacement.

Science.gov (United States)

Srinivas, Niranjan; Ouldridge, Thomas E; Sulc, Petr; Schaeffer, Joseph M; Yurke, Bernard; Louis, Ard A; Doye, Jonathan P K; Winfree, Erik

2013-12-01

Dynamic DNA nanotechnology often uses toehold-mediated strand displacement for controlling reaction kinetics. Although the dependence of strand displacement kinetics on toehold length has been experimentally characterized and phenomenologically modeled, detailed biophysical understanding has remained elusive. Here, we study strand displacement at multiple levels of detail, using an intuitive model of a random walk on a 1D energy landscape, a secondary structure kinetics model with single base-pair steps and a coarse-grained molecular model that incorporates 3D geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Two factors explain the dependence of strand displacement kinetics on toehold length: (i) the physical process by which a single step of branch migration occurs is significantly slower than the fraying of a single base pair and (ii) initiating branch migration incurs a thermodynamic penalty, not captured by state-of-the-art nearest neighbor models of DNA, due to the additional overhang it engenders at the junction. Our findings are consistent with previously measured or inferred rates for hybridization, fraying and branch migration, and they provide a biophysical explanation of strand displacement kinetics. Our work paves the way for accurate modeling of strand displacement cascades, which would facilitate the simulation and construction of more complex molecular systems.

Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression.

Science.gov (United States)

Aze, Antoine; Sannino, Vincenzo; Soffientini, Paolo; Bachi, Angela; Costanzo, Vincenzo

2016-06-01

Half of the human genome is made up of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using bacterial artificial chromosomes in Xenopus laevis egg extract. Using this approach we characterized the chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication-dependent enrichment of a network of DNA repair factors including the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR-dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to the inability of the single-stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of topoisomerase I-dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications for our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions.

Transcription-factor-mediated DNA looping probed by high-resolution, single-molecule imaging in live E. coli cells.

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Zach Hensel

Full Text Available DNA looping mediated by transcription factors plays critical roles in prokaryotic gene regulation. The "genetic switch" of bacteriophage λ determines whether a prophage stays incorporated in the E. coli chromosome or enters the lytic cycle of phage propagation and cell lysis. Past studies have shown that long-range DNA interactions between the operator sequences O(R and O(L (separated by 2.3 kb, mediated by the λ repressor CI (accession number P03034, play key roles in regulating the λ switch. In vitro, it was demonstrated that DNA segments harboring the operator sequences formed loops in the presence of CI, but CI-mediated DNA looping has not been directly visualized in vivo, hindering a deep understanding of the corresponding dynamics in realistic cellular environments. We report a high-resolution, single-molecule imaging method to probe CI-mediated DNA looping in live E. coli cells. We labeled two DNA loci with differently colored fluorescent fusion proteins and tracked their separations in real time with ∼40 nm accuracy, enabling the first direct analysis of transcription-factor-mediated DNA looping in live cells. Combining looping measurements with measurements of CI expression levels in different operator mutants, we show quantitatively that DNA looping activates transcription and enhances repression. Further, we estimated the upper bound of the rate of conformational change from the unlooped to the looped state, and discuss how chromosome compaction may impact looping kinetics. Our results provide insights into transcription-factor-mediated DNA looping in a variety of operator and CI mutant backgrounds in vivo, and our methodology can be applied to a broad range of questions regarding chromosome conformations in prokaryotes and higher organisms.

diffloop: a computational framework for identifying and analyzing differential DNA loops from sequencing data.

Science.gov (United States)

Lareau, Caleb A; Aryee, Martin J; Berger, Bonnie

2018-02-15

The 3D architecture of DNA within the nucleus is a key determinant of interactions between genes, regulatory elements, and transcriptional machinery. As a result, differences in DNA looping structure are associated with variation in gene expression and cell state. To systematically assess changes in DNA looping architecture between samples, we introduce diffloop, an R/Bioconductor package that provides a suite of functions for the quality control, statistical testing, annotation, and visualization of DNA loops. We demonstrate this functionality by detecting differences between ENCODE ChIA-PET samples and relate looping to variability in epigenetic state. Diffloop is implemented as an R/Bioconductor package available at https://bioconductor.org/packages/release/bioc/html/diffloop.html. aryee.martin@mgh.harvard.edu. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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

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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.

Senataxin Mutation Reveals How R-Loops Promote Transcription by Blocking DNA Methylation at Gene Promoters.

Science.gov (United States)

Grunseich, Christopher; Wang, Isabel X; Watts, Jason A; Burdick, Joshua T; Guber, Robert D; Zhu, Zhengwei; Bruzel, Alan; Lanman, Tyler; Chen, Kelian; Schindler, Alice B; Edwards, Nancy; Ray-Chaudhury, Abhik; Yao, Jianhua; Lehky, Tanya; Piszczek, Grzegorz; Crain, Barbara; Fischbeck, Kenneth H; Cheung, Vivian G

2018-02-01

R-loops are three-stranded nucleic acid structures found abundantly and yet often viewed as by-products of transcription. Studying cells from patients with a motor neuron disease (amyotrophic lateral sclerosis 4 [ALS4]) caused by a mutation in senataxin, we uncovered how R-loops promote transcription. In ALS4 patients, the senataxin mutation depletes R-loops with a consequent effect on gene expression. With fewer R-loops in ALS4 cells, the expression of BAMBI, a negative regulator of transforming growth factor β (TGF-β), is reduced; that then leads to the activation of the TGF-β pathway. We uncovered that genome-wide R-loops influence promoter methylation of over 1,200 human genes. DNA methyl-transferase 1 favors binding to double-stranded DNA over R-loops. Thus, in forming R-loops, nascent RNA blocks DNA methylation and promotes further transcription. Hence, our results show that nucleic acid structures, in addition to sequences, influence the binding and activity of regulatory proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Detection of DNA damage based on metal-mediated molecular beacon and DNA strands displacement reaction

Science.gov (United States)

Xiong, Yanxiang; Wei, Min; Wei, Wei; Yin, Lihong; Pu, Yuepu; Liu, Songqin

2014-01-01

DNA hairpin structure probes are usually designed by forming intra-molecular duplex based on Watson-Crick hydrogen bonds. In this paper, a molecular beacon based on silver ions-mediated cytosine-Ag+-cytosine base pairs was used to detect DNA. The inherent characteristic of the metal ligation facilitated the design of functional probe and the adjustment of its binding strength compared to traditional DNA hairpin structure probes, which make it be used to detect DNA in a simple, rapid and easy way with the help of DNA strands displacement reaction. The method was sensitive and also possesses the good specificity to differentiate the single base mismatched DNA from the complementary DNA. It was also successfully applied to study the damage effect of classic genotoxicity chemicals such as styrene oxide and sodium arsenite on DNA, which was significant in food science, environmental science and pharmaceutical science.

Strand Displacement by DNA Polymerase III Occurs through a τ-ψ-χ Link to Single-stranded DNA-binding Protein Coating the Lagging Strand Template*

OpenAIRE

Yuan, Quan; McHenry, Charles S.

2009-01-01

In addition to the well characterized processive replication reaction catalyzed by the DNA polymerase III holoenzyme on single-stranded DNA templates, the enzyme possesses an intrinsic strand displacement activity on flapped templates. The strand displacement activity is distinguished from the single-stranded DNA-templated reaction by a high dependence upon single-stranded DNA binding protein and an inability of γ-complex to support the reaction in the absence of τ. However, if γ-complex is p...

Strand displacement by DNA polymerase III occurs through a tau-psi-chi link to single-stranded DNA-binding protein coating the lagging strand template.

Science.gov (United States)

Yuan, Quan; McHenry, Charles S

2009-11-13

In addition to the well characterized processive replication reaction catalyzed by the DNA polymerase III holoenzyme on single-stranded DNA templates, the enzyme possesses an intrinsic strand displacement activity on flapped templates. The strand displacement activity is distinguished from the single-stranded DNA-templated reaction by a high dependence upon single-stranded DNA binding protein and an inability of gamma-complex to support the reaction in the absence of tau. However, if gamma-complex is present to load beta(2), a truncated tau protein containing only domains III-V will suffice. This truncated protein is sufficient to bind both the alpha subunit of DNA polymerase (Pol) III and chipsi. This is reminiscent of the minimal requirements for Pol III to replicate short single-stranded DNA-binding protein (SSB)-coated templates where tau is only required to serve as a scaffold to hold Pol III and chi in the same complex (Glover, B., and McHenry, C. (1998) J. Biol. Chem. 273, 23476-23484). We propose a model in which strand displacement by DNA polymerase III holoenzyme depends upon a Pol III-tau-psi-chi-SSB binding network, where SSB is bound to the displaced strand, stabilizing the Pol III-template interaction. The same interaction network is probably important for stabilizing the leading strand polymerase interactions with authentic replication forks. The specificity constant (k(cat)/K(m)) for the strand displacement reaction is approximately 300-fold less favorable than reactions on single-stranded templates and proceeds with a slower rate (150 nucleotides/s) and only moderate processivity (approximately 300 nucleotides). PriA, the initiator of replication restart on collapsed or misassembled replication forks, blocks the strand displacement reaction, even if added to an ongoing reaction.

Protein intercalation in DNA as one of main modes of fixation of the most stable chromatin loop domains

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М. I. Chopei

2014-08-01

Full Text Available The main mechanism of DNA track formation during comet assay of nucleoids, obtained after removal of cell membranes and most of proteins, is the extension to anode of negatively supercoiled DNA loops attached to proteins, remaining in nucleoid after lysis treatment. The composition of these residual protein structures and the nature of their strong interaction with the loop ends remain poorly studied. In this work we investigated the influence of chloroquine intercalation and denaturation of nucleoid proteins on the efficiency of electrophoretic track formation during comet assay. The results obtained suggest that even gentle protein denaturation is sufficient to reduce considerably the effectiveness of the DNA loop migration due to an increase in the loops size. The same effect was observed under local DNA unwinding upon chloroquine intercalation around the sites of the attachment of DNA to proteins. The topological interaction (protein intercalation into the double helix between DNA loop ends and nucleoid proteins is discussed.

Absence of correlation between serum CRP levels and mitochondrial D-loop DNA mutations in gastro-oesophageal adenocarcinoma

Directory of Open Access Journals (Sweden)

Benjamin H. L. Tan

2014-01-01

Full Text Available Introduction: Both inflammation and mitochondrial DNA (mtDNA mutation are thought to play a role in the many human cancers. The aim of this study was to evaluate the relationship between inflammation and accumulation of mitochondrial DNA (mtDNA mutations in the D-loop region in carcinogenesis of gastro-oesophageal adenocarcinomas. Materials and Methods: Blood samples of 20 patients with gastro-oesophageal adenocarcinoma were taken for measurement of serum C-reactive protein (CRP concentration. Direct sequencing of mtDNA in the D-loop region was done in the 20 adenocarcinoma samples and their corresponding surrounding non-cancerous tissue. Sequences were compared with existing mtDNA databases to identify mutations. Results: mtDNA mutations in the D-loop region occur commonly with almost identical frequency in both non-cancerous tissue (3.0 ΁ 1.6 and adenocarcinoma (3.1 ΁ 1.9 (P = 0.916, paired t-test. CRP levels are not predictive of the number of D-loop mutations in both adenocarcinoma (β: -0.131; 95% CI: -2.354-1.364; P = 0.583 and non-cancerous tissue samples (β: 0.130; 95% CI: -1.125-1.933; P = 0.586. Five new mutations were identified that were not recorded previously in mtDNA databases. Conclusion: D-loop mtDNA mutations are common in both gastro-oesophageal adenocarcinoma and surrounding non-cancerous tissue. However, the accumulation of such mutations appears to occur independent of systemic inflammation. The frequency of D-loop mutations is likely not useful as a marker for carcinogenesis in gastro-oesophageal adenocarcinoma.

Human ribonuclease H1 resolves R-loops and thereby enables progression of the DNA replication fork.

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Parajuli, Shankar; Teasley, Daniel C; Murali, Bhavna; Jackson, Jessica; Vindigni, Alessandro; Stewart, Sheila A

2017-09-15

Faithful DNA replication is essential for genome stability. To ensure accurate replication, numerous complex and redundant replication and repair mechanisms function in tandem with the core replication proteins to ensure DNA replication continues even when replication challenges are present that could impede progression of the replication fork. A unique topological challenge to the replication machinery is posed by RNA-DNA hybrids, commonly referred to as R-loops. Although R-loops play important roles in gene expression and recombination at immunoglobulin sites, their persistence is thought to interfere with DNA replication by slowing or impeding replication fork progression. Therefore, it is of interest to identify DNA-associated enzymes that help resolve replication-impeding R-loops. Here, using DNA fiber analysis, we demonstrate that human ribonuclease H1 (RNH1) plays an important role in replication fork movement in the mammalian nucleus by resolving R-loops. We found that RNH1 depletion results in accumulation of RNA-DNA hybrids, slowing of replication forks, and increased DNA damage. Our data uncovered a role for RNH1 in global DNA replication in the mammalian nucleus. Because accumulation of RNA-DNA hybrids is linked to various human cancers and neurodegenerative disorders, our study raises the possibility that replication fork progression might be impeded, adding to increased genomic instability and contributing to disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Mapping the structural and dynamical features of multiple p53 DNA binding domains: insights into loop 1 intrinsic dynamics.

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Suryani Lukman

Full Text Available The transcription factor p53 regulates cellular integrity in response to stress. p53 is mutated in more than half of cancerous cells, with a majority of the mutations localized to the DNA binding domain (DBD. In order to map the structural and dynamical features of the DBD, we carried out multiple copy molecular dynamics simulations (totaling 0.8 μs. Simulations show the loop 1 to be the most dynamic element among the DNA-contacting loops (loops 1-3. Loop 1 occupies two major conformational states: extended and recessed; the former but not the latter displays correlations in atomic fluctuations with those of loop 2 (~24 Å apart. Since loop 1 binds to the major groove whereas loop 2 binds to the minor groove of DNA, our results begin to provide some insight into the possible mechanism underpinning the cooperative nature of DBD binding to DNA. We propose (1 a novel mechanism underlying the dynamics of loop 1 and the possible tread-milling of p53 on DNA and (2 possible mutations on loop 1 residues to restore the transcriptional activity of an oncogenic mutation at a distant site.

In-stem labelling allows visualization of DNA strand displacements by distinct fluorescent colour change.

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Barrois, Sebastian; Wagenknecht, Hans-Achim

2013-05-21

The combination of thiazole orange (TO) and thiazole red (TR) as an internal pair of fluorescent DNA base surrogates ("DNA traffic lights") allows us to follow at least two consecutive DNA strand displacements in real time through a distinct fluorescence colour change from green to red and vice versa.

Multiplexed detection of DNA sequences using a competitive displacement assay in a microfluidic SERRS-based device.

Science.gov (United States)

Yazdi, Soroush H; Giles, Kristen L; White, Ian M

2013-11-05

We demonstrate sensitive and multiplexed detection of DNA sequences through a surface enhanced resonance Raman spectroscopy (SERRS)-based competitive displacement assay in an integrated microsystem. The use of the competitive displacement scheme, in which the target DNA sequence displaces a Raman-labeled reporter sequence that has lower affinity for the immobilized probe, enables detection of unlabeled target DNA sequences with a simple single-step procedure. In our implementation, the displacement reaction occurs in a microporous packed column of silica beads prefunctionalized with probe-reporter pairs. The use of a functionalized packed-bead column in a microfluidic channel provides two major advantages: (i) immobilization surface chemistry can be performed as a batch process instead of on a chip-by-chip basis, and (ii) the microporous network eliminates the diffusion limitations of a typical biological assay, which increases the sensitivity. Packed silica beads are also leveraged to improve the SERRS detection of the Raman-labeled reporter. Following displacement, the reporter adsorbs onto aggregated silver nanoparticles in a microfluidic mixer; the nanoparticle-reporter conjugates are then trapped and concentrated in the silica bead matrix, which leads to a significant increase in plasmonic nanoparticles and adsorbed Raman reporters within the detection volume as compared to an open microfluidic channel. The experimental results reported here demonstrate detection down to 100 pM of the target DNA sequence, and the experiments are shown to be specific, repeatable, and quantitative. Furthermore, we illustrate the advantage of using SERRS by demonstrating multiplexed detection. The sensitivity of the assay, combined with the advantages of multiplexed detection and single-step operation with unlabeled target sequences makes this method attractive for practical applications. Importantly, while we illustrate DNA sequence detection, the SERRS-based competitive

Protected DNA strand displacement for enhanced single nucleotide discrimination in double-stranded DNA

OpenAIRE

Khodakov, Dmitriy A.; Khodakova, Anastasia S.; Huang, David M.; Linacre, Adrian; Ellis, Amanda V.

2015-01-01

Single nucleotide polymorphisms (SNPs) are a prime source of genetic diversity. Discriminating between different SNPs provides an enormous leap towards the better understanding of the uniqueness of biological systems. Here we report on a new approach for SNP discrimination using toehold-mediated DNA strand displacement. The distinctiveness of the approach is based on the combination of both 3- and 4-way branch migration mechanisms, which allows for reliable discrimination of SNPs within doubl...

A Universal Fast Colorimetric Method for DNA Signal Detection with DNA Strand Displacement and Gold Nanoparticles

Directory of Open Access Journals (Sweden)

Xin Li

2015-01-01

Full Text Available DNA or gene signal detection is of great significance in many fields including medical examination, intracellular molecular monitoring, and gene disease signal diagnosis, but detection of DNA or gene signals in a low concentration with instant visual results remains a challenge. In this work, a universal fast and visual colorimetric detection method for DNA signals is proposed. Specifically, a DNA signal amplification “circuit” based on DNA strand displacement is firstly designed to amplify the target DNA signals, and then thiol modified hairpin DNA strands and gold nanoparticles are used to make signal detection results visualized in a colorimetric manner. If the target DNA signal exists, the gold nanoparticles aggregate and settle down with color changing from dark red to grey quickly; otherwise, the gold nanoparticles’ colloids remain stable in dark red. The proposed method provides a novel way to detect quickly DNA or gene signals in low concentrations with instant visual results. When applied in real-life, it may provide a universal colorimetric method for gene disease signal diagnosis.

RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain telomere integrity.

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Vannier, Jean-Baptiste; Pavicic-Kaltenbrunner, Visnja; Petalcorin, Mark I R; Ding, Hao; Boulton, Simon J

2012-05-11

T loops and telomeric G-quadruplex (G4) DNA structures pose a potential threat to genome stability and must be dismantled to permit efficient telomere replication. Here we implicate the helicase RTEL1 in the removal of telomeric DNA secondary structures, which is essential for preventing telomere fragility and loss. In the absence of RTEL1, T loops are inappropriately resolved by the SLX4 nuclease complex, resulting in loss of the telomere as a circle. Depleting SLX4 or blocking DNA replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(-/-) cells but failed to suppress telomere fragility. Conversely, stabilization of telomeric G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient cells but had no impact on TC formation or telomere loss. We propose that RTEL1 performs two distinct functions at telomeres: it disassembles T loops and also counteracts telomeric G4-DNA structures, which together ensure the dynamics and stability of the telomere. Copyright © 2012 Elsevier Inc. All rights reserved.

Programming chemical kinetics: engineering dynamic reaction networks with DNA strand displacement

Science.gov (United States)

Srinivas, Niranjan

Over the last century, the silicon revolution has enabled us to build faster, smaller and more sophisticated computers. Today, these computers control phones, cars, satellites, assembly lines, and other electromechanical devices. Just as electrical wiring controls electromechanical devices, living organisms employ "chemical wiring" to make decisions about their environment and control physical processes. Currently, the big difference between these two substrates is that while we have the abstractions, design principles, verification and fabrication techniques in place for programming with silicon, we have no comparable understanding or expertise for programming chemistry. In this thesis we take a small step towards the goal of learning how to systematically engineer prescribed non-equilibrium dynamical behaviors in chemical systems. We use the formalism of chemical reaction networks (CRNs), combined with mass-action kinetics, as our programming language for specifying dynamical behaviors. Leveraging the tools of nucleic acid nanotechnology (introduced in Chapter 1), we employ synthetic DNA molecules as our molecular architecture and toehold-mediated DNA strand displacement as our reaction primitive. Abstraction, modular design and systematic fabrication can work only with well-understood and quantitatively characterized tools. Therefore, we embark on a detailed study of the "device physics" of DNA strand displacement (Chapter 2). We present a unified view of strand displacement biophysics and kinetics by studying the process at multiple levels of detail, using an intuitive model of a random walk on a 1-dimensional energy landscape, a secondary structure kinetics model with single base-pair steps, and a coarse-grained molecular model that incorporates three-dimensional geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Our findings are consistent with previously measured or inferred rates for

Exploring the Dynamics of Propeller Loops in Human Telomeric DNA Quadruplexes Using Atomistic Simulations

Science.gov (United States)

2017-01-01

We have carried out a series of extended unbiased molecular dynamics (MD) simulations (up to 10 μs long, ∼162 μs in total) complemented by replica-exchange with the collective variable tempering (RECT) approach for several human telomeric DNA G-quadruplex (GQ) topologies with TTA propeller loops. We used different AMBER DNA force-field variants and also processed simulations by Markov State Model (MSM) analysis. The slow conformational transitions in the propeller loops took place on a scale of a few μs, emphasizing the need for long simulations in studies of GQ dynamics. The propeller loops sampled similar ensembles for all GQ topologies and for all force-field dihedral-potential variants. The outcomes of standard and RECT simulations were consistent and captured similar spectrum of loop conformations. However, the most common crystallographic loop conformation was very unstable with all force-field versions. Although the loss of canonical γ-trans state of the first propeller loop nucleotide could be related to the indispensable bsc0 α/γ dihedral potential, even supporting this particular dihedral by a bias was insufficient to populate the experimentally dominant loop conformation. In conclusion, while our simulations were capable of providing a reasonable albeit not converged sampling of the TTA propeller loop conformational space, the force-field description still remained far from satisfactory. PMID:28475322

Loop-mediated isothermal amplification (LAMP) shield for Arduino DNA detection.

Science.gov (United States)

Velders, Aldrik H; Schoen, Cor; Saggiomo, Vittorio

2018-02-01

Loop-mediated isothermal amplification (LAMP) of DNA is gaining relevance as a method to detect nucleic acids, as it is easier, faster, and more powerful than conventional Polymerase Chain Reaction. However, LAMP is still mostly used in laboratory settings, because of the lack of a cheap and easy, one-button device that can perform LAMP experiments. Here we show how to build and program an Arduino shield for a LAMP and detection of DNA. The here described Arduino Shield is cheap, easy to assemble, to program and use, it is battery operated and the detection of DNA is done by naked-eye so that it can be used in field.

Loop-mediated isothermal amplification (LAMP) shield for Arduino DNA detection

NARCIS (Netherlands)

Velders, Aldrik H.; Schoen, Cor; Saggiomo, Vittorio

2018-01-01

Objective: Loop-mediated isothermal amplification (LAMP) of DNA is gaining relevance as a method to detect nucleic acids, as it is easier, faster, and more powerful than conventional Polymerase Chain Reaction. However, LAMP is still mostly used in laboratory settings, because of the lack of a cheap

Influence of minor displacements in loops of the porcine parvovirus VP2 capsid on virus-like particles assembly and the induction of antibody responses.

Science.gov (United States)

Pan, Qunxing; He, Kongwang; Wang, Yongshan; Wang, Xiaoli; Ouyang, Wei

2013-06-01

An antigen-delivery system based on hybrid virus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of porcine parvovirus (PPV) and expressing foreign peptides offers an alternative method for vaccination. In this study, the three-dimensional structure of the PPV capsid protein and surface loops deletion mutants were analyzed to define essential domains in PPV VP2 for the assembly of VLPs. Electron microscopic analysis and SDS-PAGE analysis confirmed the presence of abundant VLPs in a loop2 deletion mutant of expected size and appropriate morphology. Loop4 and loop2-loop4 deletion mutants, however, resulted in a lower number of particles and the morphology of the particles was not well preserved. Furthermore, the green fluorescent protein (gfp) gene was used as a model. GFP was observed at the same level in displacements mutants. However, GFP displacement mutants in loop2 construct allowed better adaptation for the fusion GFP to be further displayed on the surface of the capsid-like structure. Immunogenicity study showed that there is no obvious difference in mice inoculated with rAd-VP2(Δloop2), rAd-VP2(Δloop4), rAd-VP2(Δloop2-Δloop4), and PPV inactivated vaccine. The results suggested the possibility of inserting simultaneously B and T cell epitopes in the surface loop2 and the N-terminus. The combination of different types of epitopes (B, CD4+, and CD8+) in different positions of the PPV particles opens the way to the development of highly efficient vaccines, able to stimulate at the same time the different branches of the immune system.

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks.

Science.gov (United States)

Chen, Yuan-Jyue; Rao, Sundipta D; Seelig, Georg

2015-11-25

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

DNA-mediated strand displacement facilitates sensitive electronic detection of antibodies in human serums.

Science.gov (United States)

Dou, Baoting; Yang, Jianmei; Shi, Kai; Yuan, Ruo; Xiang, Yun

2016-09-15

We describe here the development of a sensitive and convenient electronic sensor for the detection of antibodies in human serums. The sensor is constructed by self-assembly formation of a mixed monolayer containing the small molecule epitope conjugated double stranded DNA probes on gold electrode. The target antibody binds the epitope on the dsDNA probe and lowers the melting temperature of the duplex, which facilitates the displacement of the antibody-linked strand of the duplex probe by an invading methylene blue-tagged single stranded DNA (MB-ssDNA) through the strand displacement reaction and leads to the capture of many MB-ssDNA on the sensor surface. Subsequent electrochemical oxidation of the methylene blue labels results in amplified current response for sensitive monitoring of the antibodies. The antibody assay conditions are optimized and the sensor exhibits a linear range between 1.0 and 25.0nM with a detection limit of 0.67nM for the target antibody. The sensor is also selective and can be employed to detect the target antibodies in human serum samples. With the advantages of using small molecule epitope as the antibody recognition element over traditional antigen, the versatile manipulability of the DNA probes and the unique properties of the electrochemical transduction technique, the developed sensor thus hold great potential for simple and sensitive detection of different antibodies and other proteins in real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Mechanism and manipulation of DNA:RNA hybrid G-quadruplex formation in transcription of G-rich DNA.

Science.gov (United States)

Zhang, Jia-yu; Zheng, Ke-wei; Xiao, Shan; Hao, Yu-hua; Tan, Zheng

2014-01-29

We recently reported that a DNA:RNA hybrid G-quadruplex (HQ) forms during transcription of DNA that bears two or more tandem guanine tracts (G-tract) on the nontemplate strand. Putative HQ-forming sequences are enriched in the nearby 1000 nt region right downstream of transcription start sites in the nontemplate strand of warm-blooded animals, and HQ regulates transcription under both in vitro and in vivo conditions. Therefore, knowledge of the mechanism of HQ formation is important for understanding the biological function of HQ as well as for manipulating gene expression by targeting HQ. In this work, we studied the mechanism of HQ formation using an in vitro T7 transcription model. We show that RNA synthesis initially produces an R-loop, a DNA:RNA heteroduplex formed by a nascent RNA transcript and the template DNA strand. In the following round of transcription, the RNA in the R-loop is displaced, releasing the RNA in single-stranded form (ssRNA). Then the G-tracts in the RNA can jointly form HQ with those in the nontemplate DNA strand. We demonstrate that the structural cascade R-loop → ssRNA → HQ offers opportunities to intercept HQ formation, which may provide a potential method to manipulate gene expression.

Label-free fluorescence strategy for sensitive detection of adenosine triphosphate using a loop DNA probe with low background noise.

Science.gov (United States)

Lin, Chunshui; Cai, Zhixiong; Wang, Yiru; Zhu, Zhi; Yang, Chaoyong James; Chen, Xi

2014-07-15

A simple, rapid, label-free, and ultrasensitive fluorescence strategy for adenosine triphosphate (ATP) detection was developed using a loop DNA probe with low background noise. In this strategy, a loop DNA probe, which is the substrate for both ligation and digestion enzyme reaction, was designed. SYBR green I (SG I), a double-stranded specific dye, was applied for the readout fluorescence signal. Exonuclease I (Exo I) and exonuclease III (Exo III), sequence-independent nucleases, were selected to digest the loop DNA probe in order to minimize the background fluorescence signal. As a result, in the absence of ATP, the loop DNA was completely digested by Exo I and Exo III, leading to low background fluorescence owing to the weak electrostatic interaction between SG I and mononucleotides. On the other hand, ATP induced the ligation of the nicking site, and the sealed loop DNA resisted the digestion of Exo I and ExoIII, resulting in a remarkable increase of fluorescence response. Upon background noise reduction, the sensitivity of the ATP determination was improved significantly, and the detection limitation was found to be 1.2 pM, which is much lower than that in almost all the previously reported methods. This strategy has promise for wide application in the determination of ATP.

Effect of structure variation of the aptamer-DNA duplex probe on the performance of displacement-based electrochemical aptamer sensors.

Science.gov (United States)

Pang, Jie; Zhang, Ziping; Jin, Haizhu

2016-03-15

Electrochemical aptamer-based (E-AB) sensors employing electrode-immobilized, redox-tagged aptamer probes have emerged as a promising platform for the sensitive and quick detection of target analytes ranging from small molecules to proteins. Signal generation in this class of sensor is linked to change in electron transfer efficiency upon binding-induced change in flexibility/conformation of the aptamer probe. Because of this signaling mechanism, signal gains of these sensors can be improved by employing a displacement-based recognition system, which links target binding with a large-scale flexibility/conformation shift from the aptamer-DNA duplex to the single-stranded DNA or the native aptamer. Despite the relatively large number of displacement-based E-AB sensor samples, little attention has been paid to the structure variation of the aptamer-DNA duplex probe. Here we detail the effects of complementary length and position of the aptamer-DNA duplex probe on the performance of a model displacement-based E-AB sensor for ATP. We find that, greater background suppression and signal gain are observed with longer complementary length of the aptamer-DNA duplex probe. However, sensor equilibration time slows monotonically with increasing complementary length; and with too many target binding sites in aptamer sequence being occupied by the complementary DNA, the aptamer-target binding does not occur and no signal gain observed. We also demonstrate that signal gain of the displacement-based E-AB sensor is strongly dependent on the complementary position of the aptamer-DNA duplex probe, with complementary position located at the electrode-attached or redox-tagged end of the duplex probe, larger background suppression and signal increase than that of the middle position are observed. These results highlight the importance of rational structure design of the aptamer-DNA duplex probe and provide new insights into the optimization of displacement-based E-AB sensors. Copyright

Conformation and dynamics of nucleotides in bulges and symmetric internal loops in duplex DNA studied by EPR and fluorescence spectroscopies

International Nuclear Information System (INIS)

Cekan, Pavol; Sigurdsson, Snorri Th.

2012-01-01

Highlights: ► Bulges and loops were studied by both EPR and fluorescence spectroscopies using the probe Ç/Ç f . ► One-base bulge was in a temperature-dependent equilibrium between looped-out and stacked states. ► Bases in two- and three-base bulges were stacked at all temperatures, resulting in DNA bending. ► Bases were stacked in symmetrical two- to five-base internal loops, according to EPR data. ► Unexpectedly high fluorescence for the smaller loops indicated local structural perturbations. -- Abstract: The dynamics and conformation of base bulges and internal loops in duplex DNA were studied using the bifunctional spectroscopic probe Ç, which becomes fluorescent (Ç f ) upon reduction of the nitroxide functional group, along with EPR and fluorescence spectroscopies. A one-base bulge was in a conformational equilibrium between looped-out and stacked states, the former favored at higher temperature and the latter at lower temperature. Stacking of bulge bases was favored in two- and three-base bulges, independent of temperature, resulting in DNA bending as evidenced by increased fluorescence of Ç f . EPR spectra of Ç-labeled three-, four- and five-base symmetrical interior DNA bulges at 20 °C showed low mobility, indicating that the spin-label was stacked within the loop. The spin-label mobility at 37 °C increased as the loops became larger. A considerable variation in fluorescence between different loops was observed, as well as a temperature-dependence within constructs. Fluorescence unexpectedly increased as the size of the loop decreased at 2 °C. Fluorescence of the smallest loops, where a single T·T mismatch was located between the stem region and the probe, was even larger than for the single strand, indicating a considerable local structural deformation of these loops from regular B-DNA. These results show the value of combining EPR and fluorescence spectroscopy to study non-helical regions of nucleic acids.

In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level.

Science.gov (United States)

Michikawa, Yuichi; Sugahara, Keisuke; Suga, Tomo; Ohtsuka, Yoshimi; Ishikawa, Kenichi; Ishikawa, Atsuko; Shiomi, Naoko; Shiomi, Tadahiro; Iwakawa, Mayumi; Imai, Takashi

2008-12-15

The isolation and multiple genotyping of long individual DNA fragments are needed to obtain haplotype information for diploid organisms. Limiting dilution of sample DNA followed by multiple displacement amplification is a useful technique but is restricted to short (reaction (PCR)-ready form. The haplotypes of seven SNPs spanning 240 kb of the DNA surrounding the human ATM gene region on chromosome 11 were determined for 10 individuals, demonstrating the feasibility of this new method.

DNA damage mediated transcription arrest: Step back to go forward.

Science.gov (United States)

Mullenders, Leon

2015-12-01

The disturbance of DNA helix conformation by bulky DNA damage poses hindrance to transcription elongating due to stalling of RNA polymerase at transcription blocking lesions. Stalling of RNA polymerase provokes the formation of R-loops, i.e. the formation of a DNA-RNA hybrid and a displaced single stranded DNA strand as well as displacement of spliceosomes. R-loops are processed into DNA single and double strand breaks by NER factors depending on TC-NER factors leading to genome instability. Moreover, stalling of RNA polymerase induces a strong signal for cell cycle arrest and apoptosis. These toxic and mutagenic effects are counteracted by a rapid recruitment of DNA repair proteins to perform transcription coupled nucleotide excision repair (TC-NER) to remove the blocking DNA lesions and to restore transcription. Recent studies have highlighted the role of backtracking of RNA polymerase to facilitate TC-NER and identified novel factors that play key roles in TC-NER and in restoration of transcription. On the molecular level these factors facilitate stability of the repair complex by promotion and regulation of various post-translational modifications of NER factors and chromatin substrate. In addition, the continuous flow of new factors that emerge from screening assays hints to several regulatory levels to safeguard the integrity of transcription elongation after disturbance by DNA damage that have yet to be explored. Copyright © 2015 Elsevier B.V. All rights reserved.

Cooperation of DNA-PKcs and WRN helicase in the maintenance of telomeric D-loops

DEFF Research Database (Denmark)

Kusumoto-Matsuo, Rika; Opresko, Patricia L; Ramsden, Dale

2010-01-01

Werner syndrome is an inherited human progeriod syndrome caused by mutations in the gene encoding the Werner Syndrome protein, WRN. It has both 3'-5' DNA helicase and exonuclease activities, and is suggested to have roles in many aspects of DNA metabolism, including DNA repair and telomere...... D-loop model substrate. In addition, the length of telomeric G-tails decreases in DNA-PKcs knockdown cells, and this phenotype is reversed by overexpression of WRN helicase. These results suggest that WRN and DNA-PKcs may cooperatively prevent G-tail shortening in vivo....

Gener: a minimal programming module for chemical controllers based on DNA strand displacement.

Science.gov (United States)

Kahramanoğulları, Ozan; Cardelli, Luca

2015-09-01

: Gener is a development module for programming chemical controllers based on DNA strand displacement. Gener is developed with the aim of providing a simple interface that minimizes the opportunities for programming errors: Gener allows the user to test the computations of the DNA programs based on a simple two-domain strand displacement algebra, the minimal available so far. The tool allows the user to perform stepwise computations with respect to the rules of the algebra as well as exhaustive search of the computation space with different options for exploration and visualization. Gener can be used in combination with existing tools, and in particular, its programs can be exported to Microsoft Research's DSD tool as well as to LaTeX. Gener is available for download at the Cosbi website at http://www.cosbi.eu/research/prototypes/gener as a windows executable that can be run on Mac OS X and Linux by using Mono. ozan@cosbi.eu. © The Author 2015. Published by Oxford University Press.

Impact of Somatic Mutations in the D-Loop of Mitochondrial DNA on the Survival of Oral Squamous Cell Carcinoma Patients

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Lin, Jin-Ching; Wang, Chen-Chi; Jiang, Rong-San; Wang, Wen-Yi; Liu, Shih-An

2015-01-01

Objectives The aim of this study was to investigate somatic mutations in the D-loop of mitochondrial DNA (mtDNA) and their impact on survival in oral squamous cell carcinoma patients. Materials and Methods Surgical specimen confirmed by pathological examination and corresponding non-cancerous tissues were collected from 120 oral squamous cell carcinoma patients. The sequence in the D-loop of mtDNA from non-cancerous tissues was compared with that from paired cancer samples and any sequence differences were recognized as somatic mutations. Results Somatic mutations in the D-loop of mtDNA were identified in 75 (62.5%) oral squamous cell carcinoma patients and most of them occurred in the poly-C tract. Although there were no significant differences in demographic and tumor-related features between participants with and without somatic mutation, the mutation group had a better survival rate (5 year disease-specific survival rate: 64.0% vs. 43.0%, P = 0.0266). Conclusion Somatic mutation in D-loop of mtDNA was associated with a better survival in oral squamous cell carcinoma patients. PMID:25906372

[The mutations of the D-loop hypervariable region II and hypervariable region III of mitochondrial DNA in oral squamous cell carcinoma].

Science.gov (United States)

Wang, Yao-Zhong; Jia, Mu-Yun; Yuan, Rong-Tao; Han, Guo-Dong; Bu, Ling-Xue

2010-06-01

To investigate the frequency of mitochondrial DNA (mtDNA) D-loop hypervariable region II (HVR II) and hypervariable region III (HVR III) mutations in oral squamous cell carcinoma (OSCC) and their correlation to provide the new targets for the prevention and treatment of OSCC. The D-loop HVR II and HVR III regions of mtDNA in seven cases with OSCC tissues, matched with paracancerous tissues and normal mucosa tissues from the same case, were amplified by polymerase chain raction (PCR), then were detected by direct sequencing to find the mutantsites after the comparison of all sequencing results with the mtDNA Cambridge sequence in the GenBank database. 82 (56 species) nucleotide changes, with 51(26 species) nucleotide polymorphism, were found after the comparison of all sequencing results with the mtDNA Cambridge sequence in the GenBank database. 31(30 species) mutations, with 21 located within the HVR II and HVR III regions, were found in 3 tumor tissue samples, their paracancerous and normal mucosa tissue were found more polymorphic changes but no mutation. The mtDNA D-loop HVR II and HVR III regions mutation rate was 42.9% (3/7) in OSCC. The mtDNA D-loop HVR II and HVR III regions were highly polymorphic and mutable regions in OSCC. It suggested that the D-loop HVR II and HVR III regions of mtDNA might play a significant role in the tumorigenesis of OSCC. It may become new targets for the gene therapy of OSCC by regulating the above indexes.

Toehold-mediated strand displacement reaction triggered isothermal DNA amplification for highly sensitive and selective fluorescent detection of single-base mutation.

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Zhu, Jing; Ding, Yongshun; Liu, Xingti; Wang, Lei; Jiang, Wei

2014-09-15

Highly sensitive and selective detection strategy for single-base mutations is essential for risk assessment of malignancy and disease prognosis. In this work, a fluorescent detection method for single-base mutation was proposed based on high selectivity of toehold-mediated strand displacement reaction (TSDR) and powerful signal amplification capability of isothermal DNA amplification. A discrimination probe was specially designed with a stem-loop structure and an overhanging toehold domain. Hybridization between the toehold domain and the perfect matched target initiated the TSDR along with the unfolding of the discrimination probe. Subsequently, the target sequence acted as a primer to initiate the polymerization and nicking reactions, which released a great abundant of short sequences. Finally, the released strands were annealed with the reporter probe, launching another polymerization and nicking reaction to produce lots of G-quadruplex DNA, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. However, when there was even a single base mismatch in the target DNA, the TSDR was suppressed and so subsequent isothermal DNA amplification and fluorescence response process could not occur. The proposed approach has been successfully implemented for the identification of the single-base mutant sequences in the human KRAS gene with a detection limit of 1.8 pM. Furthermore, a recovery of 90% was obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of this detection strategy for single-base mutations in biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

DNA hybridization kinetics: zippering, internal displacement and sequence dependence.

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Ouldridge, Thomas E; Sulc, Petr; Romano, Flavio; Doye, Jonathan P K; Louis, Ard A

2013-10-01

Although the thermodynamics of DNA hybridization is generally well established, the kinetics of this classic transition is less well understood. Providing such understanding has new urgency because DNA nanotechnology often depends critically on binding rates. Here, we explore DNA oligomer hybridization kinetics using a coarse-grained model. Strand association proceeds through a complex set of intermediate states, with successful binding events initiated by a few metastable base-pairing interactions, followed by zippering of the remaining bonds. But despite reasonably strong interstrand interactions, initial contacts frequently dissociate because typical configurations in which they form differ from typical states of similar enthalpy in the double-stranded equilibrium ensemble. Initial contacts must be stabilized by two or three base pairs before full zippering is likely, resulting in negative effective activation enthalpies. Non-Arrhenius behavior arises because the number of base pairs required for nucleation increases with temperature. In addition, we observe two alternative pathways-pseudoknot and inchworm internal displacement-through which misaligned duplexes can rearrange to form duplexes. These pathways accelerate hybridization. Our results explain why experimentally observed association rates of GC-rich oligomers are higher than rates of AT- rich equivalents, and more generally demonstrate how association rates can be modulated by sequence choice.

Detection of HbsAg and hATIII genetically modified goats (Caprahircus) by loop-mediated isothermal amplification.

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Tao, Chenyu; Zhang, Qingde; Zhai, Shanli; Liu, Bang

2013-11-01

In this study, sensitive and rapid detection systems were designed using a loop-mediated isothermal amplification (LAMP) method to detect the genetically modified goats. A set of 4 primers were designed for each exogenous nucleic acids HBsAg and hATIII. The DNA samples were first amplified with the outer and inner primers and released a single-stranded DNA,of which both ends were stem-loop structure. Then one inner primer hybridized with the loop, and initiated displacement synthesis in less than 1 h. The result could be visualized by both agarose gel electrophoresis and unaided eyes directly after adding SYBR GREEN 1. The detection limit of LAMP was ten copies of target molecules, indicating that LAMP was tenfold more sensitive than the classical PCR. Furthermore, all the samples of genetically modified goats were tested positively by LAMP, and the results demonstrated that the LAMP was a rapid and sensitive method for detecting the genetically modified organism.

Diagnosis of brugian filariasis by loop-mediated isothermal amplification.

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Catherine B Poole

Full Text Available In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis.

Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay

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Xing, Yun-Peng; Liu, Chun; Zhou, Xiao-Hong; Shi, Han-Chang

2015-01-01

This work was the first to report that the kanamycin-binding DNA aptamer (5'-TGG GGG TTG AGG CTA AGC CGA-3') can form stable parallel G-quadruplex DNA (G4-DNA) structures by themselves and that this phenomenon can be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, we developed a novel label-free strategy for kanamycin detection based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange (TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent upon binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA-TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. Our results also served as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future.

Human mitochondrial DNA (mtDNA) types in Malaysia

International Nuclear Information System (INIS)

Lian, L.H.; Koh, C.L.; Lim, M.E.

2000-01-01

Each human cell contains hundreds of mitochondria and thousands of double-stranded circular mtDNA. The delineation of human mtDNA variation and genetics over the past decade has provided unique and often startling insights into human evolution, degenerative diseases, and aging. Each mtDNA of 16,569 base pairs, encodes 13 polypeptides essential to the enzymes of the mitochondrial energy generating pathway, plus the necessary tRNAs and rRNAs. The highly polymorphic noncoding D-(displacement) loop region, also called the control region, is approximately 1.2 kb long. It contains two well-characterized hypervariable (HV-) regions, HV1 and HV2. MtDNA identification is usually based on these sequence differences. According to the TWTGDAM (Technical Working Group for DNA Analysis Methods), the minimum requirement for a mtDNA database for HV1 is from positions 16024 to 16365 and for HV2, from positions 00073 to 00340. The targeted Malaysian population subgroups for this study were mainly the Malays, Chinese, Indians, and indigenous Ibans, Bidayuhs, Kadazan-Dusuns, and Bajaus. Research methodologies undertaken included DNA extraction of samples from unrelated individuals, amplification of the specific regions via the polymerase chain reaction (PCR), and preparation of template DNA for sequencing by using an automated DNA sequencer. Sufficient nucleotide sequence data were generated from the mtDNA analysis. When the sequences were analyzed, sequence variations were found to be caused by nucleotide substitutions, insertions, and deletions. Of the three causes of the sequence variations, nucleotide substitutions (86.1%) accounted for the vast majority of polymorphism. It is noted that transitions (83.5%) were predominant when compared to the significantly lower frequencies of transversions (2.6%). Insertions (0.9%) and deletions (13.0%) were rather rare and found only in HV2. The data generated will also form the basis of a Malaysian DNA sequence database of mtDNA D-loop

Genetic distance of Malaysian mousedeer based on mitochondrial DNA cytochrome oxidase I (COI) and D-loop region sequences

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Bakar, Mohamad-Azam Akmal Abu; Rovie-Ryan, Jeffrine Japning; Ampeng, Ahmad; Yaakop, Salmah; Nor, Shukor Md; Md-Zain, Badrul Munir

2018-04-01

Mousedeer is one of the primitive mammals that can be found mainly in Southeast-Asia region. There are two species of mousedeer in Malaysia which are Tragulus kanchil and Tragulus napu. Both species can be distinguish by size, coat coloration, and throat pattern but clear diagnosis still cannot be found. The objective of the study is to show the genetic distance relationship between T. kanchil and T. napu and their population based on mitochondrial DNA (mtDNA) cytochrome oxidase I (COI) and D-loop region. There are 42 sample of mousedeer were used in this study collected by PERHILITAN from different locality. Another 29 D-loop sequence were retrieved from Genbank for comparative analysis. All sample were amplified using universal primer and species-specific primer for COI and D-loop genes via PCR process. The amplified sequences were analyzed to determine genetic distance of T. kanchil and T. napu. From the analysis, the average genetic distance between T. kanchil and T. napu based on locus COI and D-loop were 0.145 and 0.128 respectively. The genetic distance between populations of T. kanchil based on locus COI was between 0.003-0.013. For locus D-loop, genetic distance analysis showed distance in relationship between west-coast populations to east-coast population of T. kanchil. COI and D-loop mtDNA region provided a clear picture on the relationship within the mousedeer species. Last but not least, conservation effort toward protecting this species can be done by study the molecular genetics and prevent the extinction of this species.

Volumetric contributions of loop regions of G-quadruplex DNA to the formation of the tertiary structure.

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Takahashi, Shuntaro; Sugimoto, Naoki

2017-12-01

DNA guanine-quadruplexes (G-quadruplexes) are unique DNA structures formed by guanine-rich sequences. The loop regions of G-quadruplexes play key roles in stability and topology of G-quadruplexes. Here, we investigated volumetric changes induced by pressure in the folding of the G-quadruplex formed by the thrombin binding aptamer (TBA) with mutations within the loop regions. The change of partial molar volume in the transition from coil to G-quadruplex, ∆V tr , of TBA with a mutation from T to A in the 5' most loop (TBA T3A) was 75.5cm 3 mol -1 , which was larger than that of TBA (54.6cm 3 mol -1 ). TBA with a G to T mutation in the central loop (TBA G8T) had thermal stability similar to TBA T3A but a smaller ∆V tr of 41.1cm 3 mol -1 . In the presence of poly(ethylene)glycol 200 (PEG200), ∆V tr values were 14.7cm 3 mol -1 for TBA T3A and 13.2cm 3 mol -1 for TBA G8T. These results suggest that the two mutations destabilize the G-quadruplex structure differently. Thus, volumetric data obtained using pressure-based thermodynamic analyses provides information about the dynamics of the loop regions and the roles of loops in the stabilities and folding of G-quadruplex structures. Copyright © 2017 Elsevier B.V. All rights reserved.

Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification.

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Cheng, Rui; Tao, Mangjuan; Shi, Zhilu; Zhang, Xiafei; Jin, Yan; Li, Baoxin

2015-11-15

Several fluorescence signal amplification strategies have been developed for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity, but they need fluorescence dye labeled DNA probe. We have addressed the limitation and report here a label-free strategy for sensitive detection of PNK activity by coupling DNA strand displacement reaction with enzymatic-aided amplification. A hairpin oligonucleotide (hpDNA) with blunt ends was used as the substrate for T4 PNK phosphorylation. In the presence of T4 PNK, the stem of hpDNA was phosphorylated and further degraded by lambda exonuclease (λ exo) from 5' to 3' direction to release a single-stranded DNA as a trigger of DNA strand displacement reaction (SDR). The trigger DNA can continuously displace DNA P2 from P1/P2 hybrid with the help of specific cleavage of nicking endonuclease (Nt.BbvCI). Then, DNA P2 can form G-quadruplex in the presence of potassium ions and quadruplex-selective fluorphore, N-methyl mesoporphyrin IX (NMM), resulting in a significant increase in fluorescence intensity of NMM. Thus, the accumulative release of DNA P2 led to fluorescence signal amplification for determining T4 PNK activity with a detection limit of 6.6×10(-4) U/mL, which is superior or comparative with established approaches. By ingeniously utilizing T4 PNK-triggered DNA SDR, T4 PNK activity can be specifically and facilely studied in homogeneous solution containing complex matrix without any external fluorescence labeling. Moreover, the influence of different inhibitors on the T4 PNK activity revealed that it also can be explored to screen T4 PNK inhibitors. Therefore, this label-free amplification strategy presents a facile and cost-effective approach for nucleic acid phosphorylation related research. Copyright © 2015 Elsevier B.V. All rights reserved.

Highly sensitive "signal-on" electrochemiluminescent biosensor for the detection of DNA based on dual quenching and strand displacement reaction.

Science.gov (United States)

Lou, Jing; Wang, Zhaoyin; Wang, Xiao; Bao, Jianchun; Tu, Wenwen; Dai, Zhihui

2015-10-07

A "signal-on" electrochemiluminescent DNA biosensing platform was proposed based on the dual quenching and strand displacement reaction. This novel "signal-on" detection strategy revealed its sensitivity in achieving a detection limit of 2.4 aM and its selectivity in distinguishing single nucleotide polymorphism of target DNA.

Label-free logic modules and two-layer cascade based on stem-loop probes containing a G-quadruplex domain.

Science.gov (United States)

Guo, Yahui; Cheng, Junjie; Wang, Jine; Zhou, Xiaodong; Hu, Jiming; Pei, Renjun

2014-09-01

A simple, versatile, and label-free DNA computing strategy was designed by using toehold-mediated strand displacement and stem-loop probes. A full set of logic gates (YES, NOT, OR, NAND, AND, INHIBIT, NOR, XOR, XNOR) and a two-layer logic cascade were constructed. The probes contain a G-quadruplex domain, which was blocked or unfolded through inputs initiating strand displacement and the obviously distinguishable light-up fluorescent signal of G-quadruplex/NMM complex was used as the output readout. The inputs are the disease-specific nucleotide sequences with potential for clinic diagnosis. The developed versatile computing system based on our label-free and modular strategy might be adapted in multi-target diagnosis through DNA hybridization and aptamer-target interaction. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genetic diversity of mtDNA D-loop sequences in four native Chinese chicken breeds.

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Guo, H W; Li, C; Wang, X N; Li, Z J; Sun, G R; Li, G X; Liu, X J; Kang, X T; Han, R L

2017-10-01

1. To explore the genetic diversity of Chinese indigenous chicken breeds, a 585 bp fragment of the mitochondrial DNA (mtDNA) region was sequenced in 102 birds from the Xichuan black-bone chicken, Yunyang black-bone chicken and Lushi chicken. In addition, 30 mtDNA D-loop sequences of Silkie fowls were downloaded from NCBI. The mtDNA D-loop sequence polymorphism and maternal origin of 4 chicken breeds were analysed in this study. 2. The results showed that a total of 33 mutation sites and 28 haplotypes were detected in the 4 chicken breeds. The haplotype diversity and nucleotide diversity of these 4 native breeds were 0.916 ± 0.014 and 0.012 ± 0.002, respectively. Three clusters were formed in 4 Chinese native chickens and 12 reference breeds. Both the Xichuan black-bone chicken and Yunyang black-bone chicken were grouped into one cluster. Four haplogroups (A, B, C and E) emerged in the median-joining network in these breeds. 3. It was concluded that these 4 Chinese chicken breeds had high genetic diversity. The phylogenetic tree and median network profiles showed that Chinese native chickens and its neighbouring countries had at least two maternal origins, one from Yunnan, China and another from Southeast Asia or its surrounding area.

Whole-loop mitochondrial DNA D-loop sequence variability in Egyptian Arabian equine matrilines

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Hudson, William

2017-01-01

Background Egyptian Arabian horses have been maintained in a state of genetic isolation for over a hundred years. There is only limited genetic proof that the studbook records of female lines of Egyptian Arabian pedigrees are reliable. This study characterized the mitochondrial DNA (mtDNA) signatures of 126 horses representing 14 matrilines in the Egyptian Agricultural Organization (EAO) horse-breeding program. Findings Analysis of the whole D-loop sequence yielded additional information compared to hypervariable region-1 (HVR1) analysis alone, with 42 polymorphic sites representing ten haplotypes compared to 16 polymorphic sites representing nine haplotypes, respectively. Most EAO haplotypes belonged to ancient haplogroups, suggesting origin from a wide geographical area over many thousands of years, although one haplotype was novel. Conclusions Historical families share haplotypes and some individuals from different strains belonged to the same haplogroup: the classical EAO strain designation is not equivalent to modern monophyletic matrilineal groups. Phylogenetic inference showed that the foundation mares of the historical haplotypes were highly likely to have the same haplotypes as the animals studied (p > 0.998 in all cases), confirming the reliability of EAO studbook records and providing the opportunity for breeders to confirm the ancestry of their horses. PMID:28859174

Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification.

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Wang, Yi; Wang, Yan; Ma, Ai-Jing; Li, Dong-Xun; Luo, Li-Juan; Liu, Dong-Xin; Jin, Dong; Liu, Kai; Ye, Chang-Yun

2015-07-08

We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61-65 °C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primers annealed to the template strands without a denaturing step to initiate the synthesis. For the subsequent isothermal amplification step, a series of primer binding and extension events yielded several single-stranded DNAs and single-stranded single stem-loop DNA structures. Then, these DNA products enabled the strand-displacement reaction to enter into the exponential amplification. Three mainstream methods, including colorimetric indicators, agarose gel electrophoresis and real-time turbidity, were selected for monitoring the MCDA reaction. Moreover, the practical application of the MCDA assay was successfully evaluated by detecting the target pathogen nucleic acid in pork samples, which offered advantages on quick results, modest equipment requirements, easiness in operation, and high specificity and sensitivity. Here we expounded the basic MCDA mechanism and also provided details on an alternative (Single-MCDA assay, S-MCDA) to MCDA technique.

Genetic diversity of Guangxi chicken breeds assessed with microsatellites and the mitochondrial DNA D-loop region.

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Liao, Yuying; Mo, Guodong; Sun, Junli; Wei, Fengying; Liao, Dezhong Joshua

2016-05-01

The domestic chicken (Gallus gallus domesticus) is an excellent model for genetic studies of phenotypic diversity. The Guangxi Region of China possesses several native chicken breeds displaying a broad range of phenotypes well adapted to the extreme hot-and-wet environments in the region. We thus evaluated the genetic diversity and relationships among six native chicken populations of the Guangxi region and also evaluated two commercial breeds (Arbor Acres and Roman chickens). We analyzed the sequences of the D-loop region of the mitochondrial DNA (mtDNA) and 18 microsatellite loci of 280 blood samples from six Guangxi native chicken breeds and from Arbor Acres and Roman chickens, and used the neighbor-joining method to construct the phylogenetic tree of these eight breeds. Our results showed that the genetic diversity of Guangxi native breeds was relatively rich. The phylogenetic tree using the unweighed pair-group method with arithmetic means (UPGAM) on microsatellite marks revealed two main clusters. Arbor Acres chicken and Roman chicken were in one cluster, while the Guangxi breeds were in the other cluster. Moreover, the UPGAM tree of Guangxi native breeds based on microsatellite loci was more consistent with the genesis, breeding history, differentiation and location than the mtDNA D-loop region. STRUCTURE analysis further confirmed the genetic structure of Guangxi native breeds in the Neighbor-Net dendrogram. The nomenclature of mtDNA sequence polymorphisms suggests that the Guangxi native chickens are distributed across four clades, but most of them are clustered in two main clades (B and E), with the other haplotypes within the clades A and C. The Guangxi native breeds revealed abundant genetic diversity not only on microsatellite loci but also on mtDNA D-loop region, and contained multiple maternal lineages, including one from China and another from Europe or the Middle East.

Binding of histone H1 to DNA is differentially modulated by redox state of HMGB1.

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Eva Polanská

Full Text Available HMGB1 is an architectural protein in chromatin, acting also as a signaling molecule outside the cell. Recent reports from several laboratories provided evidence that a number of both the intracellular and extracellular functions of HMGB1 may depend on redox-sensitive cysteine residues of the protein. In this study we demonstrate that redox state of HMGB1 can significantly modulate the ability of the protein to bind and bend DNA, as well as to promote DNA end-joining. We also report a high affinity binding of histone H1 to hemicatenated DNA loops and DNA minicircles. Finally, we show that reduced HMGB1 can readily displace histone H1 from DNA, while oxidized HMGB1 has limited capacity for H1 displacement. Our results suggested a novel mechanism for the HMGB1-mediated modulation of histone H1 binding to DNA. Possible biological consequences of linker histones H1 replacement by HMGB1 for the functioning of chromatin are discussed.

R-loops: targets for nuclease cleavage and repeat instability.

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Freudenreich, Catherine H

2018-01-11

R-loops form when transcribed RNA remains bound to its DNA template to form a stable RNA:DNA hybrid. Stable R-loops form when the RNA is purine-rich, and are further stabilized by DNA secondary structures on the non-template strand. Interestingly, many expandable and disease-causing repeat sequences form stable R-loops, and R-loops can contribute to repeat instability. Repeat expansions are responsible for multiple neurodegenerative diseases, including Huntington's disease, myotonic dystrophy, and several types of ataxias. Recently, it was found that R-loops at an expanded CAG/CTG repeat tract cause DNA breaks as well as repeat instability (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Two factors were identified as causing R-loop-dependent breaks at CAG/CTG tracts: deamination of cytosines and the MutLγ (Mlh1-Mlh3) endonuclease, defining two new mechanisms for how R-loops can generate DNA breaks (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Following R-loop-dependent nicking, base excision repair resulted in repeat instability. These results have implications for human repeat expansion diseases and provide a paradigm for how RNA:DNA hybrids can cause genome instability at structure-forming DNA sequences. This perspective summarizes mechanisms of R-loop-induced fragility at G-rich repeats and new links between DNA breaks and repeat instability.

R-loops cause genomic instability in T helper lymphocytes from patients with Wiskott-Aldrich syndrome.

Science.gov (United States)

Sarkar, Koustav; Han, Seong-Su; Wen, Kuo-Kuang; Ochs, Hans D; Dupré, Loïc; Seidman, Michael M; Vyas, Yatin M

2017-12-15

Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT), and X-linked neutropenia, which are caused by WAS mutations affecting Wiskott-Aldrich syndrome protein (WASp) expression or activity, manifest in immunodeficiency, autoimmunity, genomic instability, and lymphoid and other cancers. WASp supports filamentous actin formation in the cytoplasm and gene transcription in the nucleus. Although the genetic basis for XLT/WAS has been clarified, the relationships between mutant forms of WASp and the diverse features of these disorders remain ill-defined. We sought to define how dysfunctional gene transcription is causally linked to the degree of T H cell deficiency and genomic instability in the XLT/WAS clinical spectrum. In human T H 1- or T H 2-skewing cell culture systems, cotranscriptional R-loops (RNA/DNA duplex and displaced single-stranded DNA) and DNA double-strand breaks (DSBs) were monitored in multiple samples from patients with XLT and WAS and in normal T cells depleted of WASp. WASp deficiency provokes increased R-loops and R-loop-mediated DSBs in T H 1 cells relative to T H 2 cells. Mechanistically, chromatin occupancy of serine 2-unphosphorylated RNA polymerase II is increased, and that of topoisomerase 1, an R-loop preventing factor, is decreased at R-loop-enriched regions of IFNG and TBX21 (T H 1 genes) in T H 1 cells. These aberrations accompany increased unspliced (intron-retained) and decreased spliced mRNA of IFNG and TBX21 but not IL13 (T H 2 gene). Significantly, increased cellular load of R-loops and DSBs, which are normalized on RNaseH1-mediated suppression of ectopic R-loops, inversely correlates with disease severity scores. Transcriptional R-loop imbalance is a novel molecular defect causative in T H 1 immunodeficiency and genomic instability in patients with WAS. The study proposes that cellular R-loop load could be used as a potential biomarker for monitoring symptom severity and prognostic outcome in the XLT-WAS clinical spectrum

Displacement Parameter Inversion for a Novel Electromagnetic Underground Displacement Sensor

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Nanying Shentu

2014-05-01

Full Text Available Underground displacement monitoring is an effective method to explore deep into rock and soil masses for execution of subsurface displacement measurements. It is not only an important means of geological hazards prediction and forecasting, but also a forefront, hot and sophisticated subject in current geological disaster monitoring. In previous research, the authors had designed a novel electromagnetic underground horizontal displacement sensor (called the H-type sensor by combining basic electromagnetic induction principles with modern sensing techniques and established a mutual voltage measurement theoretical model called the Equation-based Equivalent Loop Approach (EELA. Based on that work, this paper presents an underground displacement inversion approach named “EELA forward modeling-approximate inversion method”. Combining the EELA forward simulation approach with the approximate optimization inversion theory, it can deduce the underground horizontal displacement through parameter inversion of the H-type sensor. Comprehensive and comparative studies have been conducted between the experimentally measured and theoretically inversed values of horizontal displacement under counterpart conditions. The results show when the measured horizontal displacements are in the 0–100 mm range, the horizontal displacement inversion discrepancy is generally tested to be less than 3 mm under varied tilt angles and initial axial distances conditions, which indicates that our proposed parameter inversion method can predict underground horizontal displacement measurements effectively and robustly for the H-type sensor and the technique is applicable for practical geo-engineering applications.

Tip-enhanced fluorescence with radially polarized illumination for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA

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Wei, Shih-Chung; Chuang, Tsung-Liang; Wang, Da-Shin; Lu, Hui-Hsin; Gu, Frank X.; Sung, Kung-Bin; Lin, Chii-Wann

2015-02-01

A tip nanobiosensor for monitoring DNA replication was presented. The effects of excitation power and polarization on tip-enhanced fluorescence (TEF) were assessed with the tip immersed in fluorescein isothiocyanate solution first. The photon count rose on average fivefold with radially polarized illumination at 50 mW. We then used polymerase-functionalized tips for monitoring loop-mediated isothermal amplification on Hepatitis C virus cDNA. The amplicon-SYBR Green I complex was detected and compared to real-time loop-mediated isothermal amplification. The signals of the reaction using 4 and 0.004 ng/μl templates were detected 10 and 30 min earlier, respectively. The results showed the potential of TEF in developing a nanobiosensor for real-time DNA amplification.

In vitro molecular machine learning algorithm via symmetric internal loops of DNA.

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Lee, Ji-Hoon; Lee, Seung Hwan; Baek, Christina; Chun, Hyosun; Ryu, Je-Hwan; Kim, Jin-Woo; Deaton, Russell; Zhang, Byoung-Tak

2017-08-01

Programmable biomolecules, such as DNA strands, deoxyribozymes, and restriction enzymes, have been used to solve computational problems, construct large-scale logic circuits, and program simple molecular games. Although studies have shown the potential of molecular computing, the capability of computational learning with DNA molecules, i.e., molecular machine learning, has yet to be experimentally verified. Here, we present a novel molecular learning in vitro model in which symmetric internal loops of double-stranded DNA are exploited to measure the differences between training instances, thus enabling the molecules to learn from small errors. The model was evaluated on a data set of twenty dialogue sentences obtained from the television shows Friends and Prison Break. The wet DNA-computing experiments confirmed that the molecular learning machine was able to generalize the dialogue patterns of each show and successfully identify the show from which the sentences originated. The molecular machine learning model described here opens the way for solving machine learning problems in computer science and biology using in vitro molecular computing with the data encoded in DNA molecules. Copyright © 2017. Published by Elsevier B.V.

PNA binding to the non-template DNA strand interferes with transcription, suggesting a blockage mechanism mediated by R-loop formation.

Science.gov (United States)

Belotserkovskii, Boris P; Hanawalt, Philip C

2015-11-01

Peptide Nucleic Acids (PNAs) are artificial DNA mimics with superior nucleic acid binding capabilities. T7 RNA polymerase (T7 RNAP) transcription upon encountering PNA bound to the non-template DNA strand was studied in vitro. A characteristic pattern of blockage signals was observed, extending downstream from the PNA binding site, similar to that produced by G-rich homopurine-homopyrimidine (hPu-hPy) sequences and likely caused by R-loop formation. Since blocked transcription complexes in association with stable R-loops may interfere with replication and in some cases trigger apoptosis, targeted R-loop formation might be employed to inactivate selected cells, such as those in tumors, based upon their unique complement of expressed genes. © 2014 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.

Microfluidic method for rapid turbidimetric detection of the DNA of Mycobacterium tuberculosis using loop-mediated isothermal amplification in capillary tubes

International Nuclear Information System (INIS)

Rafati, Adele; Gill, Pooria

2015-01-01

We describe a microfluidic method for rapid isothermal turbidimetric detection of the DNA of Mycobacterium tuberculosis. Loop-mediated isothermal amplification is accomplished in capillary tubes for amplifying DNA in less than 15 min, and sensitivity and specificity were compared to conventional loop-mediated isothermal amplification (LAMP). The method can detect as little as 1 pg mL −1 DNA in a sample. Results obtained with clinical specimens indicated 90 % sensitivity and 95 % specificity for microfluidic LAMP in comparison to culture methods. No interference occurred due to the presence of nonspecific DNAs. The findings demonstrate the power of the new microfluidic LAMP test for rapid molecular detection of microorganisms even when using bare eyes. (author)

Theory on the mechanism of distal action of transcription factors: looping of DNA versus tracking along DNA

International Nuclear Information System (INIS)

Murugan, R

2010-01-01

In this paper, we develop a theory on the mechanism of distal action of the transcription factors, which are bound at their respective cis-regulatory enhancer modules on the promoter-RNA polymerase II (PR) complexes to initiate the transcription event in eukaryotes. We consider both the looping and tracking modes of their distal communication and calculate the mean first passage time that is required for the distal interactions of the complex of enhancer and transcription factor with the PR via both these modes. We further investigate how this mean first passage time is dependent on the length of the DNA segment (L, base-pairs) that connects the cis-regulatory binding site and the respective promoter. When the radius of curvature of this connecting segment of DNA is R that was induced upon binding of the transcription factor at the cis-acting element and RNAPII at the promoter in cis-positions, our calculations indicate that the looping mode of distal action will dominate when L is such that L > 2πR and the tracking mode of distal action will be favored when L 2 bps. It seems that the free energy associated with the binding of the transcription factor with its cis-acting element and the distance of this cis-acting element from the corresponding promoter of the gene of interest is negatively correlated. Our results suggest that the looping and tracking modes of distal action are concurrently operating on the transcription activation and the physics that determines the timescales associated with the looping/tracking in the mechanism of action of these transcription factors on the initiation of the transcription event must put a selection pressure on the distribution of the distances of cis-regulatory modules from their respective promoters of the genes. The computational analysis of the upstream sequences of promoters of various genes in the human and mouse genomes for the presence of putative cis-regulatory elements for a set of known transcription factors using

Visual Detection of Potato leafroll virus by One-step Reverse Transcription Loop-Mediated Isothermal Amplification of DNA with Hydroxynaphthol Blue Dye

NARCIS (Netherlands)

Ahmadi, S.; Almasi, A.M.; Fatehi, F.; Struik, P.C.; Moradi, A.

2013-01-01

Loop-mediated isothermal amplification (LAMP) assay is a novel technique for amplifying DNA under constant temperature, with high specificity, sensitivity, rapidity and efficiency. We applied reverse transcription loop-mediated isothermal amplification (RT-LAMP) to visually detect Potato leafroll

Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance

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Haque Kashif A

2005-09-01

Full Text Available Abstract Background Whole genome amplification (WGA promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA quantity. We evaluated the performance of multiple displacement amplification (MDA WGA using gDNA extracted from lymphoblastoid cell lines (N = 27 with a range of starting gDNA input of 1–200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA was performed using three DNA quantification methods (OD, PicoGreen® and RT-PCR. Two panels of N = 15 STR (using the AmpFlSTR® Identifiler® panel and N = 49 SNP (TaqMan® genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs. Results The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates. Conclusion The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of

Design and Analysis of Compact DNA Strand Displacement Circuits for Analog Computation Using Autocatalytic Amplifiers.

Science.gov (United States)

Song, Tianqi; Garg, Sudhanshu; Mokhtar, Reem; Bui, Hieu; Reif, John

2018-01-19

A main goal in DNA computing is to build DNA circuits to compute designated functions using a minimal number of DNA strands. Here, we propose a novel architecture to build compact DNA strand displacement circuits to compute a broad scope of functions in an analog fashion. A circuit by this architecture is composed of three autocatalytic amplifiers, and the amplifiers interact to perform computation. We show DNA circuits to compute functions sqrt(x), ln(x) and exp(x) for x in tunable ranges with simulation results. A key innovation in our architecture, inspired by Napier's use of logarithm transforms to compute square roots on a slide rule, is to make use of autocatalytic amplifiers to do logarithmic and exponential transforms in concentration and time. In particular, we convert from the input that is encoded by the initial concentration of the input DNA strand, to time, and then back again to the output encoded by the concentration of the output DNA strand at equilibrium. This combined use of strand-concentration and time encoding of computational values may have impact on other forms of molecular computation.

Maternal phylogenetic relationships and genetic variation among Arabian horse populations using whole mitochondrial DNA D-loop sequencing.

Science.gov (United States)

Khanshour, Anas M; Cothran, Ernest Gus

2013-09-13

Maternal inheritance is an essential point in Arabian horse population genetics and strains classification. The mitochondrial DNA (mtDNA) sequencing is a highly informative tool to investigate maternal lineages. We sequenced the whole mtDNA D-loop of 251 Arabian horses to study the genetic diversity and phylogenetic relationships of Arabian populations and to examine the traditional strain classification system that depends on maternal family lines using native Arabian horses from the Middle East. The variability in the upstream region of the D-loop revealed additional differences among the haplotypes that had identical sequences in the hypervariable region 1 (HVR1). While the American-Arabians showed relatively low diversity, the Syrian population was the most variable and contained a very rare and old haplogroup. The Middle Eastern horses had major genetic contributions to the Western horses and there was no clear pattern of differentiation among all tested populations. Our results also showed that several individuals from different strains shared a single haplotype, and individuals from a single strain were represented in clearly separated haplogroups. The whole mtDNA D-loop sequence was more powerful for analysis of the maternal genetic diversity in the Arabian horses than using just the HVR1. Native populations from the Middle East, such as Syrians, could be suggested as a hot spot of genetic diversity and may help in understanding the evolution history of the Arabian horse breed. Most importantly, there was no evidence that the Arabian horse breed has clear subdivisions depending on the traditional maternal based strain classification system.

The cell pole: the site of cross talk between the DNA uptake and genetic recombination machinery.

Science.gov (United States)

Kidane, Dawit; Ayora, Silvia; Sweasy, Joann B; Graumann, Peter L; Alonso, Juan C

2012-01-01

Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as "guardians", protects ssDNA from degradation and limit the RecA recombinase loading. Then, the "mediators" overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by "modulators", catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or "resolver" cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the "rescuers" will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective.

The flexible loop L1 of the H3K4 demethylase JARID1B ARID domain has a crucial role in DNA-binding activity

International Nuclear Information System (INIS)

Yao, Wenming; Peng, Yu; Lin, Donghai

2010-01-01

JARID1B, a member of the JmjC demethylase family, has a crucial role in H3K4me3 demethylation. The ARID domain is a potential DNA-binding domain of JARID1B. Previous studies indicate that a GC-rich DNA motif is the specific target of the ARID domain. However, the details of the interaction between the ARID domain and duplex DNA require further study. Here, we utilized NMR spectroscopy to assign the backbone amino acids and mapped the DNA-binding sites of the human JARID1B ARID domain. Perturbations to 1 H- 15 N correlation spectra revealed that the flexible loop L1 of ARID was the main DNA-binding interface. EMSA and intrinsic fluorescence experiments demonstrated that mutations on loop L1 strongly reduced the DNA-binding activity of JARID1B ARID. Furthermore, transfection of mutant forms resulted in a distinct loss of intrinsic H3K4 demethylase activity, implying that the flexible loop L1 made a major contribution to sustaining the DNA-binding ability of JARID1B ARID domain.

Toehold strand displacement-driven assembly of G-quadruplex DNA for enzyme-free and non-label sensitive fluorescent detection of thrombin.

Science.gov (United States)

Xu, Yunying; Zhou, Wenjiao; Zhou, Ming; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

2015-02-15

Based on a new signal amplification strategy by the toehold strand displacement-driven cyclic assembly of G-quadruplex DNA, the development of an enzyme-free and non-label aptamer sensing approach for sensitive fluorescent detection of thrombin is described. The target thrombin associates with the corresponding aptamer of the partial dsDNA probes and liberates single stranded initiation sequences, which trigger the toehold strand displacement assembly of two G-quadruplex containing hairpin DNAs. This toehold strand displacement reaction leads to the cyclic reuse of the initiation sequences and the production of DNA assemblies with numerous G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binds to these G-quadruplex structures and generates significantly amplified fluorescent signals to achieve highly sensitive detection of thrombin down to 5 pM. Besides, this method shows high selectivity towards the target thrombin against other control proteins. The developed thrombin sensing method herein avoids the modification of the probes and the involvement of any enzyme or nanomaterial labels for signal amplification. With the successful demonstration for thrombin detection, our approach can be easily adopted to monitor other target molecules in a simple, low-cost, sensitive and selective way by choosing appropriate aptamer/ligand pairs. Copyright © 2014 Elsevier B.V. All rights reserved.

Enzyme-free colorimetric detection systems based on the DNA strand displacement competition reaction

Science.gov (United States)

Zhang, Z.; Birkedal, V.; Gothelf, K. V.

2016-05-01

The strand displacement competition assay is based on the dynamic equilibrium of the competitive hybridization of two oligonucleotides (A and B) to a third oligonucleotide (S). In the presence of an analyte that binds to a specific affinity-moiety conjugated to strand B, the equilibrium shifts, which can be detected by a shift in the fluorescence resonance energy transfer signal between dyes attached to the DNA strands. In the present study we have integrated an ATP aptamer in the strand B and demonstrated the optical detection of ATP. Furthermore we explore a new readout method using a split G-quadruplex DNAzyme for colorimetric readout of the detection of streptavidin by the naked eye. Finally, we integrate the whole G-quadruplex DNAzyme system in a single DNA strand and show that it is applicable to colorimetric detection.

Enzyme-free colorimetric detection systems based on the DNA strand displacement competition reaction

DEFF Research Database (Denmark)

Zhang, Zhao; Birkedal, Victoria; Gothelf, Kurt Vesterager

2016-01-01

The strand displacement competition assay is based on the dynamic equilibrium of the competitive hybridization of two oligonucleotides (A and B) to a third oligonucleotide (S). In the presence of an analyte that binds to a specific affinity-moiety conjugated to strand B, the equilibrium shifts, w...... G-quadruplex DNAzyme for colorimetric readout of the detection of streptavidin by the naked eye. Finally, we integrate the whole G-quadruplex DNAzyme system in a single DNA strand and show that it is applicable to colorimetric detection......., which can be detected by a shift in the fluorescence resonance energy transfer signal between dyes attached to the DNA strands. In the present study we have integrated an ATP aptamer in the strand B and demonstrated the optical detection of ATP. Furthermore we explore a new readout method using a split...

Digital Droplet Multiple Displacement Amplification (ddMDA for Whole Genome Sequencing of Limited DNA Samples.

Directory of Open Access Journals (Sweden)

Minsoung Rhee

Full Text Available Multiple displacement amplification (MDA is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet, ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.

Mitochondrial DNA mutation screening of male patients with obstructive sleep apnea-hypopnea syndrome.

Science.gov (United States)

Huang, Xiao-Ying; Li, Hong; Xu, Xiao-Mei; Wang, Liang-Xing

2014-08-01

The aim of the present study was to analyze the differences between the genes of the mitochondrial DNA (mtDNA) displacement loop (D-loop) region and the Cambridge Reference sequence, in order to screen the mutation sites and investigate the correlation between mutations, clinical parameters and complications associated with obstructive sleep apnea-hypopnea syndrome (OSAHS). mtDNA was obtained from male patients with OSAHS in the Zhejiang Province. In total, 60 male patients with OSAHS and 102 healthy adults were assessed to determine the levels of fasting blood glucose, total cholesterol, triglyceride (TG) and high-density and low-density lipoproteins (LDL). Furthermore, peripheral mtDNA was extracted and bidirectional sequencing was conducted to enable mutation screening. In the mtDNA D-loop region, 178 mutation sites were identified, of which 115 sites were present in the two groups. The number of non-common sites in the OSAHS group was significantly higher compared with the control group (P0.05). A total of 21 cases in the severe OSAHS group exhibited mutation rates of >10%. In the control group, there were 24 cases where the np73A-G and np263A-G mutations were predominant. The np303-np315 region was identified to be the highly variable region and various mutation forms were observed. Statistically significant differences were observed in the neck perimeter, TG and LDL levels among the OSAHS-no-mutation subgroups (P<0.05) and LDL was shown to be associated with an mtDNA mutation in the OSAHS group. Numerous polymorphic mutation sites were identified in the mtDNA D-loop region of the OSAHS group. Therefore, mtDNA mutation sites may be closely associated with the clinical manifestations and complications of OSAHS.

Theory on the mechanism of distal action of transcription factors: looping of DNA versus tracking along DNA

Energy Technology Data Exchange (ETDEWEB)

Murugan, R, E-mail: rmurugan@gmail.co [Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036 (India)

2010-10-15

In this paper, we develop a theory on the mechanism of distal action of the transcription factors, which are bound at their respective cis-regulatory enhancer modules on the promoter-RNA polymerase II (PR) complexes to initiate the transcription event in eukaryotes. We consider both the looping and tracking modes of their distal communication and calculate the mean first passage time that is required for the distal interactions of the complex of enhancer and transcription factor with the PR via both these modes. We further investigate how this mean first passage time is dependent on the length of the DNA segment (L, base-pairs) that connects the cis-regulatory binding site and the respective promoter. When the radius of curvature of this connecting segment of DNA is R that was induced upon binding of the transcription factor at the cis-acting element and RNAPII at the promoter in cis-positions, our calculations indicate that the looping mode of distal action will dominate when L is such that L > 2{pi}R and the tracking mode of distal action will be favored when L < 2{pi}R. The time required for the distal action will be minimum when L = 2{pi}R where the typical value of R for the binding of histones will be R {approx} 16 bps and L {approx} 10{sup 2} bps. It seems that the free energy associated with the binding of the transcription factor with its cis-acting element and the distance of this cis-acting element from the corresponding promoter of the gene of interest is negatively correlated. Our results suggest that the looping and tracking modes of distal action are concurrently operating on the transcription activation and the physics that determines the timescales associated with the looping/tracking in the mechanism of action of these transcription factors on the initiation of the transcription event must put a selection pressure on the distribution of the distances of cis-regulatory modules from their respective promoters of the genes. The computational analysis

DNA moves sequentially towards the nuclear matrix during DNA replication in vivo

Directory of Open Access Journals (Sweden)

Aranda-Anzaldo Armando

2011-01-01

Full Text Available Abstract Background In the interphase nucleus of metazoan cells DNA is organized in supercoiled loops anchored to a nuclear matrix (NM. There is varied evidence indicating that DNA replication occurs in replication factories organized upon the NM and that DNA loops may correspond to the actual replicons in vivo. In normal rat liver the hepatocytes are arrested in G0 but they synchronously re-enter the cell cycle after partial-hepatectomy leading to liver regeneration in vivo. We have previously determined in quiescent rat hepatocytes that a 162 kbp genomic region containing members of the albumin gene family is organized into five structural DNA loops. Results In the present work we tracked down the movement relative to the NM of DNA sequences located at different points within such five structural DNA loops during the S phase and after the return to cellular quiescence during liver regeneration. Our results indicate that looped DNA moves sequentially towards the NM during replication and then returns to its original position in newly quiescent cells, once the liver regeneration has been achieved. Conclusions Looped DNA moves in a sequential fashion, as if reeled in, towards the NM during DNA replication in vivo thus supporting the notion that the DNA template is pulled progressively towards the replication factories on the NM so as to be replicated. These results provide further evidence that the structural DNA loops correspond to the actual replicons in vivo.

C-terminal phenylalanine of bacteriophage T7 single-stranded DNA-binding protein is essential for strand displacement synthesis by T7 DNA polymerase at a nick in DNA.

Science.gov (United States)

Ghosh, Sharmistha; Marintcheva, Boriana; Takahashi, Masateru; Richardson, Charles C

2009-10-30

Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5'-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations.

C-terminal Phenylalanine of Bacteriophage T7 Single-stranded DNA-binding Protein Is Essential for Strand Displacement Synthesis by T7 DNA Polymerase at a Nick in DNA*

Science.gov (United States)

Ghosh, Sharmistha; Marintcheva, Boriana; Takahashi, Masateru; Richardson, Charles C.

2009-01-01

Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5′-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations. PMID:19726688

Comparison between Mt-DNA D-Loop and Cyt B primers for porcine DNA detection in meat products

Science.gov (United States)

Hamzah, Azhana; Mutalib, Sahilah Abd.; Babji, Abdul Salam

2013-11-01

This study was conducted to detect the presence of porcine DNA in meat products in the market using conventional polymerase chain reaction (PCR) and commercial PCR-southern hybridization analysis. Porcine DNA detection in meat products was tested due to some issues associated with the adulteration of food products in Malaysia. This is an important issue especially for Halal authentication which is required for some religious practices such as in Islam and Hinduisms. Many techniques have been developed for determining the Halal status of food products. In this paper, mt-DNA D-loop primer and cytochrome (cyt) b were used to detect the presence of porcine DNA in meat products. Positive and negative controls were always present for each batch of extraction. DNA of raw pork meat was used as a positive control while nucleus free water is used as negative control. A pair of oligonucleotide primer was used namely Pork1 and Pork2 which produced amplicon of 531 base pair (bp) in size. While, PCR-southern hybridization was conducted using primers readily supplied by commercial PCR-Southern hybridization and produced amplicon with 276 bp in size. In the present study, demonstrated that none of the samples were contaminated with porcine residuals but selected samples with pork meat were positive. The species-specific PCR amplification yielded excellent results for identification of pork derivatives in food products and it is a potentially reliable and suitable technique in routine food analysis for Halal certification.

Multiple displacement amplification of whole genomic DNA from urediospores of Puccinia striiformis f. sp. tritici.

Science.gov (United States)

Zhang, R; Ma, Z H; Wu, B M

2015-05-01

Biotrophic fungi, such as Puccinia striiformis f. sp. tritici, because they cannot be cultured on nutrient media, to obtain adequate quantity of DNA for molecular genetic analysis, are usually propagated on living hosts, wheat plants in case of P. striiformis f. sp. tritici. The propagation process is time-, space- and labor-consuming and has been a bottleneck to molecular genetic analysis of this pathogen. In this study we evaluated multiple displacement amplification (MDA) of pathogen genomic DNA from urediospores as an alternative approach to traditional propagation of urediospores followed by DNA extraction. The quantities of pathogen genomic DNA in the products were further determined via real-time PCR with a pair of primers specific for the β-tubulin gene of P. striiformis f. sp. tritici. The amplified fragment length polymorphism (AFLP) fingerprints were also compared between the DNA products. The results demonstrated that adequate genomic DNA at fragment size larger than 23 Kb could be amplified from 20 to 30 urediospores via MDA method. The real-time PCR results suggested that although fresh urediospores collected from diseased leaves were the best, spores picked from diseased leaves stored for a prolonged period could also be used for amplification. AFLP fingerprints exhibited no significant differences between amplified DNA and DNA extracted with CTAB method, suggesting amplified DNA can represent the pathogen's genomic DNA very well. Therefore, MDA could be used to obtain genomic DNA from small precious samples (dozens of spores) for molecular genetic analysis of wheat stripe rust pathogen, and other fungi that are difficult to propagate.

Histone displacement during nucleotide excision repair

DEFF Research Database (Denmark)

Dinant, C.; Bartek, J.; Bekker-Jensen, S.

2012-01-01

Nucleotide excision repair (NER) is an important DNA repair mechanism required for cellular resistance against UV light and toxic chemicals such as those found in tobacco smoke. In living cells, NER efficiently detects and removes DNA lesions within the large nuclear macromolecular complex called...... of histone variants and histone displacement (including nucleosome sliding). Here we review current knowledge, and speculate about current unknowns, regarding those chromatin remodeling activities that physically displace histones before, during and after NER....

The cytosolic DNA sensor cGAS forms an oligomeric complex with DNA and undergoes switch-like conformational changes in the activation loop.

Science.gov (United States)

Zhang, Xu; Wu, Jiaxi; Du, Fenghe; Xu, Hui; Sun, Lijun; Chen, Zhe; Brautigam, Chad A; Zhang, Xuewu; Chen, Zhijian J

2014-02-13

The presence of DNA in the cytoplasm is a danger signal that triggers immune and inflammatory responses. Cytosolic DNA binds to and activates cyclic GMP-AMP (cGAMP) synthase (cGAS), which produces the second messenger cGAMP. cGAMP binds to the adaptor protein STING and activates a signaling cascade that leads to the production of type I interferons and other cytokines. Here, we report the crystal structures of human cGAS in its apo form, representing its autoinhibited conformation as well as in its cGAMP- and sulfate-bound forms. These structures reveal switch-like conformational changes of an activation loop that result in the rearrangement of the catalytic site. The structure of DNA-bound cGAS reveals a complex composed of dimeric cGAS bound to two molecules of DNA. Functional analyses of cGAS mutants demonstrate that both the protein-protein interface and the two DNA binding surfaces are critical for cGAS activation. These results provide insights into the mechanism of DNA sensing by cGAS. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

The Cytosolic DNA Sensor cGAS Forms an Oligomeric Complex with DNA and Undergoes Switch-like Conformational Changes in the Activation Loop

Directory of Open Access Journals (Sweden)

Xu Zhang

2014-02-01

Full Text Available The presence of DNA in the cytoplasm is a danger signal that triggers immune and inflammatory responses. Cytosolic DNA binds to and activates cyclic GMP-AMP (cGAMP synthase (cGAS, which produces the second messenger cGAMP. cGAMP binds to the adaptor protein STING and activates a signaling cascade that leads to the production of type I interferons and other cytokines. Here, we report the crystal structures of human cGAS in its apo form, representing its autoinhibited conformation as well as in its cGAMP- and sulfate-bound forms. These structures reveal switch-like conformational changes of an activation loop that result in the rearrangement of the catalytic site. The structure of DNA-bound cGAS reveals a complex composed of dimeric cGAS bound to two molecules of DNA. Functional analyses of cGAS mutants demonstrate that both the protein-protein interface and the two DNA binding surfaces are critical for cGAS activation. These results provide insights into the mechanism of DNA sensing by cGAS.

DNA binding specificity of the basic-helix-loop-helix protein MASH-1.

Science.gov (United States)

Meierhan, D; el-Ariss, C; Neuenschwander, M; Sieber, M; Stackhouse, J F; Allemann, R K

1995-09-05

Despite the high degree of sequence similarity in their basic-helix-loop-helix (BHLH) domains, MASH-1 and MyoD are involved in different biological processes. In order to define possible differences between the DNA binding specificities of these two proteins, we investigated the DNA binding properties of MASH-1 by circular dichroism spectroscopy and by electrophoretic mobility shift assays (EMSA). Upon binding to DNA, the BHLH domain of MASH-1 underwent a conformational change from a mainly unfolded to a largely alpha-helical form, and surprisingly, this change was independent of the specific DNA sequence. The same conformational transition could be induced by the addition of 20% 2,2,2-trifluoroethanol. The apparent dissociation constants (KD) of the complexes of full-length MASH-1 with various oligonucleotides were determined from half-saturation points in EMSAs. MASH-1 bound as a dimer to DNA sequences containing an E-box with high affinity KD = 1.4-4.1 x 10(-14) M2). However, the specificity of DNA binding was low. The dissociation constant for the complex between MASH-1 and the highest affinity E-box sequence (KD = 1.4 x 10(-14) M2) was only a factor of 10 smaller than for completely unrelated DNA sequences (KD = approximately 1 x 10(-13) M2). The DNA binding specificity of MASH-1 was not significantly increased by the formation of an heterodimer with the ubiquitous E12 protein. MASH-1 and MyoD displayed similar binding site preferences, suggesting that their different target gene specificities cannot be explained solely by differential DNA binding. An explanation for these findings is provided on the basis of the known crystal structure of the BHLH domain of MyoD.

Molecular characterization of six sub population Indonesian local goats based on mitochondrial DNA D-loop

Directory of Open Access Journals (Sweden)

Aron Batubara

2013-03-01

Full Text Available Indonesian local goats were spread in some region, but there was still limited data’s known about the characteristics of its genetic diversity and origin. The Mitochondrial DNA D-loop sequences were used to study the genetic diversity and relationships of six sub population Indonesian local goats, namely, Kacang, Marica, Samosir, Jawarandu, Muara and Bengali goats. From 539 blood samples and DNA extraction collections were selected about 60 samples (10 samples each sub populations analyzed by PCR-RFLP methods, followed sequence analyzed about 5 PCR products each sub population. The results of the sequence analyses were edited and acquired about 957 bp of nucleotides length. After the alignment analyses were found 50 polymorphic sites which divided into 19 haplotype groups of mtDNA D-loop region. The value of nucleotide diversity was 0.014 ± 0.002. Analysis of Neighbour Joining with Kimura 2 Parameter methods and bootstrap test with 1000 replication indicated that each sub population groups was significantly different between one groups to the others. The maternal lineages origin of six breeds of Indonesian local goats was included to the group of lineage B. The Lineage B was the maternal origin of the haplogroup of goats in the region of East Asia, South Asia, China, Mongolia, North and South Africa, Malaysia, Indonesia, Pakistan and India.

Mutations reducing replication from R-loops suppress the defects of growth, chromosome segregation and DNA supercoiling in cells lacking topoisomerase I and RNase HI activity.

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Usongo, Valentine; Martel, Makisha; Balleydier, Aurélien; Drolet, Marc

2016-04-01

R-loop formation occurs when the nascent RNA hybridizes with the template DNA strand behind the RNA polymerase. R-loops affect a wide range of cellular processes and their use as origins of replication was the first function attributed to them. In Escherichia coli, R-loop formation is promoted by the ATP-dependent negative supercoiling activity of gyrase (gyrA and gyrB) and is inhibited by topoisomerase (topo) I (topA) relaxing transcription-induced negative supercoiling. RNase HI (rnhA) degrades the RNA moiety of R-loops. The depletion of RNase HI activity in topA null mutants was previously shown to lead to extensive DNA relaxation, due to DNA gyrase inhibition, and to severe growth and chromosome segregation defects that were partially corrected by overproducing topo III (topB). Here, DNA gyrase assays in crude cell extracts showed that the ATP-dependent activity (supercoiling) of gyrase but not its ATP-independent activity (relaxation) was inhibited in topA null cells lacking RNase HI. To characterize the cellular event(s) triggered by the absence of RNase HI, we performed a genetic screen for suppressors of the growth defect of topA rnhA null cells. Suppressors affecting genes in replication (holC2::aph and dnaT18::aph) nucleotide metabolism (dcd49::aph), RNA degradation (rne59::aph) and fimbriae synthesis (fimD22::aph) were found to reduce replication from R-loops and to restore supercoiling, thus pointing to a correlation between R-loop-dependent replication in topA rnhA mutants and the inhibition of gyrase activity and growth. Interestingly, the position of fimD on the E. coli chromosome corresponds to the site of one of the five main putative origins of replication from R-loops in rnhA null cells recently identified by next-generation sequencing, thus suggesting that the fimD22::aph mutation inactivated one of these origins. Furthermore, we show that topo III overproduction is unable to complement the growth defect of topA rnhA null mutants at low

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

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Gadkar, Vijay J; Filion, Martin

2013-06-01

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

Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate.

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Li, Wei; Jiang, Wei; Ding, Yongshun; Wang, Lei

2015-09-15

MicroRNAs (miRNAs) play important roles in a variety of biological processes and have been regarded as tumor biomarkers in cancer diagnosis and prognosis. In this work, a single-molecule counting method for miRNA analysis was proposed based on toehold-mediated strand displacement reaction (SDR) and DNA tetrahedron substrate. Firstly, a specially designed DNA tetrahedron was assembled with a hairpin at one of the vertex, which has an overhanging toehold domain. Then, the DNA tetrahedron was immobilized on the epoxy-functional glass slide by epoxy-amine reaction, forming a DNA tetrahedron substrate. Next, the target miRNA perhybridized with the toehold domain and initiated a strand displacement reaction along with the unfolding of the hairpin, realizing the selective recognization of miRNA. Finally, a biotin labeled detection DNA was hybridized with the new emerging single strand and the streptavidin coated QDs were used as fluorescent probes. Fluorescent images were acquired via epi-fluorescence microscopy, the numbers of fluorescence dots were counted one by one for quantification. The detection limit is 5 fM, which displayed an excellent sensitivity. Moreover, the proposed method which can accurately be identified the target miRNA among its family members, demonstrated an admirable selectivity. Furthermore, miRNA analysis in total RNA samples from human lung tissues was performed, suggesting the feasibility of this method for quantitative detection of miRNA in biomedical research and early clinical diagnostics. Copyright © 2015 Elsevier B.V. All rights reserved.

The Wilson loop and some applications

International Nuclear Information System (INIS)

Bezerra, V.B.

1983-04-01

A simple relation between the classical Wilson loop and the angular deviation in the parallel displacement is found. An example of potentials which give field copies and which suplly the same classical Wilson loop for a particular trajectory is exhibited. The asymptotic behaviour of the Wilson loop for the BPST instanton and the meron, is discussed. By using the dimensional regularization technique to calculate the second order term of the quantum Wilson loop, the influence of geometrical factors for the residue in the pole due to contact points, cuspides and intersections, in function of the space-time ν, is investigated. Charge renormalization in Quantum electrodynamics is finally calculated by using the quantum Wilson loop. (L.C.) [pt

Contribution of gold nanoparticles to the catalytic DNA strand displacement in leakage reduction and signal amplification.

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Wang, Bei; Zhou, Xiang; Yao, Dongbao; Sun, Xianbao; He, Miao; Wang, Xiaojing; Yin, Xue; Liang, Haojun

2017-10-03

A new model using a gold nanoparticle (AuNP)-DNA system to constrain leakage and improve efficiency of catalytic toehold-mediated strand displacement reactions was outlined. A 10-bp spacer on AuNPs and fourfold amount of fuels were determined for good performance of this model with an optimized toehold strategy. After the reaction at 25 °C for 10 h, a 258 pM target could be identified, which is a remarkable improvement compared with the traditional AuNP-DNA system without fuel. Moreover, this model was also studied to differentiate specific single nucleotide polymorphism on target with superior selection factors. This model may help by introducing a proposition of target detection to guide further investigation.

Accurate quantification of microRNA via single strand displacement reaction on DNA origami motif.

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Jie Zhu

Full Text Available DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs.

Accurate Quantification of microRNA via Single Strand Displacement Reaction on DNA Origami Motif

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Lou, Jingyu; Li, Weidong; Li, Sheng; Zhu, Hongxin; Yang, Lun; Zhang, Aiping; He, Lin; Li, Can

2013-01-01

DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs) play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs) labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs. PMID:23990889

Accurate quantification of microRNA via single strand displacement reaction on DNA origami motif.

Science.gov (United States)

Zhu, Jie; Feng, Xiaolu; Lou, Jingyu; Li, Weidong; Li, Sheng; Zhu, Hongxin; Yang, Lun; Zhang, Aiping; He, Lin; Li, Can

2013-01-01

DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs) play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs) labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs.

Free-energy calculations for semi-flexible macromolecules: Applications to DNA knotting and looping

International Nuclear Information System (INIS)

Giovan, Stefan M.; Scharein, Robert G.; Hanke, Andreas; Levene, Stephen D.

2014-01-01

We present a method to obtain numerically accurate values of configurational free energies of semiflexible macromolecular systems, based on the technique of thermodynamic integration combined with normal-mode analysis of a reference system subject to harmonic constraints. Compared with previous free-energy calculations that depend on a reference state, our approach introduces two innovations, namely, the use of internal coordinates to constrain the reference states and the ability to freely select these reference states. As a consequence, it is possible to explore systems that undergo substantially larger fluctuations than those considered in previous calculations, including semiflexible biopolymers having arbitrary ratios of contour length L to persistence length P. To validate the method, high accuracy is demonstrated for free energies of prime DNA knots with L/P = 20 and L/P = 40, corresponding to DNA lengths of 3000 and 6000 base pairs, respectively. We then apply the method to study the free-energy landscape for a model of a synaptic nucleoprotein complex containing a pair of looped domains, revealing a bifurcation in the location of optimal synapse (crossover) sites. This transition is relevant to target-site selection by DNA-binding proteins that occupy multiple DNA sites separated by large linear distances along the genome, a problem that arises naturally in gene regulation, DNA recombination, and the action of type-II topoisomerases

Free-energy calculations for semi-flexible macromolecules: Applications to DNA knotting and looping

Energy Technology Data Exchange (ETDEWEB)

Giovan, Stefan M. [Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas 75083 (United States); Scharein, Robert G. [Hypnagogic Software, Vancouver, British Columbia V6K 1V6 (Canada); Hanke, Andreas [Department of Physics and Astronomy, University of Texas at Brownsville, Brownsville, Texas 78520 (United States); Levene, Stephen D., E-mail: sdlevene@utdallas.edu [Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas 75083 (United States); Department of Bioengineering, University of Texas at Dallas, Richardson, Texas 75083 (United States); Department of Physics, University of Texas at Dallas, Richardson, Texas 75083 (United States)

2014-11-07

We present a method to obtain numerically accurate values of configurational free energies of semiflexible macromolecular systems, based on the technique of thermodynamic integration combined with normal-mode analysis of a reference system subject to harmonic constraints. Compared with previous free-energy calculations that depend on a reference state, our approach introduces two innovations, namely, the use of internal coordinates to constrain the reference states and the ability to freely select these reference states. As a consequence, it is possible to explore systems that undergo substantially larger fluctuations than those considered in previous calculations, including semiflexible biopolymers having arbitrary ratios of contour length L to persistence length P. To validate the method, high accuracy is demonstrated for free energies of prime DNA knots with L/P = 20 and L/P = 40, corresponding to DNA lengths of 3000 and 6000 base pairs, respectively. We then apply the method to study the free-energy landscape for a model of a synaptic nucleoprotein complex containing a pair of looped domains, revealing a bifurcation in the location of optimal synapse (crossover) sites. This transition is relevant to target-site selection by DNA-binding proteins that occupy multiple DNA sites separated by large linear distances along the genome, a problem that arises naturally in gene regulation, DNA recombination, and the action of type-II topoisomerases.

MORPHOLOGY AND GENETIC DIVERSITY OF MITOCHONDRIAL DNA D-LOOP REGION USING PCR-RFLP ANALYSIS IN MAGELANG DUCK AND OTHER NATIVE DUCK

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D. Purwantini

2014-10-01

Full Text Available The aim of this study was to investigate the different of plumage colors on morphological diversityof Magelang duck and genetic diversity using PCR-RFLP mtDNA D-loop region analysis of Magelangduck and four others native duck population (Tegal, Mojosari, Bali and Alabio duck in Indonesia. Bloodsample was taken from 50 Magelang ducks and 20 of each native ducks. Morphological characteristicsof body measurement, production ability and egg quality of Magelang duck were analyzed usingCompletely Randomized Design with 11 plumage colors as treatments. PCR technique was administeredto amplify fragments in mtDNA D-loop region and PCR products were digested with endonucleaserestriction enzyme AluI and HaeIII. The result showed that morphology diversity of Magelang duck wasstatistically affected by different plumage colors. PCR-RFLP analysis using AluI and HaeIII restrictionenzyme resulted in six combinations of restriction fragment pattern shown in six haplotypes (A, B, C, D,E and F. Haplotype difference showed genetic diversity in the population of Magelang duck and theother native ducks. In conclusion, the different plumage colors affected morphology diversity ofMagelang duck. Genetic diversity of Indonesian native duck population could be identified by usingPCR-RFLP analysis on mtDNA D-loop region.

Rapid screening method for male DNA by using the loop-mediated isothermal amplification assay.

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Kitamura, Masashi; Kubo, Seiji; Tanaka, Jin; Adachi, Tatsushi

2017-08-12

Screening for male-derived biological material from collected samples plays an important role in criminal investigations, especially those involving sexual assaults. We have developed a loop-mediated isothermal amplification (LAMP) assay targeting multi-repeat sequences of the Y chromosome for detecting male DNA. Successful amplification occurred with 0.5 ng of male DNA under isothermal conditions of 61 to 67 °C, but no amplification occurred with up to 10 ng of female DNA. Under the optimized conditions, the LAMP reaction initiated amplification within 10 min and amplified for 20 min. The LAMP reaction was sensitive at levels as low as 1-pg male DNA, and a quantitative LAMP assay could be developed because of the strong correlation between the reaction time and the amount of template DNA in the range of 10 pg to 10 ng. Furthermore, to apply the LAMP assay to on-site screening for male-derived samples, we evaluated a protocol using a simple DNA extraction method and a colorimetric intercalating dye that allows detection of the LAMP reaction by evaluating the change in color of the solution. Using this protocol, samples of male-derived blood and saliva stains were processed in approximately 30 min from DNA extraction to detection. Because our protocol does not require much hands-on time or special equipment, this LAMP assay promises to become a rapid and simple screening method for male-derived samples in forensic investigations.

Atomic force microscope cantilever as an encoding sensor for real-time displacement measurement

International Nuclear Information System (INIS)

Chen, Xiaomei; Koenders, Ludger; Wolff, Helmut; Haertig, Frank; Schilling, Meinhard

2010-01-01

A tuning fork-based atomic force microscope cantilever has been investigated for application as an encoding sensor for real-time displacement measurement. The algorithm used to encode the displacement is based on the direct count of the integer pitches of a known grating, and the calculation of the fractional parts of a pitch at the beginning and during displacement. A cross-correlation technique has been adopted and applied to the real-time signal filtering process for the determination of the pitch during scanning by using a half sinusoidal waveform template. For the first investigation, a 1D sinusoidal grating with the pitch of 300 nm is used. The repeatability of displacement measurements over a distance of 70 µm is better than 2.2 nm. As the first application, the real-time displacement of a scanning stage is measured by the new encoding principle as it is moved in an open-loop mode and closed-loop mode based on its built-in capacitance sensor

A cascade autocatalytic strand displacement amplification and hybridization chain reaction event for label-free and ultrasensitive electrochemical nucleic acid biosensing.

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Chen, Zhiqiang; Liu, Ying; Xin, Chen; Zhao, Jikuan; Liu, Shufeng

2018-04-23

Herein, an autocatalytic strand displacement amplification (ASDA) strategy was proposed for the first time, which was further ingeniously coupled with hybridization chain reaction (HCR) event for the isothermal, label-free and multiple amplification toward nucleic acid detection. During the ASDA module, the target recognition opens the immobilized hairpin probe (IP) and initiates the annealing of the auxiliary DNA strand (AS) with the opened IP for the successive polymerization and nicking reaction in the presence of DNA polymerase and nicking endonuclease. This induces the target recycling and generation of a large amount of intermediate DNA sequences, which can be used as target analogy to execute the autocatalytic strand displacement amplification. Simultaneously, the introduced AS strand can propagate the HCR between two hairpins (H1 and H2) to form a linear DNA concatamer with cytosine (C)-rich loop region, which can facilitate the in-situ synthesis of silver nanoclusters (AgNCs) as electrochemical tags for further amplification toward target responses. With current cascade ASDA and HCR strategy, the detection of target DNA could be achieved with a low detection limit of about 0.16 fM and a good selectivity. The developed biosensor also exhibits the distinct advantages of flexibility and simplicity in probe design and biosensor fabrication, and label-free electrochemical detection, thus opens a promising avenue for the detection of nucleic acid with low abundance in bioanalysis and clinical biomedicine. Copyright © 2018 Elsevier B.V. All rights reserved.

Molecular Characterization of Sudanese and Southern Sudanese Chicken Breeds Using mtDNA D-Loop

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Charles E. Wani

2014-01-01

Full Text Available The objective of this study was to assess the genetic relationships and diversity and to estimate the amount of gene flow among the five chicken populations from Sudan and South Sudan and commercial strain of egg line White Leghorn chickens. The chicken populations were genotyped using mtDNA D-loop as a molecular marker. PCR product of the mtDNA D-loop segment was 600 bp and 14 haplotypes were identified. The neighbor-joining phylogenetic tree indicated that the indigenous Sudanese chickens can be grouped into two clades, IV and IIIa only. Median joining networks analysis showed that haplotype LBB49 has the highest frequency. The hierarchal analysis of molecular variance (AMOVA showed that genetic variation within the population was 88.6% and the differentiation among the population was 11.4%. When the populations was redefined into two geographical zones, rich and poor Savanna, the results were fractioned into three genetic variations: between individuals within population 95.5%, between populations within the group 0.75%, and genetic variation between groups 3.75%. The pair wise Fst showed high genetic difference between Betwil populations and the rest with Fst ranging from 0.1492 to 0.2447. We found that there is large number of gene exchanges within the Sudanese indigenous chicken (Nm=4.622.

Voltammetric determination of attomolar levels of a sequence derived from the genom of hepatitis B virus by using molecular beacon mediated circular strand displacement and rolling circle amplification.

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Huang, Shan; Feng, Mengmeng; Li, Jiawen; Liu, Yi; Xiao, Qi

2018-03-03

The authors describe an electrochemical method for the determination of the single-stranded DNA (ssDNA) oligonucleotide with a sequence derived from the genom of hepatitis B virus (HBV). It is making use of circular strand displacement (CSD) and rolling circle amplification (RCA) strategies mediated by a molecular beacon (MB). This ssDNA hybridizes with the loop portion of the MB immobilized on the surface of a gold electrode, while primer DNA also hybridizes with the rest of partial DNA sequences of MB. This triggers the MB-mediated CSD. The RCA is then initiated to produce a long DNA strand with multiple tandem-repeat sequences, and this results in a significant increase of the differential pulse voltammetric response of the electrochemical probe Methylene Blue at a rather low working potential of -0.24 V (vs. Ag/AgCl). Under optimal experimental conditions, the assay displays an ultrahigh sensitivity (with a 2.6 aM detection limit) and excellent selectivity. Response is linear in the 10 to 700 aM DNA concentration range. Graphical abstract Schematic of a voltammetric method for the determination of attomolar levels of target DNA. It is based on molecular beacon mediated circular strand displacement and rolling circle amplification strategies. Under optimal experimental conditions, the assay displays an ultrahigh sensitivity with a 2.6 aM detection limit and excellent selectivity.

Anisotropic dislocation loop nucleation in ion-irradiated MgAl2O4

International Nuclear Information System (INIS)

Zinkle, S.J.

1992-01-01

This work is intended to investigate the effects of transmutation products and varying ionizing-to-displacive damage ratio on microstructural evolution in ceramics for fusion machine. Polycrystalline disks of stoichiometric magnesium aluminate spinel (MgAl 2 O 4 ) were irradiated with 2 MeV Al + ions at 650C and subsequently analyzed in cross section using transmission electron microscopy (TEM). Interstitial dislocation loops were observed on [110] and [111] habit planes. The population of loops on both sets of habit planes was strongly dependent on their orientation with respect to the ion beam direction. The density of loops with habit plane normals nearly perpendicular to the ion beam direction was much higher than loops with habit plane normals nearly parallel to the ion beam direction. On the other hand, the loop size was nearly independent of habit plane orientation. This anisotropic loop nucleation does not occur in ion-irradiated metals such as copper and may be associated with the structure of displacement cascades in ceramics

LAVA: An Open-Source Approach To Designing LAMP (Loop-Mediated Isothermal Amplification DNA Signatures

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Gardner Shea N

2011-06-01

Full Text Available Abstract Background We developed an extendable open-source Loop-mediated isothermal AMPlification (LAMP signature design program called LAVA (LAMP Assay Versatile Analysis. LAVA was created in response to limitations of existing LAMP signature programs. Results LAVA identifies combinations of six primer regions for basic LAMP signatures, or combinations of eight primer regions for LAMP signatures with loop primers, which can be used as LAMP signatures. The identified primers are conserved among target organism sequences. Primer combinations are optimized based on lengths, melting temperatures, and spacing among primer sites. We compare LAMP signature candidates for Staphylococcus aureus created both by LAVA and by PrimerExplorer. We also include signatures from a sample run targeting all strains of Mycobacterium tuberculosis. Conclusions We have designed and demonstrated new software for identifying signature candidates appropriate for LAMP assays. The software is available for download at http://lava-dna.googlecode.com/.

Data on haplotype diversity in the hypervariable region I, II and III of mtDNA amongst the Brahmin population of Haryana

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Kapil Verma

2018-04-01

Full Text Available Human mitochondrial DNA (mtDNA is routinely analysed for pathogenic mutations, evolutionary studies, estimation of time of divergence within or between species, phylogenetic studies and identification of degraded remains. The data on various regions of human mtDNA has added enormously to the knowledge pool of population genetics as well as forensic genetics. The displacement-loop (D-loop in the control region of mtDNA is rated as the most rapidly evolving part, due to the presence of variations in this region. The control region consists of three hypervariable regions. These hypervariable regions (HVI, HVII and HVIII tend to mutate 5–10 times faster than nuclear DNA. The high mutation rate of these hypervariable regions is used in population genetic studies and human identity testing. In the present data, potentially informative hypervariable regions of mitochondrial DNA (mtDNA i.e. HVI (np 16024–16365, HVII (np 73–340 and HVIII (np 438–576 were estimated to understand the genetic diversity amongst Brahmin population of Haryana. Blood samples had been collected from maternally unrelated individuals from the different districts of Haryana. An array of parameters comprising of polymorphic sites, transitions, transversions, deletions, gene diversity, nucleotide diversity, pairwise differences, Tajima's D test, Fu's Fs test, mismatch observed variance and expected heterozygosity were estimated. The observed polymorphisms with their respective haplogroups in comparison to rCRS were assigned. Keywords: Mitochondrial DNA, D-loop, Hypervariable regions, Forensic genetics

Mechanism of chimera formation during the Multiple Displacement Amplification reaction

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Stockwell Timothy B

2007-04-01

Full Text Available Abstract Background Multiple Displacement Amplification (MDA is a method used for amplifying limiting DNA sources. The high molecular weight amplified DNA is ideal for DNA library construction. While this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of MDA to generate chimeric DNA rearrangements in the amplified DNA. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them. Results Here, we characterize the major types of chimeras formed by carrying out an MDA whole genome amplification from a single E. coli cell and sequencing by the 454 Life Sciences method. Analysis of 475 chimeras revealed the predominant reaction mechanisms that create the DNA rearrangements. The highly branched DNA synthesized in MDA can assume many alternative secondary structures. DNA strands extended on an initial template can be displaced becoming available to prime on a second template creating the chimeras. Evidence supports a model in which branch migration can displace 3'-ends freeing them to prime on the new templates. More than 85% of the resulting DNA rearrangements were inverted sequences with intervening deletions that the model predicts. Intramolecular rearrangements were favored, with displaced 3'-ends reannealing to single stranded 5'-strands contained within the same branched DNA molecule. In over 70% of the chimeric junctions, the 3' termini had initiated priming at complimentary sequences of 2–21 nucleotides (nts in the new templates. Conclusion Formation of chimeras is an important limitation to the MDA method, particularly for whole genome sequencing. Identification of the mechanism for chimera formation provides new insight into the MDA reaction and suggests methods to reduce chimeras. The 454 sequencing approach used here will provide a rapid method to assess the utility of reaction modifications.

Accumulation of dislocation loops in the α phase of Zr Excel alloy under heavy ion irradiation

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Yu, Hongbing; Yao, Zhongwen; Idrees, Yasir; Zhang, He K.; Kirk, Mark A.; Daymond, Mark R.

2017-08-01

In-situ heavy ion irradiations were performed on the high Sn content Zr alloy 'Excel', measuring type dislocation loop accumulation up to irradiation damage doses of 10 dpa at a range of temperatures. The high content of Sn, which diffuses slowly, and the thin foil geometry of the sample provide a unique opportunity to study an extreme case where displacement cascades dominate the loop formation and evolution. The dynamic observation of dislocation loop evolution under irradiation at 200 °C reveals that type dislocation loops can form at very low dose (0.0025 dpa). The size of the dislocation loops increases slightly with irradiation damage dose. The mechanism controlling loop growth in this study is different from that in neutron irradiation; in this study, larger dislocation loops can condense directly from the interaction of displacement cascades and the high concentration of point defects in the matrix. The size of the dislocation loop is dependent on the point defect concentration in the matrix. A negative correlation between the irradiation temperature and the dislocation loop size was observed. A comparison between cascade dominated loop evolution (this study), diffusion dominated loop evolution (electron irradiation) and neutron irradiation suggests that heavy ion irradiation alone may not be enough to accurately reproduce neutron irradiation induced loop structures. An alternative method is proposed in this paper. The effects of Sn on the displacement cascades, defect yield, and the diffusion behavior of point defects are established.

Toehold-mediated strand displacement reaction-dependent fluorescent strategy for sensitive detection of uracil-DNA glycosylase activity.

Science.gov (United States)

Wu, Yushu; Wang, Lei; Jiang, Wei

2017-03-15

Sensitive detection of uracil-DNA glycosylase (UDG) activity is beneficial for evaluating the repairing process of DNA lesions. Here, toehold-mediated strand displacement reaction (TSDR)-dependent fluorescent strategy was constructed for sensitive detection of UDG activity. A single-stranded DNA (ssDNA) probe with two uracil bases and a trigger sequence were designed. A hairpin probe with toehold domain was designed, and a reporter probe was also designed. Under the action of UDG, two uracil bases were removed from ssDNA probe, generating apurinic/apyrimidinic (AP) sites. Then, the AP sites could inhibit the TSDR between ssDNA probe and hairpin probe, leaving the trigger sequence in ssDNA probe still free. Subsequently, the trigger sequence was annealed with the reporter probe, initiating the polymerization and nicking amplification reaction. As a result, numerous G-quadruplex (G4) structures were formed, which could bind with N-methyl-mesoporphyrin IX (NMM) to generate enhanced fluorescent signal. In the absence of UDG, the ssDNA probe could hybridize with the toehold domain of the hairpin probe to initiate TSDR, blocking the trigger sequence, and then the subsequent amplification reaction would not occur. The proposed strategy was successfully implemented for detecting UDG activity with a detection limit of 2.7×10 -5 U/mL. Moreover, the strategy could distinguish UDG well from other interference enzymes. Furthermore, the strategy was also applied for detecting UDG activity in HeLa cells lysate with low effect of cellular components. These results indicated that the proposed strategy offered a promising tool for sensitive quantification of UDG activity in UDG-related function study and disease prognosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Wilson loops in Kerr gravitation

International Nuclear Information System (INIS)

Bollini, C.G.; Giambiagi, J.J.; Tiomno, J.

1981-01-01

The ordered integrals for several paths in Kerr gravitation is computed in a compact form. When the path is closed its relation with the angular parallel displacement is discussed and the corresponding Wilson loop is calculated. The validity of Mandelstam relations for gauge fields is also explicitly verified. (Author) [pt

Fundamental and Harmonic Oscillations in Neighboring Coronal Loops

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Li, Hongbo; Liu, Yu; Vai Tam, Kuan

2017-06-01

We present observations of multimode (fundamental and harmonic) oscillations in a loop system, which appear to be simultaneously excited by a GOES C-class flare. Analysis of the periodic oscillations reveals that (1) the primary loop with a period of P a ≈ 4 minutes and a secondary loop with two periods of P a ≈ 4 minutes and P b ≈ 2 minutes are detected simultaneously in closely spaced loop strands; (2) both oscillation components have their peak amplitudes near the loop apex, while in the second loop the low-frequency component P a dominates in a loop segment that is two times larger than the high-frequency component P b ; (3) the harmonic mode P b shows the largest deviation from a sinusoidal loop shape at the loop apex. We conclude that multiple harmonic modes with different displacement profiles can be excited simultaneously even in closely spaced strands, similar to the overtones of a violin string.

AgNPs-3D nanostructure enhanced electrochemiluminescence of CdSe quantum dot coupled with strand displacement amplification for sensitive biosensing of DNA

International Nuclear Information System (INIS)

Jiao, Meng; Jie, Guifen; Tan, Lu; Niu, Shuyan

2017-01-01

A novel strategy using Ag nanoparticles-3D (AgNPs-3D) nanostructure enhanced electrochemiluminescence (ECL) of CdSe quantum dots (QDs) coupled with strand displacement amplification (SDA) for sensitive biosensing of DNA was successfully designed. The prepared CdSe QDs with intense ECL were assembled on the poly (diallyldimethylammonium chloride) (PDDA) graphene oxide (GO) nanocomposites modified electrode, then gold nanoparticles (NPs) as the quenching probe was conjugated to the QDs, ECL signal was efficiently quenched. The target DNA induced cycling SDA and generated a large number of DNA s1. The released DNA s1 could open the hairpin DNA with quenching probe. So the presence of low levels of target DNA can potentially result in a significant enhancement of ECL signal. Furthermore, large number of AgNPs were then in situ reduced in the 3D DNA skeleton on the electrode, which dramaticlly enhanced ECL signal of QDs owing to the excellent electrical conductivity, and the much amplified ECL signal change has a quantitative relation with the target DNA. So by combining the AgNPs-3D nanostructure and cycling SDA to achieve greatly amplified detection of DNA, the promising ECL strategy could provide a highly sensitive platform for various biomolecules and has a good prospect for clinical diagnosis in the future. - Graphical abstract: A novel strategy using AgNPs-3D nanostructure enhanced electrochemiluminescence of CdSe quantum dot coupled with DNA strand displacement amplification for sensitive biosensing of DNA was successfully designed, the proposed biosensor can be expected to be an emerging alternative for straightforward nucleic acid detection in complex samples with an easy and rapid way. - Highlights: • AgNPs-3D nanostructure for enhancing ECL signal of CdSe QDs was successfully designed. • A new dual amplification strategy for detection of DNA by using AgNPs-3D nanostructure coupled with SDA was developed. • It is for the first time AgNPs-3D nanostructure

Ultrasensitive electrochemical biosensor for detection of DNA from Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification.

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Hu, Yuhua; Xu, Xueqin; Liu, Qionghua; Wang, Ling; Lin, Zhenyu; Chen, Guonan

2014-09-02

A simple, ultrasensitive, and specific electrochemical biosensor was designed to determine the given DNA sequence of Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification. The target DNA (TD, the DNA sequence from the hypervarient region of 16S rDNA of Bacillus subtilis) could be detected by the differential pulse voltammetry (DPV) in a range from 0.1 fM to 20 fM with the detection limit down to 0.08 fM at the 3s(blank) level. This electrochemical biosensor exhibits high distinction ability to single-base mismatch, double-bases mismatch, and noncomplementary DNA sequence, which may be expected to detect single-base mismatch and single nucleotide polymorphisms (SNPs). Moreover, the applicability of the designed biosensor for detecting the given DNA sequence from Bacillus subtilis was investigated. The result obtained by electrochemical method is approximately consistent with that by a real-time quantitative polymerase chain reaction detecting system (QPCR) with SYBR Green.

Detection of bovine mastitis pathogens by loop-mediated isothermal amplification and an electrochemical DNA chip.

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Kawai, Kazuhiro; Inada, Mika; Ito, Keiko; Hashimoto, Koji; Nikaido, Masaru; Hata, Eiji; Katsuda, Ken; Kiku, Yoshio; Tagawa, Yuichi; Hayashi, Tomohito

2017-12-22

Bovine mastitis causes significant economic losses in the dairy industry. Effective prevention of bovine mastitis requires an understanding of the infection status of a pathogenic microorganism in a herd that has not yet shown clinical signs of mastitis and appropriate treatment specific for the pathogenic microorganism. However, bacterial identification by culture has drawbacks in that the sensitivity may be low and the procedure can be complex. In this study, we developed a genetic detection method to identify mastitis pathogens using a simple and highly sensitive electrochemical DNA chip which can specifically detect bacterial DNA in milk specimens. First, we selected microorganisms belonging to 12 families and/or genera associated with mastitis for which testing should be performed. Next, we optimized the conditions for amplifying microorganism DNA by loop-mediated isothermal amplification (LAMP) using 32 primers and the use of a DNA chip capable of measuring all pathogens simultaneously. Sample detection could be completed in just a few hours using this method. Comparison of the results obtained with our DNA chip method and those obtained by bacterial culture verified that when the culture method was set to 100%, the total positive concordance rate of the DNA chip was 85.0% and the total negative concordance rate was 86.9%. Furthermore, the proposed method allows both rapid and highly sensitive detection of mastitis pathogens. We believe that this method will contribute to the development of an effective mastitis control program.

Phylogenetic relationships of German heavy draught horse breeds inferred from mitochondrial DNA D-loop variation.

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Aberle, K S; Hamann, H; Drögemüller, C; Distl, O

2007-04-01

We analysed a 610-bp mitochondrial (mt)DNA D-loop fragment in a sample of German draught horse breeds and compared the polymorphic sites with sequences from Arabian, Hanoverian, Exmoor, Icelandic, Sorraia and Przewalski's Horses as well as with Suffolk, Shire and Belgian horses. In a total of 65 horses, 70 polymorphic sites representing 47 haplotypes were observed. The average percentage of polymorphic sites was 11.5% for the mtDNA fragment analysed. In the nine different draught horse breeds including South German, Mecklenburg, Saxon Thuringa coldblood, Rhenisch German, Schleswig Draught Horse, Black Forest Horse, Shire, Suffolk and Belgian, 61 polymorphic sites and 24 haplotypes were found. The phylogenetic analysis failed to show monophyletic groups for the draught horses. The analysis indicated that the draught horse populations investigated consist of diverse genetic groups with respect to their maternal lineage.

Phylogenetic relationships of Scomberomorus commerson using sequence analysis of the mtDNA D-loop region in the Persian Gulf, Oman Sea and Arabian Sea

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Ana Mansourkiaei

2016-04-01

Full Text Available Abstract Narrow-barred Spanish mackerel, Scomberomorus commerson, is an epipelagic and migratory species of family Scombridae which have a significant role in terms of ecology and fishery. 100 samples were collected from the Persian Gulf, Oman Sea and Arabian Sea. Part of their dorsal fins was snipped and transferred to micro-tubes containing ethanol; then, DNAs were extracted and HRM-Real Time PCR was performed to designate representative specimens for sequencing. Phylogenetic relationships of S. commerson from Persian Gulf, Oman Sea and Arabian Sea were investigated using sequence data of mitochondrial DNA D-loop region. None clustered Neighbor Joining tree indicated the proximity amid S. commerson in four sites. As numbers demonstrated in sequence analyses of mitochondrial DNA D-Loop region a sublimely high degree of genetic similarity among S. commerson from the Persian Gulf and Oman Sea were perceived, thereafter, having one stock structure of S. commerson in four regions were proved, and this approximation can be merely justified by their migration process along the coasts of Oman Sea and Persian Gulf. Therefore, the assessment of distribution patterns of 20 haplotypes in the constructed phylogenetic tree using mtDNA D-Loop sequences ascertained that no significant clustering according to the sampling sites was concluded.

Improved multiple displacement amplification (iMDA) and ultraclean reagents.

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Motley, S Timothy; Picuri, John M; Crowder, Chris D; Minich, Jeremiah J; Hofstadler, Steven A; Eshoo, Mark W

2014-06-06

Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA). A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance

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

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Margarita Salas

2016-08-01

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

Comparison of DNA Microarray, Loop-Mediated Isothermal Amplification (LAMP) and Real-Time PCR with DNA Sequencing for Identification of Fusarium spp. Obtained from Patients with Hematologic Malignancies.

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de Souza, Marcela; Matsuzawa, Tetsuhiro; Sakai, Kanae; Muraosa, Yasunori; Lyra, Luzia; Busso-Lopes, Ariane Fidelis; Levin, Anna Sara Shafferman; Schreiber, Angélica Zaninelli; Mikami, Yuzuru; Gonoi, Tohoru; Kamei, Katsuhiko; Moretti, Maria Luiza; Trabasso, Plínio

2017-08-01

The performance of three molecular biology techniques, i.e., DNA microarray, loop-mediated isothermal amplification (LAMP), and real-time PCR were compared with DNA sequencing for properly identification of 20 isolates of Fusarium spp. obtained from blood stream as etiologic agent of invasive infections in patients with hematologic malignancies. DNA microarray, LAMP and real-time PCR identified 16 (80%) out of 20 samples as Fusarium solani species complex (FSSC) and four (20%) as Fusarium spp. The agreement among the techniques was 100%. LAMP exhibited 100% specificity, while DNA microarray, LAMP and real-time PCR showed 100% sensitivity. The three techniques had 100% agreement with DNA sequencing. Sixteen isolates were identified as FSSC by sequencing, being five Fusarium keratoplasticum, nine Fusarium petroliphilum and two Fusarium solani. On the other hand, sequencing identified four isolates as Fusarium non-solani species complex (FNSSC), being three isolates as Fusarium napiforme and one isolate as Fusarium oxysporum. Finally, LAMP proved to be faster and more accessible than DNA microarray and real-time PCR, since it does not require a thermocycler. Therefore, LAMP signalizes as emerging and promising methodology to be used in routine identification of Fusarium spp. among cases of invasive fungal infections.

Fiber-optic couplers as displacement sensors

Science.gov (United States)

Baruch, Martin C.; Gerdt, David W.; Adkins, Charles M.

2003-04-01

We introduce the novel concept of using a fiber-optic coupler as a versatile displacement sensor. Comparatively long fiber-optic couplers, with a coupling region of approximately 10 mm, are manufactured using standard communication SM fiber and placed in a looped-back configuration. The result is a displacement sensor, which is robust and highly sensitive over a wide dynamic range. This displacement sensor resolves 1-2 μm over distances of 1-1.5 mm and is characterized by the essential absence of a 'spring constant' plaguing other strain gauge-type sensors. Consequently, it is possible to couple to extremely weak vibrations, such as the skin displacement affected by arterial heart beat pulsations. Used as a wrist-worn heartbeat monitor, the fidelity of the arterial pulse signal has been shown to be so high that it is possible to not only determine heartbeat and breathing rates, but to implement a new single-point blood pressure measurement scheme which does not squeeze the arm. In an application as a floor vibration sensor for the non-intrusive monitoring of independently living elderly, the sensor has been shown to resolve the distinct vibration spectra of different persons and different events.

Fluorometric determination of nucleic acids based on the use of polydopamine nanotubes and target-induced strand displacement amplification.

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Ge, Jia; Bai, Dong-Mei; -Geng, Xin; Hu, Ya-Lei; Cai, Qi-Yong; Xing, Ke; Zhang, Lin; Li, Zhao-Hui

2018-01-10

The authors describe a fluorometric method for the quantitation of nucleic acids by combining (a) cycled strand displacement amplification, (b) the unique features of the DNA probe SYBR Green, and (c) polydopamine nanotubes. SYBR Green undergoes strong fluorescence enhancement upon intercalation into double-stranded DNA (dsDNA). The polydopamine nanotubes selectively adsorb single-stranded DNA (ssDNA) and molecular beacons. In the absence of target DNA, the molecular beacon, primer and SYBR Green are adsorbed on the surface of polydopamine nanotubes. This results in quenching of the fluorescence of SYBR Green, typically measured at excitation/emission wavelengths of 488/518 nm. Upon addition of analyte (target DNA) and polymerase, the stem of the molecular beacon is opened so that it can bind to the primer. This triggers target strand displacement polymerization, during which dsDNA is synthesized. The hybridized target is then displaced due to the strand displacement activity of the polymerase. The displaced target hybridizes with another molecular beacon. This triggers the next round of polymerization. Consequently, a large amount of dsDNA is formed which is detected by addition of SYBR Green. Thus, sensitive and selective fluorometric detection is realized. The fluorescent sensing strategy shows very good analytical performances towards DNA detection, such as a wide linear range from 0.05 to 25 nM with a low limit of detection of 20 pM. Graphical abstract Schematic of a fluorometric strategy for highly sensitive and selective determination of nucleic acids by combining strand displacement amplification and the unique features of SYBR Green I (SG) and polydopamine nanotubes.

A Looping-Based Model for Quenching Repression.

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Yaroslav Pollak

2017-01-01

Full Text Available We model the regulatory role of proteins bound to looped DNA using a simulation in which dsDNA is represented as a self-avoiding chain, and proteins as spherical protrusions. We simulate long self-avoiding chains using a sequential importance sampling Monte-Carlo algorithm, and compute the probabilities for chain looping with and without a protrusion. We find that a protrusion near one of the chain's termini reduces the probability of looping, even for chains much longer than the protrusion-chain-terminus distance. This effect increases with protrusion size, and decreases with protrusion-terminus distance. The reduced probability of looping can be explained via an eclipse-like model, which provides a novel inhibitory mechanism. We test the eclipse model on two possible transcription-factor occupancy states of the D. melanogaster eve 3/7 enhancer, and show that it provides a possible explanation for the experimentally-observed eve stripe 3 and 7 expression patterns.

Subspecies identification of captive Orang Utan in Melaka based on D-loop mitochondria DNA

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Kamaluddin, Siti Norsyuhada; Yaakop, Salmah; Idris, Wan Mohd Razi; Rovie-Ryan, Jeffrine Japning; Md-Zain, Badrul Munir

2018-04-01

Mitochondrial DNA of Bornean Orang Utan populations suggests that there are three different subspecies (Pongo pygmaeus pygmaeus; Sarawak & Northwest Kalimantan, P. p. wurmbii; Southern West Kalimantan and Central Kalimantan, P. p. morio; East Kalimantan and Sabah). The subspecies of Orang Utans in captivity are difficult to determine through morphological observation. Thus, misidentification by ranger or zoo staffs leads to unwanted consequences especially towards conservation efforts of Orang Utan. The main objective of this study was to identify the subspecies and the geographic origin of 10 Orang Utans in Zoo Melaka and A' Famosa by using partial mitochondrial D-loop gene sequences. DNA of all individuals was extracted from FTA Card. Data analyses were performed using Maximum Parsimony, MP and Neighbor Joining, NJ. Molecular phylogeny analysis revealed that all the samples likely belong to one species of Sumatran Orang Utan (P. abelii) and three different subspecies of Bornean Orang Utans (P. p. pygmaeus, P. p. morio, and P. p. wurmbii). The results obtained in this study indirectly help the management of zoos in term of conservation and visitor's education.

Detection of Panton-Valentine Leukocidin DNA from methicillin-resistant Staphylococcus aureus by resistive pulse sensing and loop-mediated isothermal amplification with gold nanoparticles

International Nuclear Information System (INIS)

Yang, Alice Kar Lai; Lu, Haifei; Wu, Shu Yuen; Kwok, Ho Chin; Ho, Ho Pui; Yu, Samuel; Cheung, Anthony Ka Lun; Kong, Siu Kai

2013-01-01

Graphical abstract: -- Highlights: •A novel diagnostic assay is developed to detect the MRSA's Panton-Valentine Leukocidin toxin. •Detection is based on target DNA amplification at one single temperature at 65 °C by LAMP. •Amplicons are then hybridized with 2 Au-nanoparticles with specific DNA probes for sensing. •The supra-assemblies are subsequently sensed by resistive pulse sensing. •Detection limit: ∼200 copies of DNA; time for detection: completed within 2 h. -- Abstract: This report describes a novel diagnostic assay for rapid detection of the Panton-Valentine Leukocidin (PVL) toxin of methicillin-resistant Staphylococcus aureus (MRSA) utilizing resistive pulse sensing (RPS), loop-mediated isothermal DNA amplification (LAMP) in combination with gold nanoparticles (AuNPs). The PVL DNA from MRSA was specifically amplified by LAMP using four primers at one temperature (65 °C). The DNA products with biotin were then conjugated to a first AuNP1 (55 ± 2 nm) through biotin–avidin binding. A second AuNP2 (30 ± 1.5 nm) coated with a specific DNA probe hybridized with the LAMP DNA products at the loop region to enhance assay sensitivity and specificity, to generate supra-AuNP1-DNA-AuNP2 assemblies. Scanning electron microscopy confirmed the presence of these supra-assemblies. Using RPS, detection and quantitation of the agglomerated AuNPs were performed by a tunable fluidic nanopore sensor. The results demonstrate that the LAMP-based RPS sensor is sensitive and rapid for detecting the PVL DNA. This technique could achieve a limit of detection (LOD) up to about 500 copies of genomic DNA from the bacteria MRSA MW2 and the detection can be completed within two hours with a straightforward signal-to-readout setup. It is anticipated that this LAMP-based AuNP RPS may become an effective tool for MRSA detection and a potential platform in clinical laboratory to report the presence or absence of other types of infectious agents

An ATR-dependent function for the Ddx19 RNA helicase in nuclear R-loop metabolism.

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Hodroj, Dana; Recolin, Bénédicte; Serhal, Kamar; Martinez, Susan; Tsanov, Nikolay; Abou Merhi, Raghida; Maiorano, Domenico

2017-05-02

Coordination between transcription and replication is crucial in the maintenance of genome integrity. Disturbance of these processes leads to accumulation of aberrant DNA:RNA hybrids (R-loops) that, if unresolved, generate DNA damage and genomic instability. Here we report a novel, unexpected role for the nucleopore-associated mRNA export factor Ddx19 in removing nuclear R-loops formed upon replication stress or DNA damage. We show, in live cells, that Ddx19 transiently relocalizes from the nucleopore to the nucleus upon DNA damage, in an ATR/Chk1-dependent manner, and that Ddx19 nuclear relocalization is required to clear R-loops. Ddx19 depletion induces R-loop accumulation, proliferation-dependent DNA damage and defects in replication fork progression. Further, we show that Ddx19 resolves R-loops in vitro via its helicase activity. Furthermore, mutation of a residue phosphorylated by Chk1 in Ddx19 disrupts its interaction with Nup214 and allows its nuclear relocalization. Finally, we show that Ddx19 operates in resolving R-loops independently of the RNA helicase senataxin. Altogether these observations put forward a novel, ATR-dependent function for Ddx19 in R-loop metabolism to preserve genome integrity in mammalian cells. © 2017 The Authors.

Effects of internal hydrogen on the vacancy loop formation probability in Al

International Nuclear Information System (INIS)

Bui, T.X.; Sirois, E.; Robertson, I.M.

1990-04-01

The effect of internal hydrogen on the formation of vacancy dislocation loops from heavy-ion generated displacement cascades in Al has been investigated. Samples of high-purity aluminum and aluminum containing 900 and 1300 appM of hydrogen were irradiated at room temperature with 50 keV Kr+ ions. The ion dose rate was typically 2 x 10 10 ions cm -2 sec -1 and the ion dose was between 10 11 and 10 13 ion cm -2 . Under these irradiation conditions, dislocation loops were observed in all compositions, although the formation probability was relatively low (less than 10 percent of the displacement cascades produced a vacancy loop). The loop formation probability was further reduced by the presence of hydrogen. No difference in the geometry or the size of the loops created in the hydrogen free and hydrogen charged samples was found. These results are difficult to interpret, and the explanation may lie in the distribution and form of the hydrogen. To account for the large hydrogen concentrations and from calculations of the energy associated with hydrogen entry into aluminum, it has been suggested that the hydrogen enters the aluminum lattice with an accompanying vacancy. This will create hydrogen-vacancy complexes in the material; two dimensional complexes have been detected in the hydrogen-charged, but unirradiated, samples by the small-angle x-ray scattering technique. The possibility of these complexes trapping the vacancies produced by the cascade process exists thus lowering the formation probability. However, such a mechanism must occur within the lifetime of the cascade. Alternatively, if a displacement cascade overlaps with the hydrogen-vacancy complexes, the lower atomic density of the region will result in an increase in the cascade volume (decrease in the local vacancy concentration) which will also reduce the loop formation probability

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

Getting in (and out of) the loop: regulating higher order telomere structures.

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Luke-Glaser, Sarah; Poschke, Heiko; Luke, Brian

2012-01-01

The DNA at the ends of linear chromosomes (the telomere) folds back onto itself and forms an intramolecular lariat-like structure. Although the telomere loop has been implicated in the protection of chromosome ends from nuclease-mediated resection and unscheduled DNA repair activities, it potentially poses an obstacle to the DNA replication machinery during S-phase. Therefore, the coordinated regulation of telomere loop formation, maintenance, and resolution is required in order to establish a balance between protecting the chromosome ends and promoting their duplication prior to cell division. Until recently, the only factor known to influence telomere looping in human cells was TRF2, a component of the shelterin complex. Recent work in yeast and mouse cells has uncovered additional regulatory factors that affect the loop structure at telomeres. In the following "perspective" we outline what is known about telomere looping and highlight the latest results regarding the regulation of this chromosome end structure. We speculate about how the manipulation of the telomere loop may have therapeutic implications in terms of diseases associated with telomere dysfunction and uncontrolled proliferation.

Abrupt suppression of the transcription in the mammalian cells by X-irradiation at the violation of topological contraint of DNA superhelical loops

International Nuclear Information System (INIS)

Luchnik, A.N.; Dubinina, E.N.; Zbarskij, I.B.; Georgiev, G.P.; AN SSSR, Moscow. Inst. Molekulyarnoj Biologii)

1987-01-01

It is supposed that the whole transcription in mammalian cells depends on elastic stresses in constraint DNA loops. It is assumed that a specific molecular mechanism of transcription stop under irradiation is explained as follows: conformation of nucleosomes is changed (including DNA, histon octamer) in actively elastic-stressed chromatin. Elastically stressed DNA can promote stabilization of unfolded nucleosomes. Such nucleosomes do not interfere in RNA-polymerase conducting transcription. Nucleosomes after losing elastic stresses take wither globulra (non-active) conformation or histons dissociate with DNA, and instead of it non-active ocatmers of nucleosomes precipitate on it. The globular octamer prevents RNA-polymerase from conducting transcription through the whole length of nucleoprotein fibril. This hypothesis is substantiated by the facts that X-radiation removes High- and super-sensitivity of active chromatin to DNA-aze I as well as dissociates histons from active minichromosoms

[Investigation of RNA viral genome amplification by multiple displacement amplification technique].

Science.gov (United States)

Pang, Zheng; Li, Jian-Dong; Li, Chuan; Liang, Mi-Fang; Li, De-Xin

2013-06-01

In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.

R-loops in bacterial transcription: their causes and consequences.

Science.gov (United States)

Gowrishankar, J; Leela, J Krishna; Anupama, K

2013-01-01

Nascent untranslated transcripts in bacteria are prone to generating RNA-DNA hybrids (R-loops); Rho-dependent transcription termination acts to reduce their prevalence. Here we discuss the mechanisms of R-loop formation and growth inhibition in bacteria.

Boosted PWM open loop control of hydraulic proportional valves

International Nuclear Information System (INIS)

Amirante, R.; Innone, A.; Catalano, L.A.

2008-01-01

This paper presents an innovative open loop control technique for direct single stage hydraulic proportional valves whose response rate is significantly higher than that obtained by standard open loop control techniques, even comparable to more costly commercial closed loop systems. Different from standard open loop techniques, which provide the coil with a constant current proportional to the target position, the control strategy proposed in this paper employs the peak and hold (P and H) technique, widely used in Diesel engine modern supply systems, to boost the duty cycle value of the pulse width modulation (PWM) signal for a short time, namely during the spool displacement, while maintaining a lower duty cycle for holding the spool in the required opening position. The developed 'boosted PWM' technique only requires a low cost microcontroller, such as a peripheral interface controller (PIC) equipped with a metal oxide semiconductor (MOS) power driver. The PWM parameters are calibrated as a function of the spool displacement so as to maximize the response rate without introducing overshoots: the collected data are stored in the PIC. Different valve opening procedures with step response have been compared to demonstrate the merits of the proposed boosted PWM technique. No overshoots have been registered. Moreover, the proposed method is characterized by a significantly higher response rate with respect to a standard open loop control, which approximately has the same cost. Similar experimental tests show that the proposed boosted PWM technique has a response rate even higher than that provided by the more costly commercial closed loop system mounted on the valve, and it produces no overshoots

Boosted PWM open loop control of hydraulic proportional valves

Energy Technology Data Exchange (ETDEWEB)

Amirante, R.; Catalano, L.A. [Dipartimento di Ingegneria Meccanica e Gestionale, Politecnico di Bari, Via Re David 200, 70125 Bari (Italy); Innone, A. [Universita degli Studi di Foggia, via Napoli, 25 Foggia (Italy)

2008-08-15

This paper presents an innovative open loop control technique for direct single stage hydraulic proportional valves whose response rate is significantly higher than that obtained by standard open loop control techniques, even comparable to more costly commercial closed loop systems. Different from standard open loop techniques, which provide the coil with a constant current proportional to the target position, the control strategy proposed in this paper employs the peak and hold (P and H) technique, widely used in Diesel engine modern supply systems, to boost the duty cycle value of the pulse width modulation (PWM) signal for a short time, namely during the spool displacement, while maintaining a lower duty cycle for holding the spool in the required opening position. The developed 'boosted PWM' technique only requires a low cost microcontroller, such as a peripheral interface controller (PIC) equipped with a metal oxide semiconductor (MOS) power driver. The PWM parameters are calibrated as a function of the spool displacement so as to maximize the response rate without introducing overshoots: the collected data are stored in the PIC. Different valve opening procedures with step response have been compared to demonstrate the merits of the proposed boosted PWM technique. No overshoots have been registered. Moreover, the proposed method is characterized by a significantly higher response rate with respect to a standard open loop control, which approximately has the same cost. Similar experimental tests show that the proposed boosted PWM technique has a response rate even higher than that provided by the more costly commercial closed loop system mounted on the valve, and it produces no overshoots. (author)

Loop-Mediated Isothermal Amplification Targeting Actin DNA of Trichomonas vaginalis.

Science.gov (United States)

Goo, Youn-Kyoung; Shin, Won-Sik; Yang, Hye-Won; Joo, So-Young; Song, Su-Min; Ryu, Jae-Sook; Kong, Hyun-Hee; Lee, Won-Ki; Chung, Dong-Il; Hong, Yeonchul

2016-06-01

Trichomoniasis caused by Trichomonas vaginalis is a common sexually transmitted disease. Its association with several health problems, including preterm birth, pelvic inflammatory disease, cervical cancer, and transmission of human immunodeficiency virus, emphasizes the importance of improved access to early and accurate detection of T. vaginalis. In this study, a rapid and efficient loop-mediated isothermal amplification-based method for the detection of T. vaginalis was developed and validated, using vaginal swab specimens from subjects suspected to have trichomoniasis. The LAMP assay targeting the actin gene was highly sensitive with detection limits of 1 trichomonad and 1 pg of T. vaginalis DNA per reaction, and specifically amplified the target gene only from T. vaginalis. Validation of this assay showed that it had the highest sensitivity and better agreement with PCR (used as the gold standard) compared to microscopy and multiplex PCR. This study showed that the LAMP assay, targeting the actin gene, could be used to diagnose early infections of T. vaginalis. Thus, we have provided an alternative molecular diagnostic tool and a point-of-care test that may help to prevent trichomoniasis transmission and associated complications.

Second-strand cDNA synthesis: classical method

International Nuclear Information System (INIS)

Gubler, U.

1987-01-01

The classical scheme for the synthesis of double-stranded cDNA as it was reported in 1976 is described. Reverse transcription of mRNA with oligo(dT) as the primer generates first strands with a small loop at the 3' end of the cDNA (the end that corresponds to the 5' end of the mRNA). Subsequent removal of the mRNA by alkaline hydrolysis leaves single-stranded cDNA molecules again with a small 3' loop. This loop can be used by either reverse transcriptase or Klenow fragment of DNA polymerase I as a primer for second-strand synthesis. The resulting products are double-stranded cDNA molecules that are covalently closed at the end corresponding to the 5' end of the original mRNA. Subsequent cleavage of the short piece of single-stranded cDNA within the loop with the single-strand-specific S 1 nuclease generate open double-stranded molecules that can be used for molecular cloning in plasmids or in phage. Useful variations of this scheme have been described

Genetic instability associated with loop or stem–loop structures within transcription units can be independent of nucleotide excision repair

Science.gov (United States)

Burns, John A; Chowdhury, Moinuddin A; Cartularo, Laura; Berens, Christian; Scicchitano, David A

2018-01-01

Abstract Simple sequence repeats (SSRs) are found throughout the genome, and under some conditions can change in length over time. Germline and somatic expansions of trinucleotide repeats are associated with a series of severely disabling illnesses, including Huntington's disease. The underlying mechanisms that effect SSR expansions and contractions have been experimentally elusive, but models suggesting a role for DNA repair have been proposed, in particular the involvement of transcription-coupled nucleotide excision repair (TCNER) that removes transcription-blocking DNA damage from the transcribed strand of actively expressed genes. If the formation of secondary DNA structures that are associated with SSRs were to block RNA polymerase progression, TCNER could be activated, resulting in the removal of the aberrant structure and a concomitant change in the region's length. To test this, TCNER activity in primary human fibroblasts was assessed on defined DNA substrates containing extrahelical DNA loops that lack discernible internal base pairs or DNA stem–loops that contain base pairs within the stem. The results show that both structures impede transcription elongation, but there is no corresponding evidence that nucleotide excision repair (NER) or TCNER operates to remove them. PMID:29474673

Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella.

Science.gov (United States)

Papenfort, Kai; Espinosa, Elena; Casadesús, Josep; Vogel, Jörg

2015-08-25

Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution but constitutes a complex process that requires synchronization with the physiological state of the host bacteria. Although several host transcription factors are known to regulate plasmid-borne transfer genes, RNA-based regulatory circuits for host-plasmid communication remain unknown. We describe a posttranscriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two separate seed-pairing domains to activate the mRNAs of both the sigma-factor σ(S) and the RicI protein, a previously uncharacterized membrane protein here shown to inhibit conjugation. Transcription of ricI requires σ(S) and, together, RprA and σ(S) orchestrate a coherent feedforward loop with AND-gate logic to tightly control the activation of RicI synthesis. RicI interacts with the conjugation apparatus protein TraV and limits plasmid transfer under membrane-damaging conditions. To our knowledge, this study reports the first small RNA-controlled feedforward loop relying on posttranscriptional activation of two independent targets and an unexpected role of the conserved RprA small RNA in controlling extrachromosomal DNA transfer.

Getting in (and out of the loop: regulating higher order telomere structures

Directory of Open Access Journals (Sweden)

Sarah eLuke-Glaser

2012-11-01

Full Text Available The DNA at the ends of linear chromosomes (the telomere folds back onto itself and forms an intramolecular lariat-like structure. Although the telomere loop has been implicated in the protection of chromosome ends from nuclease-mediated resection and unscheduled DNA repair activities, it potentially poses an obstacle to the DNA replication machinery during S phase. Therefore, the coordinated regulation of telomere loop formation, maintenance and resolution is required in order to establish a balance between protecting the chromosome ends and promoting their duplication prior to cell division. Until recently, the only factor know to influence telomere looping in human cells was TRF2, a component of the shelterin complex. Recent work in yeast and mouse cells has uncovered additional regulatory factors that affect the loop structure at telomeres. In the following perspective we will outline what is known about telomere looping and highlight the latest results regarding the regulation of this chromosome end structure. We will speculate about how the manipulation of the telomere loop may have therapeutic implications in terms of diseases associated with telomere dysfunction and uncontrolled proliferation.

Time Course of Detection of Human Male DNA from Stained Blood Sample on Various Surfaces by Loop Mediated Isothermal Amplification and Polymerase Chain Reaction

OpenAIRE

Panan Kanchanaphum

2018-01-01

This study explores determining the sex of humans from blood stains taken from different surfaces and compares the time course of detection with the conventional PCR, Conventional Loop Mediated Isothermal Amplification (LAMP), and LAMP-Lateral Flow Dipstick (LFD). For the DNA templates, 7 male and 7 female blood stained samples were extracted and added to LAMP and PCR reaction solution to amplify the SRY gene. The DNA samples were extracted from the following blood stained materials: cloth, w...

Analysis of the distribution of DNA repair patches in the DNA-nuclear matrix complex from human cells

International Nuclear Information System (INIS)

Mullenders, L.H.F.

1983-01-01

The distribution of ultraviolet-induced repair patches along DNA loops attached to the nuclear matrix, was investigated by digestion with DNA-degrading enzymes and neutral sucrose gradient centrifugation. When DNA was gradually removed by DNAase 1, pulse label incorporated by ultraviolet-irradiated cells during 10 min in the presence of hydroxyurea or hydroxyurea/arabinosylcytosine showed similar degradation kinetics as prelabelled DNA. No preferential association of pulse label with the nuclear matrix was observed, neither within 30 min nor 13 h after iiradiation. When the pulse label was incorporated by replicative synthesis under the same conditions, a preferential association of newly-synthesized DNA with the nuclear matrix was observed. Single-strand specific digestion with nuclease S 1 of nuclear lysates from ultraviolet-irradiated cells, pulse labelled in the presence of hydroxyurea/arabinosylcytosine, caused a release of about 70% of the prelabelled DNA and 90% of the pulse-labelled DNA from the rapidly sedimenting material in sucrose gradients. The results suggest no specific involvement of the nuclear matrix in repair synthesis, a random distribution of repair patches along the DNA loops, and simultaneously multiple incision events per DNA loop. (Auth.)

Analysis of the distribution of DNA repair patches in the DNA-nuclear matrix complex from human cells

Energy Technology Data Exchange (ETDEWEB)

Mullenders, L.H.F. (Rijksuniversiteit Leiden (Netherlands). Lab. voor Stralengenetica en Chemische Mutagenese); Zeeland, A.A. van; Natarajan, A.T. (Cohen (J.A.) Inst. voor Radiopathologie en Stralenbescherming, Leiden (Netherlands))

1983-09-09

The distribution of ultraviolet-induced repair patches along DNA loops attached to the nuclear matrix, was investigated by digestion with DNA-degrading enzymes and neutral sucrose gradient centrifugation. When DNA was gradually removed by DNAase 1, pulse label incorporated by ultraviolet-irradiated cells during 10 min in the presence of hydroxyurea or hydroxyurea/arabinosylcytosine showed similar degradation kinetics as prelabelled DNA. No preferential association of pulse label with the nuclear matrix was observed, neither within 30 min nor 13 h after irradiation. When the pulse label was incorporated by replicative synthesis under the same conditions, a preferential association of newly-synthesized DNA with the nuclear matrix was observed. Single-strand specific digestion with nuclease S/sub 1/ of nuclear lysates from ultraviolet-irradiated cells, pulse labelled in the presence of hydroxyurea/arabinosylcytosine, caused a release of about 70% of the prelabelled DNA and 90% of the pulse-labelled DNA from the rapidly sedimenting material in sucrose gradients. The results suggest no specific involvement of the nuclear matrix in repair synthesis, a random distribution of repair patches along the DNA loops, and simultaneously multiple incision events per DNA loop.

An optimized protocol for DNA extraction from wheat seeds and Loop-Mediated Isothermal Amplification (LAMP) to detect Fusarium graminearum contamination of wheat grain.

Science.gov (United States)

Abd-Elsalam, Kamel; Bahkali, Ali; Moslem, Mohamed; Amin, Osama E; Niessen, Ludwig

2011-01-01

A simple, rapid, and efficient method for isolating genomic DNA from germinated seeds of wheat that is free from polysaccharides and polyphenols is reported. DNA was extracted, treated with RNase, measured and tested for completeness using agarose gel electrophoresis. DNA purification from wheat grains yielded abundant, amplifiable DNA with yields typically between 100 and 200 ng DNA/mg. The effectiveness and reliability of the method was tested by assessing quantity and quality of the isolated DNA using three PCR-based markers. Inter-simple sequence repeats (ISSRs) were used to assess the genetic diversity between different wheat varieties. Specific PCR primer pair Tox5-1/Tox5-2 and a loop-mediated isothermal amplification (LAMP) procedure were used to detect genomic DNA of Fusarium graminearum in contaminated wheat seeds. In this method there is no need to use liquid nitrogen for crushing germinated seedlings. The protocol takes approximately one hour to prepare high quality DNA. In combination with the LAMP assay it is a fast and cost-effective alternative to traditional diagnostic methods for the early detection of toxigenic fusaria in cereals.

Subthreshold displacement damage in copper--aluminum alloys during electron irradiation

International Nuclear Information System (INIS)

Drosd, R.; Kosel, T.; Washburn, J.

1976-12-01

During electron irradiation at low energies which results in a negligible damage rate in a pure material, lighter solute atoms are displaced, which may in turn indirectly displace solvent atoms by a focussed replacement collision or an interstitial diffusion jump. The extent to which lighter solute atoms contribute to the subthreshold damage rate has been examined by irradiating copper--aluminum alloys at high temperatures in a high voltage electron microscope. The damage rate, as measured by monitoring the growth rate of dislocation loops, at 300 kV was found to increase linearly with the aluminum concentration

DNA aptamer selection and aptamer-based fluorometric displacement assay for the hepatotoxin microcystin-RR

International Nuclear Information System (INIS)

Wu, Shijia; Li, Qi; Duan, Nuo; Wang, Zhouping; Ma, Haile

2016-01-01

Microcystin-RR (MC-RR) is a highly acute hepatotoxin produced by cyanobacteria. It is harmful to both humans and the environment. A novel aptamer was identified by the systemic evolution of ligands by exponential enrichment (SELEX) method as a recognition element for determination of MC-RR in aquatic products. The graphene oxide (GO) SELEX strategy was adopted to generate aptamers with high affinity and specificity. Of the 50 aptamer candidates tested, sequence RR-33 was found to display high affinity and selectivity, with a dissociation constant of 45.7 ± 6.8 nM. Aptamer RR-33 therefore was used as the recognition element in a fluorometric assay that proceeds as follows: (1) Biotinylated aptamer RR-33 is immobilized on the streptavidinylated wells of a microtiterplate, and carboxyfluorescein (FAM) labelled complementary DNA is then allowed to hybridize. (2) After removal of excess (unbound) cDNA, sample containing MC-RR is added and incubated at 37 °C for 2 h. (3) Displaced free cDNA is washed away and fluorescence intensity measured at excitation/emission wavelengths of 490/515 nm. The calibration plot is linear in the 0.20 to 2.5 ng·mL −1 concentration range, and the limit of detection is 80 pg·mL −1 . The results indicate that the GO-SELEX technology is appropriate for the screening of aptamers against small-molecule toxins. The detection scheme was applied to the determination of MC-RR in (spiked) water, mussel and fish and gave recoveries between 91 and 98 %. The method compares favorably to a known ELISA. Conceivably, this kind of assay is applicable to other toxins for which appropriate aptamers are available. (author)

Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples.

Science.gov (United States)

Abbasi, Ibrahim; Kirstein, Oscar D; Hailu, Asrat; Warburg, Alon

2016-10-01

Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Elastic field of approaching dislocation loop in isotropic bimaterial

International Nuclear Information System (INIS)

Wu, Wenwang; Xu, Shucai; Zhang, Jinhuan; Xia, Re; Qian, Guian

2015-01-01

A semi-analytical solution is developed for calculating interface traction stress (ITS) fields due to elastic modulus mismatch across the interface plane of isotropic perfectly bounded bimaterial system. Based on the semi-analytical approaches developed, ITS is used to correct the bulk elastic field of dislocation loop within infinite homogenous medium, and to produce continuous displacement and stress fields across the perfectly-bounded interface. Firstly, calculation examples of dislocation loops in Al–Cu bimaterial system are performed to demonstrate the efficiency of the developed semi-analytical approach; Then, the elastic fields of dislocation loops in twinning Cu and Cu–Nb bimaterial are analyzed; Finally, the effect of modulus mismatch across interface plane on the elastic field of bimaterial system is investigated, it is found that modulus mismatch has a drastic impact on the elastic fields of dislocation loops within bimaterial system. (paper)

Detection of phytoplasma by loop-mediated isothermal amplification of DNA (LAMP).

Science.gov (United States)

Obura, E; Masiga, D; Wachira, F; Gurja, B; Khan, Z R

2011-02-01

Napier stunt phytoplasma (16SrXI and 16SrIII) in eastern Africa is a serious threat to the expansion of Napier grass (Pennisetum purpureum) farming in the region, where it is widely cultivated as fodder in zero grazing livestock systems. The grass has high potential for bio-fuel production, and has been adopted by farmers as a countermeasure to cereal stem borer Lepidoptera, since it attracts and traps the insect. Diagnosis of stunt phytoplasma have been largely by nested polymerase chain reaction (nPCR) targeting the 16S rRNA gene. However, the method is laborious, costly and technically demanding. This investigation has developed a simpler but effective phytoplasma diagnostic tool, called; loop-mediated isothermal amplification of DNA (LAMP). The assay was tested on 8 symptomatic and 8 asymptomatic plants, while its detection limit was compared to nested PCR using samples serially diluted from 3 ng/μl to 0.38 pg/μl. Molecular typing of LAMP products was determined by BsrI restriction digestion and Southern blot analysis. The assay sensitivity, positive and negative predictive values were estimated, while the specificity was tested on 11 phytoplasma groups. LAMP was specific to 5 phytoplasma groups: 16SrVI, X, XI and XVI. BsrI restriction digestion produced two predicted fragments, and there was specific binding of probe DNA to the LAMP amplicons in Southern blot analysis. The assay sensitivity was 100%, while the positive and negative predictive values were 63 and 100% respectively. LAMP was 20-fold more sensitive than nested PCR. This study validates LAMP for routine diagnosis of Napier stunt and other closely related phytoplasmas. Copyright © 2010 Elsevier B.V. All rights reserved.

Selectivity in ligand recognition of G-quadruplex loops.

Science.gov (United States)

Campbell, Nancy H; Patel, Manisha; Tofa, Amina B; Ghosh, Ragina; Parkinson, Gary N; Neidle, Stephen

2009-03-03

A series of disubstituted acridine ligands have been cocrystallized with a bimolecular DNA G-quadruplex. The ligands have a range of cyclic amino end groups of varying size. The crystal structures show that the diagonal loop in this quadruplex results in a large cavity for these groups, in contrast to the steric constraints imposed by propeller loops in human telomeric quadruplexes. We conclude that the nature of the loop has a significant influence on ligand selectivity for particular quadruplex folds.

Assay for Human Rad51-Mediated DNA Displacement Loop Formation

OpenAIRE

sprotocols

2014-01-01

Authors: Steven Raynard and Patrick Sung Corresponding author ([]()) ### INTRODUCTION Homologous recombination is an important mechanism for the repair of damaged chromosomes, for preventing the demise of damaged replication forks, and for several other aspects of chromosome metabolism and maintenance. The homologous recombination reaction is mediated by the Rad51 recombinase. In the presence of ATP, Rad51 polymerizes on single-stranded D...

Signal-off Electrochemiluminescence Biosensor Based on Phi29 DNA Polymerase Mediated Strand Displacement Amplification for MicroRNA Detection.

Science.gov (United States)

Chen, Anyi; Gui, Guo-Feng; Zhuo, Ying; Chai, Ya-Qin; Xiang, Yun; Yuan, Ruo

2015-06-16

A target induced cycling strand displacement amplification (SDA) mediated by phi29 DNA polymerase (phi29) was first investigated and applied in a signal-off electrochemiluminescence (ECL) biosensor for microRNA (miRNA) detection. Herein, the target miRNA triggered the phi29-mediated SDA which could produce amounts of single-stranded DNA (assistant probe) with accurate and comprehensive nucleotide sequence. Then, the assistant probe hybridized with the capture probe and the ferrocene-labeled probe (Fc-probe) to form a ternary "Y" structure for ECL signal quenching by ferrocene. Therefore, the ECL intensity would decrease with increasing concentration of the target miRNA, and the sensitivity of biosensor would be promoted on account of the efficient signal amplification of the target induced cycling reaction. Besides, a self-enhanced Ru(II) ECL system was designed to obtain a stable and strong initial signal to further improve the sensitivity. The ECL assay for miRNA-21 detection is developed with excellent sensitivity of a concentration variation from 10 aM to 1.0 pM and limit of detection down to 3.3 aM.

A programming language for composable DNA circuits.

Science.gov (United States)

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.

Interplay of protein and DNA structure revealed in simulations of the lac operon.

Directory of Open Access Journals (Sweden)

Luke Czapla

Full Text Available The E. coli Lac repressor is the classic textbook example of a protein that attaches to widely spaced sites along a genome and forces the intervening DNA into a loop. The short loops implicated in the regulation of the lac operon suggest the involvement of factors other than DNA and repressor in gene control. The molecular simulations presented here examine two likely structural contributions to the in-vivo looping of bacterial DNA: the distortions of the double helix introduced upon association of the highly abundant, nonspecific nucleoid protein HU and the large-scale deformations of the repressor detected in low-resolution experiments. The computations take account of the three-dimensional arrangements of nucleotides and amino acids found in crystal structures of DNA with the two proteins, the natural rest state and deformational properties of protein-free DNA, and the constraints on looping imposed by the conformation of the repressor and the orientation of bound DNA. The predicted looping propensities capture the complex, chain-length-dependent variation in repression efficacy extracted from gene expression studies and in vitro experiments and reveal unexpected chain-length-dependent variations in the uptake of HU, the deformation of repressor, and the folding of DNA. Both the opening of repressor and the presence of HU, at levels approximating those found in vivo, enhance the probability of loop formation. HU affects the global organization of the repressor and the opening of repressor influences the levels of HU binding to DNA. The length of the loop determines whether the DNA adopts antiparallel or parallel orientations on the repressor, whether the repressor is opened or closed, and how many HU molecules bind to the loop. The collective behavior of proteins and DNA is greater than the sum of the parts and hints of ways in which multiple proteins may coordinate the packaging and processing of genetic information.

Interplay of protein and DNA structure revealed in simulations of the lac operon.

Science.gov (United States)

Czapla, Luke; Grosner, Michael A; Swigon, David; Olson, Wilma K

2013-01-01

The E. coli Lac repressor is the classic textbook example of a protein that attaches to widely spaced sites along a genome and forces the intervening DNA into a loop. The short loops implicated in the regulation of the lac operon suggest the involvement of factors other than DNA and repressor in gene control. The molecular simulations presented here examine two likely structural contributions to the in-vivo looping of bacterial DNA: the distortions of the double helix introduced upon association of the highly abundant, nonspecific nucleoid protein HU and the large-scale deformations of the repressor detected in low-resolution experiments. The computations take account of the three-dimensional arrangements of nucleotides and amino acids found in crystal structures of DNA with the two proteins, the natural rest state and deformational properties of protein-free DNA, and the constraints on looping imposed by the conformation of the repressor and the orientation of bound DNA. The predicted looping propensities capture the complex, chain-length-dependent variation in repression efficacy extracted from gene expression studies and in vitro experiments and reveal unexpected chain-length-dependent variations in the uptake of HU, the deformation of repressor, and the folding of DNA. Both the opening of repressor and the presence of HU, at levels approximating those found in vivo, enhance the probability of loop formation. HU affects the global organization of the repressor and the opening of repressor influences the levels of HU binding to DNA. The length of the loop determines whether the DNA adopts antiparallel or parallel orientations on the repressor, whether the repressor is opened or closed, and how many HU molecules bind to the loop. The collective behavior of proteins and DNA is greater than the sum of the parts and hints of ways in which multiple proteins may coordinate the packaging and processing of genetic information.

Directional R-Loop Formation by the CRISPR-Cas Surveillance Complex Cascade Provides Efficient Off-Target Site Rejection

Directory of Open Access Journals (Sweden)

Marius Rutkauskas

2015-03-01

Full Text Available CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against foreign nucleic acids. In type I CRISPR-Cas systems, invading DNA is detected by a large ribonucleoprotein surveillance complex called Cascade. The crRNA component of Cascade is used to recognize target sites in foreign DNA (protospacers by formation of an R-loop driven by base-pairing complementarity. Using single-molecule supercoiling experiments with near base-pair resolution, we probe here the mechanism of R-loop formation and detect short-lived R-loop intermediates on off-target sites bearing single mismatches. We show that R-loops propagate directionally starting from the protospacer-adjacent motif (PAM. Upon reaching a mismatch, R-loop propagation stalls and collapses in a length-dependent manner. This unambiguously demonstrates that directional zipping of the R-loop accomplishes efficient target recognition by rapidly rejecting binding to off-target sites with PAM-proximal mutations. R-loops that reach the protospacer end become locked to license DNA degradation by the auxiliary Cas3 nuclease/helicase without further target verification.

Revealing the uncultivated majority: combining DNA stable-isotope probing, multiple displacement amplification and metagenomic analyses of uncultivated Methylocystis in acidic peatlands.

Science.gov (United States)

Chen, Yin; Dumont, Marc G; Neufeld, Josh D; Bodrossy, Levente; Stralis-Pavese, Nancy; McNamara, Niall P; Ostle, Nick; Briones, Maria J I; Murrell, J Colin

2008-10-01

Peatlands represent an enormous carbon reservoir and have a potential impact on the global climate because of the active methanogenesis and methanotrophy in these soils. Uncultivated methanotrophs from seven European peatlands were studied using a combination of molecular methods. Screening for methanotroph diversity using a particulate methane monooxygenase-based diagnostic gene array revealed that Methylocystis-related species were dominant in six of the seven peatlands studied. The abundance and methane oxidation activity of Methylocystis spp. were further confirmed by DNA stable-isotope probing analysis of a sample taken from the Moor House peatland (England). After ultracentrifugation, (13)C-labelled DNA, containing genomic DNA of these Methylocystis spp., was separated from (12)C DNA and subjected to multiple displacement amplification (MDA) to generate sufficient DNA for the preparation of a fosmid metagenomic library. Potential bias of MDA was detected by fingerprint analysis of 16S rRNA using denaturing gradient gel electrophoresis for low-template amplification (0.01 ng template). Sufficient template (1-5 ng) was used in MDA to circumvent this bias and chimeric artefacts were minimized by using an enzymatic treatment of MDA-generated DNA with S1 nuclease and DNA polymerase I. Screening of the metagenomic library revealed one fosmid containing methanol dehydrogenase and two fosmids containing 16S rRNA genes from these Methylocystis-related species as well as one fosmid containing a 16S rRNA gene related to that of Methylocella/Methylocapsa. Sequencing of the 14 kb methanol dehydrogenase-containing fosmid allowed the assembly of a gene cluster encoding polypeptides involved in bacterial methanol utilization (mxaFJGIRSAC). This combination of DNA stable-isotope probing, MDA and metagenomics provided access to genomic information of a relatively large DNA fragment of these thus far uncultivated, predominant and active methanotrophs in peatland soil.

Nanoscale dislocation shear loops at static equilibrium and finite temperature

Science.gov (United States)

Dang, Khanh; Capolungo, Laurent; Spearot, Douglas E.

2017-12-01

Atomistic simulations are used to determine the resolved shear stress necessary for equilibrium and the resulting geometry of nanoscale dislocation shear loops in Al. Dislocation loops with different sizes and shapes are created via superposition of elemental triangular dislocation displacement fields in the presence of an externally imposed shear stress. First, a bisection algorithm is developed to determine systematically the resolved shear stress necessary for equilibrium at 0 K. This approach allows for the identification of dislocation core structure and a correlation between dislocation loop size, shape and the computed shear stress for equilibrium. It is found, in agreement with predictions made by Scattergood and Bacon, that the equilibrium shape of a dislocation loop becomes more circular with increasing loop size. Second, the bisection algorithm is extended to study the influence of temperature on the resolved shear stress necessary for stability. An approach is presented to compute the effective lattice friction stress, including temperature dependence, for dislocation loops in Al. The temperature dependence of the effective lattice friction stress can be reliably computed for dislocation loops larger than 16.2 nm. However, for dislocation loops smaller than this threshold, the effective lattice friction stress shows a dislocation loop size dependence caused by significant overlap of the stress fields on the interior of the dislocation loops. Combined, static and finite temperature atomistic simulations provide essential data to parameterize discrete dislocation dynamics simulations.

An Optimized Protocol for DNA Extraction from Wheat Seeds and Loop-Mediated Isothermal Amplification (LAMP to Detect Fusarium graminearum Contamination of Wheat Grain

Directory of Open Access Journals (Sweden)

Mohamed Moslem

2011-06-01

Full Text Available A simple, rapid, and efficient method for isolating genomic DNA from germinated seeds of wheat that is free from polysaccharides and polyphenols is reported. DNA was extracted, treated with RNase, measured and tested for completeness using agarose gel electrophoresis. DNA purification from wheat grains yielded abundant, amplifiable DNA with yields typically between 100 and 200 ng DNA/mg. The effectiveness and reliability of the method was tested by assessing quantity and quality of the isolated DNA using three PCR-based markers. Inter-simple sequence repeats (ISSRs were used to assess the genetic diversity between different wheat varieties. Specific PCR primer pair Tox5-1/Tox5-2 and a loop-mediated isothermal amplification (LAMP procedure were used to detect genomic DNA of Fusarium graminearum in contaminated wheat seeds. In this method there is no need to use liquid nitrogen for crushing germinated seedlings. The protocol takes approximately one hour to prepare high quality DNA. In combination with the LAMP assay it is a fast and cost-effective alternative to traditional diagnostic methods for the early detection of toxigenic fusaria in cereals.

Exchange bias and asymmetric hysteresis loops from a microscopic model of core/shell nanoparticles

International Nuclear Information System (INIS)

Iglesias, Oscar; Batlle, Xavier; Labarta, Amilcar

2007-01-01

We present Monte Carlo simulations of hysteresis loops of a model of a magnetic nanoparticle with a ferromagnetic core and an antiferromagnetic shell with varying values of the core/shell interface exchange coupling which aim to clarify the microscopic origin of exchange bias observed experimentally. We have found loop shifts in the field direction as well as displacements along the magnetization axis that increase in magnitude when increasing the interfacial exchange coupling. Overlap functions computed from the spin configurations along the loops have been obtained to explain the origin and magnitude of these features microscopically

Repair of abasic sites in DNA

Energy Technology Data Exchange (ETDEWEB)

Dianov, Grigory L.; Sleeth, Kate M.; Dianova, Irina I.; Allinson, Sarah L

2003-10-29

Repair of both normal and reduced AP sites is activated by AP endonuclease, which recognizes and cleaves a phosphodiester bond 5' to the AP site. For a short period of time an incised AP site is occupied by poly(ADP-ribose) polymerase and then DNA polymerase {beta} adds one nucleotide into the repair gap and simultaneously removes the 5'-sugar phosphate. Finally, the DNA ligase III/XRCC1 complex accomplishes repair by sealing disrupted DNA ends. However, long-patch BER pathway, which is involved in the removal of reduced abasic sites, requires further DNA synthesis resulting in strand displacement and the generation of a damage-containing flap that is later removed by the flap endonuclease. Strand-displacement DNA synthesis is accomplished by DNA polymerase {delta}/{epsilon} and DNA ligase I restores DNA integrity. DNA synthesis by DNA polymerase {delta}/{epsilon} is dependent on proliferating cell nuclear antigen, which also stimulates the DNA ligase I and flap endonuclease. These repair events are supported by multiple protein-protein interactions.

The development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of abalone herpesvirus DNA.

Science.gov (United States)

Chen, M H; Kuo, S T; Renault, T; Chang, P H

2014-02-01

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of abalone herpesvirus DNA. Two pairs of primers were designed, based on the sequence of the DNA polymerase gene of abalone herpesvirus. The reaction temperature and time were optimized to 63°C and 60min, respectively. LAMP amplicons were analyzed by 2% agarose gel electrophoresis or by visual inspection of a colour change emitted by fluorescent dye. The method developed was specific for the detection of abalone herpesvirus, without cross-reactions with other tested herpesviruses including ostreid herpesvirus 1 (OsHV-1), European eel herpesvirus, koi herpesvirus (KHV) and an avian herpesvirus. The LAMP assay was 100 folds more sensitive than a conventional PCR and 10 folds less sensitive than a SYBR Green PCR. These results indicate that the developed LAMP assay is a simple, rapid, sensitive, specific and reliable technique for the detection of abalone herpesvirus. Copyright © 2013 Elsevier B.V. All rights reserved.

An optimal open/closed-loop control method with application to a pre-stressed thin duralumin plate

Science.gov (United States)

Nadimpalli, Sruthi Raju

The excessive vibrations of a pre-stressed duralumin plate, suppressed by a combination of open-loop and closed-loop controls, also known as open/closed-loop control, is studied in this thesis. The two primary steps involved in this process are: Step (I) with an assumption that the closed-loop control law is proportional, obtain the optimal open-loop control by direct minimization of the performance measure consisting of energy at terminal time and a penalty on open-loop control force via calculus of variations. If the performance measure also involves a penalty on closed-loop control effort then a Fourier based method is utilized. Step (II) the energy at terminal time is minimized numerically to obtain optimal values of feedback gains. The optimal closed-loop control gains obtained are used to describe the displacement and the velocity of open-loop, closed-loop and open/closed-loop controlled duralumin plate.

Distribution of u.v.-induced repair events in higher-order chromatin loops in human and hamster fibroblasts

International Nuclear Information System (INIS)

Mullenders, L.H.F.; Zeeland, A.A. van; Natarajan, A.T.; Kesteren, A.C. van; Bussmann, C.J.M.

1986-01-01

The repair of u.v.-induced damage in human and rodent cells was investigated at the level of DNA loops attached to the nuclear matrix. After 2 h post-u.v. incubation, DNase I digestion studies revealed a 3- to 4-fold enrichment of repair-labeled DNA at the nuclear matrix in four xeroderma pigmentosum cell strains belonging to complementation group C. Two xeroderma pigmentosum cell strains of complementation group D and Syrian hamster embryonic cells, as well as in HeLa cells and normal human fibroblasts, no enrichment of repair-labeled DNA at the nuclear matrix was observed. Visualization of repair events in DNA loops by autoradiography of DNA halo - matrix structures confirmed the biochemical observations. The presence or absence of preferential repair of nuclear matrix-associated DNA paralleled the presence or absence of inhomogeneity in the distribution of T4 endonuclease-V-sensitive sites. In xeroderma pigmentosum cells of complementation group C showed that after 2 h post-u.v. incubation, repair events were found at both attachment sites in a limited number of loops and that large domains of loops were not subjected to repair. (author)

Noncanonical ATM Activation and Signaling in Response to Transcription-Blocking DNA Damage.

Science.gov (United States)

Marteijn, Jurgen A; Vermeulen, Wim; Tresini, Maria

2017-01-01

Environmental genotoxins and metabolic byproducts generate DNA lesions that can cause genomic instability and disrupt tissue homeostasis. To ensure genomic integrity, cells employ mechanisms that convert signals generated by stochastic DNA damage into organized responses, including activation of repair systems, cell cycle checkpoints, and apoptotic mechanisms. DNA damage response (DDR) signaling pathways coordinate these responses and determine cellular fates in part, by transducing signals that modulate RNA metabolism. One of the master DDR coordinators, the Ataxia Telangiectasia Mutated (ATM) kinase, has a fundamental role in mediating DNA damage-induced changes in mRNA synthesis. ATM acts by modulating a variety of RNA metabolic pathways including nascent RNA splicing, a process catalyzed by the spliceosome. Interestingly, ATM and the spliceosome influence each other's activity in a reciprocal manner by a pathway that initiates when transcribing RNA polymerase II (RNAPII) encounters DNA lesions that prohibit forward translocation. In response to stalling of RNAPII assembly of late-stage spliceosomes is disrupted resulting in increased splicing factor mobility. Displacement of spliceosomes from lesion-arrested RNA polymerases facilitates formation of R-loops between the nascent RNA and DNA adjacent to the transcription bubble. R-loops signal for noncanonical ATM activation which in quiescent cells occurs in absence of detectable dsDNA breaks. In turn, activated ATM signals to regulate spliceosome dynamics and AS genome wide.This chapter describes the use of fluorescence microscopy methods that can be used to evaluate noncanonical ATM activation by transcription-blocking DNA damage. First, we present an immunofluorescence-detection method that can be used to evaluate ATM activation by autophosphorylation, in fixed cells. Second, we present a protocol for Fluorescence Recovery After Photobleaching (FRAP) of GFP-tagged splicing factors, a highly sensitive and

TSC plasma halo simulation of a DIII-D vertical displacement episode

International Nuclear Information System (INIS)

Sayer, R.O.; Peng, Y.K.M.; Jardin, S.C.

1993-01-01

A benchmark of the Tokamak Simulation Code (TSC) plasma halo model has been achieved by calibration against a DIII-D vertical displacement episode (VDE) consisting of vertical drift, thermal quench and current quench. With a suitable halo surrounding the main plasma, the TSC predictions are in good agreement with experimental results for the plasma current decay, plasma trajectory, toroidal and poloidal vessel currents, and for the magnetic probe and flux loop values for the entire VDE. Simulations with no plasma halo yield much faster vertical motion and significantly worse agreement with the magnetics and flux loop data than do halo simulations. (author). 12 refs, 13 figs

R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters.

Science.gov (United States)

Chen, Liang; Chen, Jia-Yu; Zhang, Xuan; Gu, Ying; Xiao, Rui; Shao, Changwei; Tang, Peng; Qian, Hao; Luo, Daji; Li, Hairi; Zhou, Yu; Zhang, Dong-Er; Fu, Xiang-Dong

2017-11-16

R-loop, a three-stranded RNA/DNA structure, has been linked to induced genome instability and regulated gene expression. To enable precision analysis of R-loops in vivo, we develop an RNase-H-based approach; this reveals predominant R-loop formation near gene promoters with strong G/C skew and propensity to form G-quadruplex in non-template DNA, corroborating with all biochemically established properties of R-loops. Transcription perturbation experiments further indicate that R-loop induction correlates to transcriptional pausing. Interestingly, we note that most mapped R-loops are each linked to a nearby free RNA end; by using a ribozyme to co-transcriptionally cleave nascent RNA, we demonstrate that such a free RNA end coupled with a G/C-skewed sequence is necessary and sufficient to induce R-loop. These findings provide a topological solution for RNA invasion into duplex DNA and suggest an order for R-loop initiation and elongation in an opposite direction to that previously proposed. Copyright © 2017 Elsevier Inc. All rights reserved.

A rolling circle replication mechanism produces multimeric lariats of mitochondrial DNA in Caenorhabditis elegans.

Directory of Open Access Journals (Sweden)

Samantha C Lewis

2015-02-01

Full Text Available Mitochondrial DNA (mtDNA encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elongation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans.

Loop-mediated isothermal amplification (LAMP) assay-A rapid detection tool for identifying red fox (Vulpes vulpes) DNA in the carcasses of harbour porpoises (Phocoena phocoena).

Science.gov (United States)

Heers, Teresa; van Neer, Abbo; Becker, André; Grilo, Miguel Luca; Siebert, Ursula; Abdulmawjood, Amir

2017-01-01

Carcasses of wild animals are often visited by different scavengers. However, determining which scavenger caused certain types of bite marks is particularly difficult and knowledge thereof is lacking. Therefore, a loop-mediated isothermal amplification (LAMP) assay (target sequence cytochrome b) was developed to detect red fox DNA in carcasses of harbour porpoises. The MSwab™ method for direct testing without prior DNA isolation was validated. As a detection device, the portable real-time fluorometer Genie® II was used, which yields rapid results and can be used in field studies without huge laboratory equipment. In addition to in vitro evaluation and validation, a stranded and scavenged harbour porpoise carcass was successfully examined for red fox DNA residues. The developed LAMP method is a valuable diagnostic tool for confirming presumable red fox bite wounds in harbour porpoises without further DNA isolation steps.

In situ TEM study on elastic interaction between a prismatic loop and a gliding dislocation

International Nuclear Information System (INIS)

Matsukawa, Yoshitaka; Liu, Grace S.

2012-01-01

In situ straining in a transmission electron microscope was performed in order to investigate dislocation interactions with a prismatic loop, which as a mobile obstacle is expected to be displaced by the strain-field of dislocation prior to physical contact. It was found that when a gliding dislocation approached a critical distance, the prismatic loop was certainly attracted to the dislocation. The captured loop disrupted the dislocation motion and was not dragged along with the mobile dislocation. Instead, the dislocation bypassed the loop via cross-slip to another slip plane with a resolved shear stress estimated to be 40% lower than that of the original plane.

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

Directory of Open Access Journals (Sweden)

Marijn T J van Loenhout

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

Displacer Diameter Effect in Displacer Pulse Tube Refrigerator

Science.gov (United States)

Zhu, Shaowei

2017-12-01

Gas driving displacer pulse tube refrigerators are one of the work recovery type of pulse tube refrigerators whose theoretical efficiency is the same as Stirling refrigerators'. Its cooling power is from the displacement of the displacer. Displace diameter, rod diameter and pressure drop of the regenerator influence the displacement, which are investigated by numerical simulation. It is shown that the displacement ratio of the displacer over the piston is almost not affected by the displacer diameter at the same rod diameter ratio, or displacer with different diameters almost has the same performance.

Investigating the dynamics of surface-immobilized DNA nanomachines

Science.gov (United States)

Dunn, Katherine E.; Trefzer, Martin A.; Johnson, Steven; Tyrrell, Andy M.

2016-07-01

Surface-immobilization of molecules can have a profound influence on their structure, function and dynamics. Toehold-mediated strand displacement is often used in solution to drive synthetic nanomachines made from DNA, but the effects of surface-immobilization on the mechanism and kinetics of this reaction have not yet been fully elucidated. Here we show that the kinetics of strand displacement in surface-immobilized nanomachines are significantly different to those of the solution phase reaction, and we attribute this to the effects of intermolecular interactions within the DNA layer. We demonstrate that the dynamics of strand displacement can be manipulated by changing strand length, concentration and G/C content. By inserting mismatched bases it is also possible to tune the rates of the constituent displacement processes (toehold-binding and branch migration) independently, and information can be encoded in the time-dependence of the overall reaction. Our findings will facilitate the rational design of surface-immobilized dynamic DNA nanomachines, including computing devices and track-based motors.

In situ detection of tandem DNA repeat length

Energy Technology Data Exchange (ETDEWEB)

Yaar, R.; Szafranski, P.; Cantor, C.R.; Smith, C.L. [Boston Univ., MA (United States)

1996-11-01

A simple method for scoring short tandem DNA repeats is presented. An oligonucleotide target, containing tandem repeats embedded in a unique sequence, was hybridized to a set of complementary probes, containing tandem repeats of known lengths. Single-stranded loop structures formed on duplexes containing a mismatched (different) number of tandem repeats. No loop structure formed on duplexes containing a matched (identical) number of tandem repeats. The matched and mismatched loop structures were enzymatically distinguished and differentially labeled by treatment with S1 nuclease and the Klenow fragment of DNA polymerase. 7 refs., 4 figs.

Phosphate-methylated DNA aimed at HIV-1 RNA loops and integrated DNA inhibits viral infectivity

NARCIS (Netherlands)

Buck, H. M.; Koole, L. H.; van Genderen, M. H.; Smit, L.; Geelen, J. L.; Jurriaans, S.; Goudsmit, J.

1990-01-01

Phosphate-methylated DNA hybridizes strongly and specifically to natural DNA and RNA. Hybridization to single-stranded and double-stranded DNA leads to site-selective blocking of replication and transcription. Phosphate-methylated DNA was used to interrupt the life cycle of the human

Population genetic structure of skipjack tuna Katsuwonus pelamis from the Indian coast using sequence analysis of the mitochondrial DNA D-loop region

Digital Repository Service at National Institute of Oceanography (India)

Menezes, M.R.; Kumar, G.; Kunal, S.P.

Biology (2012) 80, 2198–2212 doi:10.1111/j.1095-8649.2012.03270.x, available online at wileyonlinelibrary.com Population genetic structure of skipjack tuna Katsuwonus pelamis from the Indian coast using sequence analysis of the mitochondrial DNA D...-loop region M. R. Menezes*, G. Kumar and S. P. Kunal Biological Oceanography Division, National Institute of Oceanography (CSIR), Dona Paula, Goa 403 004, India (Received 26 May 2011, Accepted 14 February 2012) Genetic structure of skipjack tuna Katsuwonus...

Involvement of DNA mismatch repair in the maintenance of heterochromatic DNA stability in Saccharomyces cerevisiae.

Directory of Open Access Journals (Sweden)

Basanta K Dahal

2017-10-01

Full Text Available Heterochromatin contains a significant part of nuclear DNA. Little is known about the mechanisms that govern heterochromatic DNA stability. We show here that in the yeast Saccharomyces cerevisiae (i DNA mismatch repair (MMR is required for the maintenance of heterochromatic DNA stability, (ii MutLα (Mlh1-Pms1 heterodimer, MutSα (Msh2-Msh6 heterodimer, MutSβ (Msh2-Msh3 heterodimer, and Exo1 are involved in MMR at heterochromatin, (iii Exo1-independent MMR at heterochromatin frequently leads to the formation of Pol ζ-dependent mutations, (iv MMR cooperates with the proofreading activity of Pol ε and the histone acetyltransferase Rtt109 in the maintenance of heterochromatic DNA stability, (v repair of base-base mismatches at heterochromatin is less efficient than repair of base-base mismatches at euchromatin, and (vi the efficiency of repair of 1-nt insertion/deletion loops at heterochromatin is similar to the efficiency of repair of 1-nt insertion/deletion loops at euchromatin.

Lift, drag, and guidance forces on alternating polarity magnets, using loop guideways

International Nuclear Information System (INIS)

Lindenbaum, S.D.; Lee, M.S.

1975-01-01

Exact solutions of track current, lift force, and drag force, together with their velocity dependence, have been computed for a vehicle carrying a finite number of fixed current alternating polarity superconducting magnets, suspended at various heights over structured track guideways of the single- and double-loop (''null'') types. Results for the double-loop case are compared with those of a previously reported approximate analysis. The analytical method is then applied to a study of a low-drag guidance loop guideway which is integrable with lift loop guideways utilizing a common set of vehicle magnets. Solutions are obtained for guidance track restoring forces, lateral destabilization forces, and lift force degradation as functions of lateral displacement from symmetry. The dependence of lift, drag, and lift-to-drag on track loop parameters is studied and the linear dependence of lift-to-drag on loop time constant confirmed. The contribution to the forces made by successive addition of alternating polarity magnets is calculated and the marked reduction in lift force pulsation noted

A mitosis-specific and R loop-driven ATR pathway promotes faithful chromosome segregation.

Science.gov (United States)

Kabeche, Lilian; Nguyen, Hai Dang; Buisson, Rémi; Zou, Lee

2018-01-05

The ataxia telangiectasia mutated and Rad3-related (ATR) kinase is crucial for DNA damage and replication stress responses. Here, we describe an unexpected role of ATR in mitosis. Acute inhibition or degradation of ATR in mitosis induces whole-chromosome missegregation. The effect of ATR ablation is not due to altered cyclin-dependent kinase 1 (CDK1) activity, DNA damage responses, or unscheduled DNA synthesis but to loss of an ATR function at centromeres. In mitosis, ATR localizes to centromeres through Aurora A-regulated association with centromere protein F (CENP-F), allowing ATR to engage replication protein A (RPA)-coated centromeric R loops. As ATR is activated at centromeres, it stimulates Aurora B through Chk1, preventing formation of lagging chromosomes. Thus, a mitosis-specific and R loop-driven ATR pathway acts at centromeres to promote faithful chromosome segregation, revealing functions of R loops and ATR in suppressing chromosome instability. Copyright © 2018, American Association for the Advancement of Science.

Real-time monitoring of enzyme-free strand displacement cascades by colorimetric assays

Science.gov (United States)

Duan, Ruixue; Wang, Boya; Hong, Fan; Zhang, Tianchi; Jia, Yongmei; Huang, Jiayu; Hakeem, Abdul; Liu, Nannan; Lou, Xiaoding; Xia, Fan

2015-03-01

The enzyme-free toehold-mediated strand displacement reaction has shown potential for building programmable DNA circuits, biosensors, molecular machines and chemical reaction networks. Here we report a simple colorimetric method using gold nanoparticles as signal generators for the real-time detection of the product of the strand displacement cascade. During the process the assembled gold nanoparticles can be separated, resulting in a color change of the solution. This assay can also be applied in complex mixtures, fetal bovine serum, and to detect single-base mismatches. These results suggest that this method could be of general utility to monitor more complex enzyme-free strand displacement reaction-based programmable systems or for further low-cost diagnostic applications.The enzyme-free toehold-mediated strand displacement reaction has shown potential for building programmable DNA circuits, biosensors, molecular machines and chemical reaction networks. Here we report a simple colorimetric method using gold nanoparticles as signal generators for the real-time detection of the product of the strand displacement cascade. During the process the assembled gold nanoparticles can be separated, resulting in a color change of the solution. This assay can also be applied in complex mixtures, fetal bovine serum, and to detect single-base mismatches. These results suggest that this method could be of general utility to monitor more complex enzyme-free strand displacement reaction-based programmable systems or for further low-cost diagnostic applications. Electronic supplementary information (ESI) available: Experimental procedures and analytical data are provided. See DOI: 10.1039/c5nr00697j

NSC666715 and Its Analogs Inhibit Strand-Displacement Activity of DNA Polymerase β and Potentiate Temozolomide-Induced DNA Damage, Senescence and Apoptosis in Colorectal Cancer Cells.

Directory of Open Access Journals (Sweden)

Aruna S Jaiswal

Full Text Available Recently approved chemotherapeutic agents to treat colorectal cancer (CRC have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase β (Pol-β activity. Temozolomide (TMZ, an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-β-directed base excision repair (BER pathway. In previous studies, we used structure-based molecular docking of Pol-β and identified a potent small molecule inhibitor (NSC666715. In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-β-directed LP-BER, cause apurinic/apyrimidinic (AP site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC50 of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients.

Molecular dynamics simulations of electrostatics and hydration distributions around RNA and DNA motifs

Science.gov (United States)

Marlowe, Ashley E.; Singh, Abhishek; Semichaevsky, Andrey V.; Yingling, Yaroslava G.

2009-03-01

Nucleic acid nanoparticles can self-assembly through the formation of complementary loop-loop interactions or stem-stem interactions. Presence and concentration of ions can significantly affect the self-assembly process and the stability of the nanostructure. In this presentation we use explicit molecular dynamics simulations to examine the variations in cationic distributions and hydration environment around DNA and RNA helices and loop-loop interactions. Our simulations show that the potassium and sodium ionic distributions are different around RNA and DNA motifs which could be indicative of ion mediated relative stability of loop-loop complexes. Moreover in RNA loop-loop motifs ions are consistently present and exchanged through a distinct electronegative channel. We will also show how we used the specific RNA loop-loop motif to design a RNA hexagonal nanoparticle.

Rate of displacement for Jakob Type 1 lateral condyle fractures treated with a cast.

Science.gov (United States)

Zale, C; Winthrop, Z A; Hennrikus, W

2018-04-01

The aim of this retrospective study is to report the rate of displacement of Jakob Type 1 lateral condyle fractures that were initially treated in a cast. We performed a retrospective review of all patients that were treated for a non-displaced (Jakob Type 1 displaced and were converted to a closed pinning treatment plan with a conversion rate of 8.5%. There was a mean of 13.2 days (4 to 21) between treatment by initial casting and closed pinning. This study demonstrates an 8.5% displacement and conversion rate from cast treatment to closed pinning for initially non-displaced Jakob Type 1 lateral condyle fractures of the humerus. The internal oblique radiograph is most accurate to determine displacement. We recommend obtaining an internal oblique view at initial evaluation and at follow-up in the cast for lateral condyle fractures. To minimize movement at the fracture site, we recommend treating Jakob Type 1 lateral condyle fractures with a long arm cast with the elbow at 90° and the forearm in the supine position with a sling-loop design. IV - retrospective therapeutic study.

An IPMC microgripper with integrated actuator and sensing for constant finger-tip displacement

International Nuclear Information System (INIS)

Gonzalez, Carlos; Lumia, Ron

2015-01-01

Ionic polymer metal composite (IPMC) is a type of smart material that has gained the interest of many researchers due to its ability to achieve large displacements under small input voltages, usually less than 2.5 V. This has motivated the use of these materials in microsystems and systems in the millimeter scale, such as microgrippers. However, few of the control techniques developed thus far have considered the feasibility of using IPMCs in closed loop systems without the need of oversized external sensors. This paper presents a control scheme for a two-finger IPMC microgripper that accomplishes constant finger-tip displacements without external sensors. This scheme generates a displacement-dependent, time varying reference signal to obtain constant finger-tip displacements applied by a separate actuated IPMC. This actuator uses a PID controller tuned with a model-free approach, and is gain scheduled to span up to 1 mm finger-tip displacements. The microgripper achieves zero steady state error for finger-tip displacements on the tuned values of the PID controller. The gain scheduled PID controller is tested and results show zero steady state error to 0.25 mm displacements, and 15 and 20% steady state error when referenced to deflection of 0.45 and 0.75 mm, respectively. This shows that there is great confidence and validity of the control scheme, especially when tracking small reference deflections. (paper)

Three genetic stocks of frigate tuna Auxis thazard thazard (Lacepede, 1800) along the Indian coast revealed from sequence analyses of mitochondrial DNA D-loop region

Digital Repository Service at National Institute of Oceanography (India)

GirishKumar; Kunal, S.P.; Menezes, M.R.; Meena, R.M.

revealed from sequence analyses of mitochondrial DNA D-loop region Name of authors: 1. Girish Kumar* Biological Oceanography Division (BOD) National Institute of Oceanography (NIO) Dona Paula, Goa 403004, India. Email: girishkumar....nio@gmail.com Tel: +919766548060 2. Swaraj Priyaranjan Kunal Biological Oceanography Division (BOD) National Institute of Oceanography (NIO) Dona Paula, Goa 403004, India. Email: swar.mbt@gmail.com 3. Maria Rosalia Menezes Biological Oceanography...

Monitoring of pipe displacements in French LMFBR SUPERPHENIX

International Nuclear Information System (INIS)

Foucher, N.; Debaene, J.P.; Renault, Y.; Blin, B.

1993-01-01

In order to check that pipe supports work properly and that the locking of snubbers or the loss of supports do not put a pipe in unacceptable loading conditions, a monitoring of the behaviour of the main pipes of SUPERPHENIX is planned. This monitoring system consists in measuring the displacements at selected points of the pipe by means of measuring rods and checking that these displacements remain inside allowable domains. These allowable domains are defined so that, if the displacements of the pipe are inside all these domains, the plant operator is sure that the stresses verify the allowable limits and then no additional inspection is carried out. In the opposite case, the operator will inspect the pipe in detail in order to determine the consequences and repair if necessary before restarting. Selection of points for monitoring was done with the to minimize the number of measures to be carried out and to use as far as possible the measuring rods that were installed to check that pipe displacements were consistent with what has been obtained in design calculations. However, it appears necessary to ensure that any incident occurring at any point of the pipe can be detected and, if necessary, additional measuring rods may be installed. An incident is said detectable if it induces on at least one measuring rod a deviation with respect to expected displacement not lower than 5 mm. It has been chosen so that small normal changes in measured displacements are not mistaken as incidents. The incidents that are supposed likely to occur are: 1) loss of a support which induces mainly primary stresses, 2) locking of a snubber which induces mainly secondary stresses. Monitoring of pipe displacements is a simple and effective way of checking that no damaging perturbation has occurred on the pipe. Calculations carried out on the DHR loops of SUPERPHENIX show that allowable domains of acceptable size may be obtained using a relatively small number of measuring rods. The method

A “four-ferrocene” modified stem-loop structure as a probe for sensitive detection and single-base mismatch discrimination of DNA

International Nuclear Information System (INIS)

Chatelain, Grégory; Ripert, Micaël; Farre, Carole; Ansanay-Alex, Salomé; Chaix, Carole

2012-01-01

We report the use of a four-ferrocene modified oligonucleotide as a probe for DNA detection with a gold electrode microsystem. This oligonucleotide is synthesized by automated solid-phase synthesis with four successive ferrocene moieties at the 5′-end and a C6-thiol modifier group at the 3′-end. The grafting of this 4Fc-DNA probe on a gold electrode microsystem results in the appearance of the ferrocene redox couple in cyclic voltammetry. The probe sequence is a stem-loop structure that folds efficiently on the electrode, thus optimizing electron transfer. Such architecture serves as sensor for DNA detection which is based on hybridization. The resulting disposable voltammetric sensor allowed direct, reagentless DNA detection in a small volume (20 μL). Electrochemical response upon hybridization with complementary short sequence (30-base length) and long sequence (50-base length) strands was observed by differential pulse voltammetry. Current variations were compared. The longer the sequence, the greater the decrease in current. The system's detection limit was estimated at 3.5 pM (0.07 fmol in 20 μL) with the 50-base length target and provided a dynamic detection range between 3.5 pM and 5 nM. Single mismatch detection showed a good level of sensitivity. The system was regenerated twice with no significant loss of Fc signal. Finally, 1 pM sensitivity was reached with a long chain analog of DNA PCR products of Influenza virus.

Preferential repair of nuclear matrix associated DNA in xeroderma pigmentosum complementation group C

International Nuclear Information System (INIS)

Mullenders, L.H.F.; Kesteren, A.C. van; Bussmann, C.J.M.; Zeeland, A.A. van; Natarajan, A.T.

1984-01-01

The distribution of ultraviolet-induced DNA repair patches in the genome of xeroderma pigmentosum cells of complementation group C was investigated by determining the molecular weight distribution of repair labeled DNA and prelabeled DNA in alkaline sucrose gradients after treatment with the dimer-specific endonuclease V of bacteriophage T 4 . The results suggest that DNA-repair synthesis in xeroderma pigmentosum cells of complementation group C occurs in localized regions of the genome. Analysis of the spatial distribution of ultraviolet-induced repair patches in DNA loops attached to the nuclear matrix revealed that in xeroderma pigmentosum cells of complementation group C repair patches are preferentially situated near the attachment sites of DNA loops at the nuclear matrix. In normal human fibroblasts the authors observed no enrichment of repair-labeled DNA at the nuclear matrix and repair patches appeared to be distributed randomly along the DNA loops. The enrichment of repair-labeled DNA at the nuclear matrix in xeroderma pigmentosum cells of complementation group C may indicate that the residual DNA-repair synthesis in these cells occurs preferentially in regions of the genome. (Auth.)

Superconducting inductive displacement detection of a microcantilever

Science.gov (United States)

Vinante, A.

2014-07-01

We demonstrate a superconducting inductive technique to measure the displacement of a micromechanical resonator. In our scheme, a type I superconducting microsphere is attached to the free end of a microcantilever and approached to the loop of a dc Superconducting Quantum Interference Device (SQUID) microsusceptometer. A local magnetic field as low as 100 μT, generated by a field coil concentric to the SQUID, enables detection of the cantilever thermomechanical noise at 4.2 K. The magnetomechanical coupling and the magnetic spring are in good agreement with image method calculations assuming pure Meissner effect. These measurements are relevant to recent proposals of quantum magnetomechanics experiments based on levitating superconducting microparticles.

Mitochondrial D-loop sequence variation among Italian horse breeds

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Zanotti Marta

2004-11-01

Full Text Available Abstract The genetic variability of the mitochondrial D-loop DNA sequence in seven horse breeds bred in Italy (Giara, Haflinger, Italian trotter, Lipizzan, Maremmano, Thoroughbred and Sarcidano was analysed. Five unrelated horses were chosen in each breed and twenty-two haplotypes were identified. The sequences obtained were aligned and compared with a reference sequence and with 27 mtDNA D-loop sequences selected in the GenBank database, representing Spanish, Portuguese, North African, wild horses and an Equus asinus sequence as the outgroup. Kimura two-parameter distances were calculated and a cluster analysis using the Neighbour-joining method was performed to obtain phylogenetic trees among breeds bred in Italy and among Italian and foreign breeds. The cluster analysis indicates that all the breeds but Giara are divided in the two trees, and no clear relationships were revealed between Italian populations and the other breeds. These results could be interpreted as showing the mixed origin of breeds bred in Italy and probably indicate the presence of many ancient maternal lineages with high diversity in mtDNA sequences.

Dna2 nuclease-helicase structure, mechanism and regulation by Rpa.

Science.gov (United States)

Zhou, Chun; Pourmal, Sergei; Pavletich, Nikola P

2015-11-02

The Dna2 nuclease-helicase maintains genomic integrity by processing DNA double-strand breaks, Okazaki fragments and stalled replication forks. Dna2 requires ssDNA ends, and is dependent on the ssDNA-binding protein Rpa, which controls cleavage polarity. Here we present the 2.3 Å structure of intact mouse Dna2 bound to a 15-nucleotide ssDNA. The nuclease active site is embedded in a long, narrow tunnel through which the DNA has to thread. The helicase domain is required for DNA binding but not threading. We also present the structure of a flexibly-tethered Dna2-Rpa interaction that recruits Dna2 to Rpa-coated DNA. We establish that a second Dna2-Rpa interaction is mutually exclusive with Rpa-DNA interactions and mediates the displacement of Rpa from ssDNA. This interaction occurs at the nuclease tunnel entrance and the 5' end of the Rpa-DNA complex. Hence, it only displaces Rpa from the 5' but not 3' end, explaining how Rpa regulates cleavage polarity.

Two modes of interaction of the single-stranded DNA-binding protein of bacteriophage T7 with the DNA polymerase-thioredoxin complex

KAUST Repository

Ghosh, Sharmistha; Hamdan, Samir; Richardson, Charles C.

2010-01-01

The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD.gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Two modes of interaction of the single-stranded DNA-binding protein of bacteriophage T7 with the DNA polymerase-thioredoxin complex

KAUST Repository

Ghosh, Sharmistha

2010-04-06

The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD.gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Interaction of the host protein NbDnaJ with Potato virus X minus-strand stem-loop 1 RNA and capsid protein affects viral replication and movement.

Science.gov (United States)

Cho, Sang-Yun; Cho, Won Kyong; Sohn, Seong-Han; Kim, Kook-Hyung

2012-01-06

Plant viruses must interact with host cellular components to replicate and move from cell to cell. In the case of Potato virus X (PVX), it carries stem-loop 1 (SL1) RNA essential for viral replication and movement. Using two-dimensional electrophoresis northwestern blot analysis, we previously identified several host proteins that bind to SL1 RNA. Of those, we further characterized a DnaJ-like protein from Nicotiana benthamiana named NbDnaJ. An electrophoretic mobility shift assay confirmed that NbDnaJ binds only to SL1 minus-strand RNA, and bimolecular fluorescence complementation (BiFC) indicated that NbDnaJ interacts with PVX capsid protein (CP). Using a series of deletion mutants, the C-terminal region of NbDnaJ was found to be essential for the interaction with PVX CP. The expression of NbDnaJ significantly changed upon infection with different plant viruses such as PVX, Tobacco mosaic virus, and Cucumber mosaic virus, but varied depending on the viral species. In transient experiments, both PVX replication and movement were inhibited in plants that over-expressed NbDnaJ but accelerated in plants in which NbDnaJ was silenced. In summary, we suggest that the newly identified NbDnaJ plays a role in PVX replication and movement by interacting with SL1(-) RNA and PVX CP. Copyright © 2011 Elsevier Inc. All rights reserved.

Cascade DNA nanomachine and exponential amplification biosensing.

Science.gov (United States)

Xu, Jianguo; Wu, Zai-Sheng; Shen, Weiyu; Xu, Huo; Li, Hongling; Jia, Lee

2015-11-15

DNA is a versatile scaffold for the assembly of multifunctional nanostructures, and potential applications of various DNA nanodevices have been recently demonstrated for disease diagnosis and treatment. In the current study, a powerful cascade DNA nanomachine was developed that can execute the exponential amplification of p53 tumor suppressor gene. During the operation of the newly-proposed DNA nanomachine, dual-cyclical nucleic acid strand-displacement polymerization (dual-CNDP) was ingeniously introduced, where the target trigger is repeatedly used as the fuel molecule and the nicked fragments are dramatically accumulated. Moreover, each displaced nicked fragment is able to activate the another type of cyclical strand-displacement amplification, increasing exponentially the value of fluorescence intensity. Essentially, one target binding event can induce considerable number of subsequent reactions, and the nanodevice was called cascade DNA nanomachine. It can implement several functions, including recognition element, signaling probe, polymerization primer and template. Using the developed autonomous operation of DNA nanomachine, the p53 gene can be quantified in the wide concentration range from 0.05 to 150 nM with the detection limit of 50 pM. If taking into account the final volume of mixture, the detection limit is calculated as lower as 6.2 pM, achieving an desirable assay ability. More strikingly, the mutant gene can be easily distinguished from the wild-type one. The proof-of-concept demonstrations reported herein is expected to promote the development and application of DNA nanomachine, showing great potential value in basic biology and medical diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Platinated DNA oligonucleotides: new probes forming ultrastable conjugates with graphene oxide

Science.gov (United States)

Wang, Feng; Liu, Juewen

2014-05-01

Metal containing polymers have expanded the property of polymers by involving covalently associated metal complexes. DNA is a special block copolymer. While metal ions are known to influence DNA, little is explored on its polymer property when strong metal complexes are associated. In this work, we study cisplatin modified DNA as a new polymer and probe. Out of the complexes formed between cisplatin-A15, HAuCl4-A15, Hg2+-T15 and Ag+-C15, only the cisplatin adduct is stable under the denaturing gel electrophoresis condition. Each Pt-nucleobase bond gives a positive charge and thus makes DNA a zwitterionic polymer. This allows ultrafast adsorption of DNA by graphene oxide (GO) and the adsorbed complex is highly stable. Non-specific DNA, protein, surfactants and thiolated compounds cannot displace platinated DNA from GO, while non-modified DNA is easily displaced in most cases. The stable GO/DNA conjugate is further tested for surface hybridization. This is the first demonstration of using metallated DNA as a polymeric material for interfacing with nanoscale materials.Metal containing polymers have expanded the property of polymers by involving covalently associated metal complexes. DNA is a special block copolymer. While metal ions are known to influence DNA, little is explored on its polymer property when strong metal complexes are associated. In this work, we study cisplatin modified DNA as a new polymer and probe. Out of the complexes formed between cisplatin-A15, HAuCl4-A15, Hg2+-T15 and Ag+-C15, only the cisplatin adduct is stable under the denaturing gel electrophoresis condition. Each Pt-nucleobase bond gives a positive charge and thus makes DNA a zwitterionic polymer. This allows ultrafast adsorption of DNA by graphene oxide (GO) and the adsorbed complex is highly stable. Non-specific DNA, protein, surfactants and thiolated compounds cannot displace platinated DNA from GO, while non-modified DNA is easily displaced in most cases. The stable GO/DNA conjugate

Loop-Mediated Isothermal Amplification on Crude DNA as a Point-of-Care Test for the Diagnosis of Mycoplasma-Related Vaginitis During Early Pregnancy.

Science.gov (United States)

Wang, Yichao; Zhang, Bumei; Sun, Yan; Liu, Yunde; Gu, Yajun

2017-12-20

Mycoplasma-related vaginitis gradually has been growing as a threat in adults-genitourinary infection contributes to funisitis, spontaneous abortion, and low birth weight. Until now, use of loop-mediated isothermal amplification (LAMP) to detect Ureaplasma urealyticum (UU), Mycoplasma hominis (MH), or Mycoplasma genitalium (MG) has been reported by some researchers. However, previous studies focused on purified DNA as the template for LAMP assay, which is usually extracted via commercial kit. We developed a LAMP assay for rapid detection of UU, MH, and MG genital mycoplasmas using a simple boiling method for DNA extraction, in a cohort of pregnant women with mycoplasma-related vaginitis. We monitored amplicons with the naked eye using SYBR Green I. The cohort in our study showed a prevalence of 22.6% in pregnant women, as detected by UU-LAMP assay. Compared to the polymerase chain reaction (PCR) test with purified DNA, the sensitivity of the UU-LAMP in clinical specimens with crude DNA was 87.5% (95% confidence interval [CI], 64.6%->99.9). For crude DNA specimens, UU-LAMP was more sensitive and reliable than PCR, with a higher agreement rate (96.8%) and Youden index value (0.88). As a point-of-care test, LAMP is a useful, specific, and efficient way to detect genital mycoplasmas in resource-limited settings, especially for crude DNA. © American Society for Clinical Pathology 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Importance of protamine phosphorylation to histone displacement in spermatids: can the disruption of this process be used for male contraception

Energy Technology Data Exchange (ETDEWEB)

Balhorn, R.; Hud, N.V.; Corzett, M.; Mazrimas, J.

1995-06-01

Protamine is a small protein that packages DNA in the sperm of most vertebrates. Shortly after its synthesis, the serine and threonine residues in each protamine are phosphorylated and the modified proteins are deposited onto DNA, displacing the histones and other chromatin proteins. We have hypothesized that the phosphorylation of protamine 1 induces protamine dimerization and these dimers are required for efficient histone displacement. Histone displacement by protamines in late-step spermatids appears to be essential for the production of fertile sperm in man and other mammals, and the disruption of this process could provide a new approach for male contraception. As a first step towards testing this theory, we have initiated a set of in vitro experiments to determine whether of not protamine phosphorylation is essential for histone displacement. Thee results of these experiments, although incomplete, confirm that unphosphorylated protamine cannot effectively displace histone from DNA. Polyarginine molecules twice the size of a protamine molecule and salmine dimer were found to be more effective. These results are consistent with the theory that the disruption of protamine phosphorylation may prove to be a useful new approach for male contraception if it can be shown to facilitate or induce protamine dimerization.

Anterior mandibular displacement and condylar growth. An experimental study in the rat

International Nuclear Information System (INIS)

Tonge, E.A.; Heath, J.K.; Meikle, M.C.

1982-01-01

Anterior displacement of the mandible was produced in twenty-eight 1-month-old female rats by two methods: (1) cast-gold splints cemented to the maxillary incisor teeth and (2) a removable stainless steel mesh appliance worn 6 hours each day, during which time the animals were sedated. The controls were littermates without appliances and in the mesh group were also sedated. Animals in the splint group were killed after 24 hours, 1 week, and 1 month; those in the mesh group were killed after 24 hours and after 1 week. the condyles were removed and cultured for 24 hours in medium containing 3 H-thymidine. One condyle from each animal was processed for routine histologic and autoradiographic study. The other was digested in phosphate-buffered saline containing RNA-ase and pronase, and the specific activity of 3 H-thymidine incorporation expressed as dpm/microgramDNA. Anterior mandibular displacement produced by both methods failed to result in a significant increase in the incorporation of 3 H-thymidine into explant DNA. In the 7-day mesh experiment, however, there was a significant increase in the DNA content of the condylar explants from the displacement group, suggesting an increase in the cell population. This finding should be treated with caution because of the small numbers of animals involved, but it indicates an important area for further study. Changes in the distribution of labeled cells within the proliferative zone (PZ) were also observed autoradiographically in the mesh group, but there was little to suggest that mandibular displacement was accompanied by a significant increase in cell division within the PZ. Remodeling changes affecting both the articular tissue and the subchondral bone were a characteristic feature of the 1-month bit plane group

A cryptic targeting signal creates a mitochondrial FEN1 isoform with tailed R-Loop binding properties.

Directory of Open Access Journals (Sweden)

Lawrence Kazak

Full Text Available A growing number of DNA transacting proteins is found in the nucleus and in mitochondria, including the DNA repair and replication protein Flap endonuclease 1, FEN1. Here we show a truncated FEN1 isoform is generated by alternative translation initiation, exposing a mitochondrial targeting signal. The shortened form of FEN1, which we term FENMIT, localizes to mitochondria, based on import into isolated organelles, immunocytochemistry and subcellular fractionation. In vitro FENMIT binds to flap structures containing a 5' RNA flap, and prefers such substrates to single-stranded RNA. FENMIT can also bind to R-loops, and to a lesser extent to D-loops. Exposing human cells to ethidium bromide results in the generation of RNA/DNA hybrids near the origin of mitochondrial DNA replication. FENMIT is recruited to the DNA under these conditions, and is released by RNase treatment. Moreover, high levels of recombinant FENMIT expression inhibit mtDNA replication, following ethidium bromide treatment. These findings suggest FENMIT interacts with RNA/DNA hybrids in mitochondrial DNA, such as those found at the origin of replication.

Hairpin DNA-Templated Silver Nanoclusters as Novel Beacons in Strand Displacement Amplification for MicroRNA Detection.

Science.gov (United States)

Zhang, Jingpu; Li, Chao; Zhi, Xiao; Ramón, Gabriel Alfranca; Liu, Yanlei; Zhang, Chunlei; Pan, Fei; Cui, Daxiang

2016-01-19

MicroRNA (miRNA) biomarkers display great potential for cancer diagnosis and prognosis. The development of rapid and specific methods for miRNA detection has become a hotspot. Herein, hairpin DNA-templated silver nanoclusters (AgNCs/HpDNA) were prepared and integrated into strand-displacement amplification (SDA) as a novel beacon for miRNA detection. The light-up platform was established based on guanine (G)-rich fluorescence enhancement that essentially converted the excitation/emission pair of AgNCs/HpDNAs from a shorter wavelength to a longer wavelength, and then achieved fluorescent enhancement at longer wavelength. On the basis of the validation of the method, the single and duplex detection were conducted in two plasma biomarkers (miR-16-5p and miR-19b-3p) for the diagnosis of gastric cancer. The probe (AgNCs/RED 16(7s)C) utilized for miR-16-5p detection adopted a better conformation with high specificity to recognize single-base mismatches by producing dramatically opposite signals (increase or decrease at 580 nm ex/640 nm em) while the probe (AgNCs/GRE 19b(5s)C) for miR-19b-3p generated dual signals (increase at 490 nm ex/570 nm em and decrease at 430 nm ex/530 nm em) with bright fluorescence in one reaction during the amplification, but unexpectedly was partially digested. This is for the first time to allow the generation of enhanced fluorescent AgNCs and the target recognition integrated into a single process, which offers great opportunity for specific miRNA detection in an easy and rapid way.

Dynamic Effects of Topoisomerase I Inhibition on R-Loops and Short Transcripts at Active Promoters.

Directory of Open Access Journals (Sweden)

Jessica Marinello

Full Text Available Topoisomerase I-DNA-cleavage complexes (Top1cc stabilized by camptothecin (CPT have specific effects at transcriptional levels. We recently reported that Top1cc increase antisense transcript (aRNAs levels at divergent CpG-island promoters and, transiently, DNA/RNA hybrids (R-loop in nuclear and mitochondrial genomes of colon cancer HCT116 cells. However, the relationship between R-loops and aRNAs was not established. Here, we show that aRNAs can form R-loops in N-TERA-2 cells under physiological conditions, and that promoter-associated R-loops are somewhat increased and extended in length immediately upon cell exposure to CPT. In contrast, persistent Top1ccs reduce the majority of R-loops suggesting that CPT-accumulated aRNAs are not commonly involved in R-loops. The enhancement of aRNAs by Top1ccs is present both in human colon cancer HCT116 cells and WI38 fibroblasts suggesting a common response of cancer and normal cells. Although Top1ccs lead to DSB and DDR kinases activation, we do not detect a dependence of aRNA accumulation on ATM or DNA-PK activation. However, we showed that the cell response to persistent Top1ccs can involve an impairment of aRNA turnover rather than a higher synthesis rate. Finally, a genome-wide analysis shows that persistent Top1ccs also determine an accumulation of sense transcripts at 5'-end gene regions suggesting an increased occurrence of truncated transcripts. Taken together, the results indicate that Top1 may regulate transcription initiation by modulating RNA polymerase-generated negative supercoils, which can in turn favor R-loop formation at promoters, and that transcript accumulation at TSS is a response to persistent transcriptional stress by Top1 poisoning.

Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes

Science.gov (United States)

Sanborn, Adrian; Rao, Suhas; Huang, Su-Chen; Durand, Neva; Huntley, Miriam; Jewett, Andrew; Bochkov, Ivan; Chinnappan, Dharmaraj; Cutkosky, Ashok; Li, Jian; Geeting, Kristopher; McKenna, Doug; Stamenova, Elena; Gnirke, Andreas; Melnikov, Alexandre; Lander, Eric; Aiden, Erez

Our recent kilobase-resolution genome-wide maps of DNA self-contacts demonstrated that mammalian genomes are organized into domains and loops demarcated by the DNA-binding protein CTCF. Here, we combine these maps with new Hi-C, microscopy, and genome-editing experiments to study the physical structure of chromatin fibers, domains, and loops. We find that domains are inconsistent with equilibrium and fractal models. Instead, we use physical simulations to study two models of genome folding. In one, intermonomer attraction during condensation leads to formation of an anisotropic ``tension globule.'' In the other, CTCF and cohesin act together to extrude unknotted loops. Both models are consistent with the observed domains and loops. However, the extrusion model explains a far wider array of observations, such as why the CTCF-binding motifs at pairs of loop anchors lie in the convergent orientation. Finally, we perform 13 genome-editing experiments examining the effect of altering CTCF-binding sites on chromatin folding. The extrusion model predicts in silico the experimental maps using only CTCF-binding sites. Thus, we show that it is possible to disrupt, restore, and move loops and domains using targeted mutations as small as a single base pair.

Understanding the Elementary Steps in DNA Tile-Based Self-Assembly.

Science.gov (United States)

Jiang, Shuoxing; Hong, Fan; Hu, Huiyu; Yan, Hao; Liu, Yan

2017-09-26

Although many models have been developed to guide the design and implementation of DNA tile-based self-assembly systems with increasing complexity, the fundamental assumptions of the models have not been thoroughly tested. To expand the quantitative understanding of DNA tile-based self-assembly and to test the fundamental assumptions of self-assembly models, we investigated DNA tile attachment to preformed "multi-tile" arrays in real time and obtained the thermodynamic and kinetic parameters of single tile attachment in various sticky end association scenarios. With more sticky ends, tile attachment becomes more thermostable with an approximately linear decrease in the free energy change (more negative). The total binding free energy of sticky ends is partially compromised by a sequence-independent energy penalty when tile attachment forms a constrained configuration: "loop". The minimal loop is a 2 × 2 tetramer (Loop4). The energy penalty of loops of 4, 6, and 8 tiles was analyzed with the independent loop model assuming no interloop tension, which is generalizable to arbitrary tile configurations. More sticky ends also contribute to a faster on-rate under isothermal conditions when nucleation is the rate-limiting step. Incorrect sticky end contributes to neither the thermostability nor the kinetics. The thermodynamic and kinetic parameters of DNA tile attachment elucidated here will contribute to the future improvement and optimization of tile assembly modeling, precise control of experimental conditions, and structural design for error-free self-assembly.

One simple DNA extraction device and its combination with modified visual loop-mediated isothermal amplification for rapid on-field detection of genetically modified organisms.

Science.gov (United States)

Zhang, Miao; Liu, Yinan; Chen, Lili; Quan, Sheng; Jiang, Shimeng; Zhang, Dabing; Yang, Litao

2013-01-02

Quickness, simplicity, and effectiveness are the three major criteria for establishing a good molecular diagnosis method in many fields. Herein we report a novel detection system for genetically modified organisms (GMOs), which can be utilized to perform both on-field quick screening and routine laboratory diagnosis. In this system, a newly designed inexpensive DNA extraction device was used in combination with a modified visual loop-mediated isothermal amplification (vLAMP) assay. The main parts of the DNA extraction device included a silica gel membrane filtration column and a modified syringe. The DNA extraction device could be easily operated without using other laboratory instruments, making it applicable to an on-field GMO test. High-quality genomic DNA (gDNA) suitable for polymerase chain reaction (PCR) and isothermal amplification could be quickly isolated from plant tissues using this device within 15 min. In the modified vLAMP assay, a microcrystalline wax encapsulated detection bead containing SYBR green fluorescent dye was introduced to avoid dye inhibition and cross-contaminations from post-LAMP operation. The system was successfully applied and validated in screening and identification of GM rice, soybean, and maize samples collected from both field testing and the Grain Inspection, Packers, and Stockyards Administration (GIPSA) proficiency test program, which demonstrated that it was well-adapted to both on-field testing and/or routine laboratory analysis of GMOs.

LoopIng: a template-based tool for predicting the structure of protein loops.

KAUST Repository

Messih, Mario Abdel

2015-08-06

Predicting the structure of protein loops is very challenging, mainly because they are not necessarily subject to strong evolutionary pressure. This implies that, unlike the rest of the protein, standard homology modeling techniques are not very effective in modeling their structure. However, loops are often involved in protein function, hence inferring their structure is important for predicting protein structure as well as function.We describe a method, LoopIng, based on the Random Forest automated learning technique, which, given a target loop, selects a structural template for it from a database of loop candidates. Compared to the most recently available methods, LoopIng is able to achieve similar accuracy for short loops (4-10 residues) and significant enhancements for long loops (11-20 residues). The quality of the predictions is robust to errors that unavoidably affect the stem regions when these are modeled. The method returns a confidence score for the predicted template loops and has the advantage of being very fast (on average: 1 min/loop).www.biocomputing.it/loopinganna.tramontano@uniroma1.itSupplementary data are available at Bioinformatics online.

Displaced Sense: Displacement, Religion and Sense-making

OpenAIRE

Naidu, Maheshvari

2016-01-01

Whether formally categorized as refugees or not, displaced migrants experience varying degrees of vulnerability in relation to where they find themselves displaced. The internally displaced furthermore squat invisibly and outside the boundaries of the legal framework and incentive structures accorded to those classified as 'refugee'. They are thus arguably, by and large, left to source sustaining solutions for themselves. This article works through the theoretical prism of sense-making theory...

Development of a real-time closed-loop micro-/nano-positioning system embedded with a capacitive sensor

International Nuclear Information System (INIS)

Shiou, Fang-Jung; Chiang, Chia-Jui; Liou, Ke-Jhen; Liao, Shu-Chung; Chen, Chao-Jung; Liou, Huay-Chung

2010-01-01

The hysteresis and nonlinearity of the PZT is an actual problem in the piezo-driven micro-/nano-positioning stage, especially for the open-loop positioning stage. The study presents the development of an NI cRIO9074-based real-time closed-loop micro-/nano-positioning system, to overcome the problem of the hysteresis and nonlinearity of a PZT and to increase the positioning speed of the positioning stage. The developed system mainly consists of a piezoelectric actuator, a bridge-type hinge mechanism for displacement magnification, a micro-/nano-positioning stage body, a capacitive sensor system, an NI cRIO9074 real-time control unit with FPGA chip and a PC. After executing the optimization analysis of the displacement, stress and the frequency, using the ANSYS software, the dimensions of the stage body have been designed and determined. A set of software written with the LabView programming language was developed to construct the real-time PID closed-loop control of the developed positioning system. Based on the test results, the designed closed-loop micro-/nano-positioning system was capable of precision positioning within the travel of 119.08 µm with maximum stage tilting angle at 25 µrad. The steady-state positioning deviation of the stage is about ±2 nm in the step-positioning test. In the transient slope-tracing test at a tracing speed of 5 µm s −1 , an error of about ±100 nm is observed

Experimental and simulated displacement of oil in sand berea cores using aqueous solutions

International Nuclear Information System (INIS)

Ramirez, A.; Gonzalez, J.L.; Hernandez, F.; Hernandez, H.

2009-01-01

The development of a mathematical algorithm to simulate the displacement of a resident fluid using a displacing one in a saturated anisotropic porous media is shown in this work. The algorithm was included in the simulator developed by the present authors in previous works to represent the anisotropic distribution of the porous media properties and the fluid injection [Ramirez A et al. Mathematical simulation of oil reservoir properties. Chaos, solitons and Fractals 2008;38:778-88, Ramirez A et al. Simulation of uncompressible fluid flow through a porous media. Chaos, Solitons and Fractals 2009;39:1753-63] in a nested loop to analyze the participant nodes in the transport process and calculate the volumes of the resident and new fluids. The new routine developed takes in count the mobility of both fluids. Additionally experimental fluid displacement tests were done using heavy oil from Mexican reservoirs as a resident fluid in sand berea cores. The injection of new fluids in natural oil reservoirs is a part of the Enhanced oil recovery (EOR) methods used to improve the oil displacement and increase production after the primary stage of the oil recovery has been finished. Water is an available and economical resource to be used as a displacing fluid due to many of the producers (off-shores) of the oil industries are placed in the sea near the coast.

Noncoding transcription by alternative rna polymerases dynamically regulates an auxin-driven chromatin loop

KAUST Repository

Ariel, Federico D.; Jé gu, Teddy; Latrasse, David; Romero-Barrios, Natali; Christ, Auré lie; Benhamed, Moussa; Crespi, Martí n D.

2014-01-01

The eukaryotic epigenome is shaped by the genome topology in three-dimensional space. Dynamic reversible variations in this epigenome structure directly influence the transcriptional responses to developmental cues. Here, we show that the Arabidopsis long intergenic noncoding RNA (lincRNA) APOLO is transcribed by RNA polymerases II and V in response to auxin, a phytohormone controlling numerous facets of plant development. This dual APOLO transcription regulates the formation of a chromatin loop encompassing the promoter of its neighboring gene PID, a key regulator of polar auxin transport. Altering APOLO expression affects chromatin loop formation, whereas RNA-dependent DNA methylation, active DNA demethylation, and Polycomb complexes control loop dynamics. This dynamic chromatin topology determines PID expression patterns. Hence, the dual transcription of a lincRNA influences local chromatin topology and directs dynamic auxin-controlled developmental outputs on neighboring genes. This mechanism likely underscores the adaptive success of plants in diverse environments and may be widespread in eukaryotes. © 2014 Elsevier Inc.

Noncoding transcription by alternative rna polymerases dynamically regulates an auxin-driven chromatin loop

KAUST Repository

Ariel, Federico D.

2014-08-01

The eukaryotic epigenome is shaped by the genome topology in three-dimensional space. Dynamic reversible variations in this epigenome structure directly influence the transcriptional responses to developmental cues. Here, we show that the Arabidopsis long intergenic noncoding RNA (lincRNA) APOLO is transcribed by RNA polymerases II and V in response to auxin, a phytohormone controlling numerous facets of plant development. This dual APOLO transcription regulates the formation of a chromatin loop encompassing the promoter of its neighboring gene PID, a key regulator of polar auxin transport. Altering APOLO expression affects chromatin loop formation, whereas RNA-dependent DNA methylation, active DNA demethylation, and Polycomb complexes control loop dynamics. This dynamic chromatin topology determines PID expression patterns. Hence, the dual transcription of a lincRNA influences local chromatin topology and directs dynamic auxin-controlled developmental outputs on neighboring genes. This mechanism likely underscores the adaptive success of plants in diverse environments and may be widespread in eukaryotes. © 2014 Elsevier Inc.

Detection of novel polymorphisms in the mitochondrial DNA D-Loop ...

African Journals Online (AJOL)

aghomotsegin

2015-04-08

Apr 8, 2015 ... 1Department of Molecular Biology, Babylon University, Hilla City, Iraq. 2Babylon ... Mitochondrial DNA (mtDNA) is a useful genetic marker for answering .... to you for choosing the project, your enthusiasm for helping us ...

Caffeine inhibits gene conversion by displacing Rad51 from ssDNA

Science.gov (United States)

Tsabar, Michael; Mason, Jennifer M.; Chan, Yuen-Ling; Bishop, Douglas K.; Haber, James E.

2015-01-01

Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1ATR/Tel1ATM-dependent DNA damage response or caffeine's inhibition of 5′ to 3′ resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments. PMID:26019181

Structural and dynamic characterization of the upper part of the HIV-1 cTAR DNA hairpin

OpenAIRE

Zargarian, Loussin?; Kanevsky, Igor; Bazzi, Ali; Boynard, Jonathan; Chaminade, Fran?oise; Foss?, Philippe; Mauffret, Olivier

2009-01-01

First strand transfer is essential for HIV-1 reverse transcription. During this step, the TAR RNA hairpin anneals to the cTAR DNA hairpin; this annealing reaction is promoted by the nucleocapsid protein and involves an initial loop?loop interaction between the apical loops of TAR and cTAR. Using NMR and probing methods, we investigated the structural and dynamic properties of the top half of the cTAR DNA (mini-cTAR). We show that the upper stem located between the apical and the internal loop...

Mechanisms of defect production and atomic mixing in high energy displacement cascades: A molecular dynamics study

International Nuclear Information System (INIS)

Diaz de la Rubia, T.; Guinan, M.W.

1991-01-01

We have performed molecular dynamics computer simulation studies of displacement cascades in Cu at low temperature. For 25 keV recoils we observe the splitting of a cascade into subcascades and show that cascades in Cu may lead to the formation of vacancy and interstitial dislocation loops. We discuss a new mechanism of defect production based on the observation of interstitial prismatic dislocation loop punching from cascades at 10 K. We also show that below the subcascade threshold, atomic mixing in the cascade is recoil-energy dependent and obtain a mixing efficiency that scales as the square root of the primary recoil energy. 44 refs., 12 figs

Loop-mediated isothermal amplification (LAMP) assays for rapid detection and differentiation of Nosema apis and N. ceranae in honeybees.

Science.gov (United States)

Ptaszyńska, Aneta A; Borsuk, Grzegorz; Woźniakowski, Grzegorz; Gnat, Sebastian; Małek, Wanda

2014-08-01

Nosemosis is a contagious disease of honeybees (Apis mellifera) manifested by increased winter mortality, poor spring build-up and even the total extinction of infected bee colonies. In this paper, loop-mediated isothermal amplifications (LAMP) were used for the first time to identify and differentiate N. apis and N. ceranae, the causative agents of nosemosis. LAMP assays were performed at a constant temperature of 60 °C using two sets of six species-specific primers, recognising eight distinct fragments of 16S rDNA gene and GspSSD polymerase with strand displacement activity. The optimal time for LAMP and its Nosema species sensitivity and specificity were assessed. LAMP only required 30 min for robust identification of the amplicons. Ten-fold serial dilutions of total DNA isolated from bees infected with microsporidia were used to determine the detection limit of N. apis and N. ceranae DNAs by LAMP and standard PCR assays. LAMP appeared to be 10(3) -fold more sensitive than a standard PCR in detecting N. apis and N. ceranae. LAMP methods developed by us are highly Nosema species specific and allow to identify and differentiate N. apis and N. ceranae. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Biomolecular and structural analyses of cauliflower-like DNAs by ultraviolet, circular dichroism, and fluorescence spectroscopies in comparison with natural DNA.

Science.gov (United States)

Gill, Pooria; Ranjbar, Bijan; Saber, Reza; Khajeh, Khosro; Mohammadian, Mehdi

2011-07-01

Cauliflower-like DNAs are stem-loop DNAs that are fabricated periodically in inverted repetitions from deoxyribonucleic acid phosphates (dNTPs) by loop-mediated isothermal amplification (LAMP). Cauliflower-like DNAs have ladder-shape behaviors on gel electrophoresis, and increasing the time of LAMP leads to multiplying the repetitions, stem-loops, and electrophoretic bands. Cauliflower-like DNAs were fabricated via LAMP using two loop primers, two bumper primers, dNTPs, a λ-phage DNA template, and a Bst DNA polymerase in 75- and 90-min periods. These times led to manufacturing two types of cauliflower-like DNAs with different contents of inverted repetitions and stem-loops, which were clearly indicated by two comparable electrophoresis patterns in agarose gel. LAMP-fabricated DNAs and natural dsB-DNA (salmon genomic DNA) were dialyzed in Gomori phosphate buffer (10 mM, pH 7.4) to be isolated from salts, nucleotides, and primers. Dialyzed DNAs were studied using UV spectroscopy, circular dichroism spectropolarimetry, and fluorescence spectrophotometry. Structural analyses indicated reduction of the molecular ellipticity and extinction coefficients in comparison with B-DNA. Also, cauliflower-like DNAs demonstrated less intrinsic and more extrinsic fluorescence in comparison with natural DNA. The overwinding and lengthening of the cauliflower-like configurations of LAMP DNAs led to changes in physical parameters of this type of DNA in comparison with natural DNA. The results obtained introduced new biomolecular characteristics of DNA macromolecules fabricated within a LAMP process and show the effects of more inverted repeats and stem-loops, which are manufactured by lengthening the process.

Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA.

Science.gov (United States)

Herzner, Anna-Maria; Hagmann, Cristina Amparo; Goldeck, Marion; Wolter, Steven; Kübler, Kirsten; Wittmann, Sabine; Gramberg, Thomas; Andreeva, Liudmila; Hopfner, Karl-Peter; Mertens, Christina; Zillinger, Thomas; Jin, Tengchuan; Xiao, Tsan Sam; Bartok, Eva; Coch, Christoph; Ackermann, Damian; Hornung, Veit; Ludwig, Janos; Barchet, Winfried; Hartmann, Gunther; Schlee, Martin

2015-10-01

Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon-inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

Variable Displacement Control of the Concrete Pumping System Based on Dynamic Programming

Directory of Open Access Journals (Sweden)

Ye Min

2017-01-01

Full Text Available To solve the problems of cylinder piston striking cylinder and the hydraulic shocking of the main pump, and causing energy waste problem, the method of variable displacement control of piston stroke was proposed. In order to achieve effective control of the piston stroke, variable displacement control model was established under the physical constraint condition of non-collision between piston and cylinder. And the control process was realized by Dynamic Programming(DP, the simulation and test results show that piston of concrete pumping system don’t strike cylinder and reduce the hydraulic shock of the main pump outlet, meanwhile improve the response speed of the cylinder and achieve energy-saving purposes under varying loads. This control model built in the integration design space of structure variable and control variable is of guiding significance for solving open-loop system’s engineering problems.

Autosomal mutations affecting Y chromosome loops in Drosophila melanogaster

Directory of Open Access Journals (Sweden)

Petrucci Romano

2008-04-01

Full Text Available Abstract Background The Y chromosome of Drosophila melanogaster harbors several genes required for male fertility. The genes for these fertility factors are very large in size and contain conspicuous amounts of repetitive DNA and transposons. Three of these loci (ks-1, kl-3 and kl-5 have the ability to develop giant lampbrush-like loops in primary spermatocytes, a cytological manifestation of their active state in these cells. Y-loops bind a number of non-Y encoded proteins, but the mechanisms regulating their development and their specific functions are still to be elucidated. Results Here we report the results of a screen of 726 male sterile lines to identify novel autosomal genes controlling Y-loop function. We analyzed mutant testis preparations both in vivo and by immunofluorescence using antibodies directed against Y-loop-associated proteins. This screen enabled us to isolate 17 mutations at 15 loci whose wild-type function is required for proper Y-loop morphogenesis. Six of these loci are likely to specifically control loop development, while the others display pleiotropic effects on both loops and meiotic processes such as spermiogenesis, sperm development and maturation. We also determined the map position of the mutations affecting exclusively Y-loop morphology. Conclusion Our cytological screening permitted us to identify novel genetic functions required for male spermatogenesis, some of which show pleiotropic effects. Analysis of these mutations also shows that loop development can be uncoupled from meiosis progression. These data represent a useful framework for the characterization of Y-loop development at a molecular level and for the study of the genetic control of heterochromatin.

Flexible DNA Path in the MCM Double Hexamer Loaded on DNA.

Science.gov (United States)

Hizume, Kohji; Kominami, Hiroaki; Kobayashi, Kei; Yamada, Hirofumi; Araki, Hiroyuki

2017-05-16

The formation of the pre-replicative complex (pre-RC) during the G1 phase, which is also called the licensing of DNA replication, is the initial and essential step of faithful DNA replication during the subsequent S phase. It is widely accepted that in the pre-RC, double-stranded DNA passes through the holes of two ring-shaped minichromosome maintenance (MCM) 2-7 hexamers; however, the spatial organization of the DNA and proteins involved in pre-RC formation is unclear. Here we reconstituted the pre-RC from purified DNA and proteins and visualized the complex using atomic force microscopy (AFM). AFM revealed that the MCM double hexamers formed elliptical particles on DNA. Analysis of the angle of binding of DNA to the MCM double hexamer suggests that the DNA does not completely pass through both holes of the MCM hexamers, possibly because the DNA exited from the gap between Mcm2 and Mcm5. A DNA loop fastened by the MCM double hexamer was detected in pre-RC samples reconstituted from purified proteins as well as those purified from yeast cells, suggesting a higher-order architecture of the loaded MCM hexamers and DNA strands.

Structural basis for sequence-specific recognition of DNA by TAL effectors

KAUST Repository

Deng, Dong

2012-01-05

TAL (transcription activator-like) effectors, secreted by phytopathogenic bacteria, recognize host DNA sequences through a central domain of tandem repeats. Each repeat comprises 33 to 35 conserved amino acids and targets a specific base pair by using two hypervariable residues [known as repeat variable diresidues (RVDs)] at positions 12 and 13. Here, we report the crystal structures of an 11.5-repeat TAL effector in both DNA-free and DNA-bound states. Each TAL repeat comprises two helices connected by a short RVD-containing loop. The 11.5 repeats form a right-handed, superhelical structure that tracks along the sense strand of DNA duplex, with RVDs contacting the major groove. The 12th residue stabilizes the RVD loop, whereas the 13th residue makes a base-specific contact. Understanding DNA recognition by TAL effectors may facilitate rational design of DNA-binding proteins with biotechnological applications.

Chemo-mechanical pushing of proteins along single-stranded DNA.

Science.gov (United States)

Sokoloski, Joshua E; Kozlov, Alexander G; Galletto, Roberto; Lohman, Timothy M

2016-05-31

Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3'-Cy3-labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5' to 3' ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5' to 3') pushing of the SSB toward the 3' ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3' to 5') direction, are observed with EcRep and EcUvrD (both 3' to 5' ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA.

Timing, coordination, and rhythm: Acrobatics at the DNA replication fork

KAUST Repository

Hamdan, Samir

2010-04-09

In DNA replication, the antiparallel nature of the parental duplex imposes certain constraints on the activity of the DNA polymerases that synthesize new DNA. The leading-strand polymerase advances in a continuous fashion, but the lagging-strand polymerase is forced to restart at short intervals. In several prokaryotic systems studied so far, this problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. The timing of Okazaki fragment synthesis and loop formation is determined by a subtle interplay of enzymatic activities at the fork. Recent developments in single-molecule techniques have enabled the direct observation of these processes and have greatly contributed to a better understanding of the dynamic nature of the replication fork. Here, we will review recent experimental advances, present the current models, and discuss some of the exciting developments in the field. 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Timing, coordination, and rhythm: Acrobatics at the DNA replication fork

KAUST Repository

Hamdan, Samir; van Oijen, Antoine M.

2010-01-01

In DNA replication, the antiparallel nature of the parental duplex imposes certain constraints on the activity of the DNA polymerases that synthesize new DNA. The leading-strand polymerase advances in a continuous fashion, but the lagging-strand polymerase is forced to restart at short intervals. In several prokaryotic systems studied so far, this problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. The timing of Okazaki fragment synthesis and loop formation is determined by a subtle interplay of enzymatic activities at the fork. Recent developments in single-molecule techniques have enabled the direct observation of these processes and have greatly contributed to a better understanding of the dynamic nature of the replication fork. Here, we will review recent experimental advances, present the current models, and discuss some of the exciting developments in the field. 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

The evolution mechanism of the dislocation loops in irradiated lanthanum doped cerium oxide

International Nuclear Information System (INIS)

Miao, Yinbin; Aidhy, Dilpuneet; Chen, Wei-Ying; Mo, Kun; Oaks, Aaron; Wolf, Dieter; Stubbins, James F.

2014-01-01

Cerium dioxide, a non-radioactive surrogate of uranium dioxide, is useful for simulating the radiation responses of uranium dioxide and mixed oxide fuel (MOX). Controlled additions of lanthanum can also be used to form various levels of lattice oxide or anion vacancies. In previous transmission electron microscopy (TEM) experimental studies, the growth rate of dislocation loops in irradiated lanthanum doped ceria was reported to vary with lanthanum concentration. This work reports findings of the evolution mechanisms of the dislocation loops in cerium oxide with and without lanthanum dopants based on a combination of molecular statics and molecular dynamics simulations. These dislocation loops are found to be b=1/3〈111〉 interstitial type Frank loops. Calculations of the defect energy profiles of the dislocation loops with different structural configurations and radii reveal the basis for preference of nucleation as well as the driving force of growth. Frenkel pair evolution simulations and displacement cascade overlaps simulations were conducted for a variety of lanthanum doping conditions. The nucleation and growth processes of the Frank loop were found to be controlled by the mobility of cation interstitials, which is significantly influenced by the lanthanum doping concentration. Competition mechanisms coupled with the mobility of cation point defects were discovered, and can be used to explain the lanthanum effects observed in experiments

Isothermal circular-strand-displacement polymerization of DNA and microRNA in digital microfluidic devices.

Science.gov (United States)

Giuffrida, Maria Chiara; Zanoli, Laura Maria; D'Agata, Roberta; Finotti, Alessia; Gambari, Roberto; Spoto, Giuseppe

2015-02-01

Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use of digital microfluidic devices to miniaturize amplification techniques reduces the required sample volume and the analysis time and offers new possibilities for process automation and integration in a single device. The recently introduced droplet polymerase-chain-reaction (PCR) amplification methods require repeated cycles of two or three temperature-dependent steps during the amplification of the nucleic-acid target sequence. In contrast, low-temperature isothermal-amplification methods have no need for thermal cycling, thus requiring simplified microfluidic-device features. Here, the combined use of digital microfluidics and molecular-beacon (MB)-assisted isothermal circular-strand-displacement polymerization (ICSDP) to detect microRNA-210 sequences is described. MicroRNA-210 has been described as the most consistently and predominantly upregulated hypoxia-inducible factor. The nmol L(-1)-pmol L(-1) detection capabilities of the method were first tested by targeting single-stranded DNA sequences from the genetically modified Roundup Ready soybean. The ability of the droplet-ICSDP method to discriminate between full-matched, single-mismatched, and unrelated sequences was also investigated. The detection of a range of nmol L(-1)-pmol L(-1) microRNA-210 solutions compartmentalized in nanoliter-sized droplets was performed, establishing the ability of the method to detect as little as 10(-18) mol of microRNA target sequences compartmentalized in 20 nL droplets. The suitability of the method for biological samples was tested by detecting microRNA-210 from transfected K562 cells.

TERRA and hnRNPA1 orchestrate an RPA-to-POT1 switch on telomeric single-stranded DNA.

Science.gov (United States)

Flynn, Rachel Litman; Centore, Richard C; O'Sullivan, Roderick J; Rai, Rekha; Tse, Alice; Songyang, Zhou; Chang, Sandy; Karlseder, Jan; Zou, Lee

2011-03-24

Maintenance of telomeres requires both DNA replication and telomere 'capping' by shelterin. These two processes use two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomeres 1 (POT1). Although RPA and POT1 each have a critical role at telomeres, how they function in concert is not clear. POT1 ablation leads to activation of the ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase at telomeres, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. Unexpectedly, we found that purified POT1 and its functional partner TPP1 are unable to prevent RPA binding to telomeric ssDNA efficiently. In cell extracts, we identified a novel activity that specifically displaces RPA, but not POT1, from telomeric ssDNA. Using purified protein, here we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) recapitulates the RPA displacing activity. The RPA displacing activity is inhibited by the telomeric repeat-containing RNA (TERRA) in early S phase, but is then unleashed in late S phase when TERRA levels decline at telomeres. Interestingly, TERRA also promotes POT1 binding to telomeric ssDNA by removing hnRNPA1, suggesting that the re-accumulation of TERRA after S phase helps to complete the RPA-to-POT1 switch on telomeric ssDNA. Together, our data suggest that hnRNPA1, TERRA and POT1 act in concert to displace RPA from telomeric ssDNA after DNA replication, and promote telomere capping to preserve genomic integrity.

Structure of a preternary complex involving a prokaryotic NHEJ DNA polymerase.

Science.gov (United States)

Brissett, Nigel C; Martin, Maria J; Pitcher, Robert S; Bianchi, Julie; Juarez, Raquel; Green, Andrew J; Fox, Gavin C; Blanco, Luis; Doherty, Aidan J

2011-01-21

In many prokaryotes, a specific DNA primase/polymerase (PolDom) is required for nonhomologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). Here, we report the crystal structure of a catalytically active conformation of Mycobacterium tuberculosis PolDom, consisting of a polymerase bound to a DNA end with a 3' overhang, two metal ions, and an incoming nucleotide but, significantly, lacking a primer strand. This structure represents a polymerase:DNA complex in a preternary intermediate state. This polymerase complex occurs in solution, stabilizing the enzyme on DNA ends and promoting nucleotide extension of short incoming termini. We also demonstrate that the invariant Arg(220), contained in a conserved loop (loop 2), plays an essential role in catalysis by regulating binding of a second metal ion in the active site. We propose that this NHEJ intermediate facilitates extension reactions involving critically short or noncomplementary DNA ends, thus promoting break repair and minimizing sequence loss during DSB repair. Copyright © 2011 Elsevier Inc. All rights reserved.

Human DNA polymerase delta double-mutant D316A;E318A interferes with DNA mismatch repair in vitro

DEFF Research Database (Denmark)

Liu, Dekang; Frederiksen, Jane H.; Liberti, Sascha Emilie

2017-01-01

DNA mismatch repair (MMR) is a highly-conserved DNA repair mechanism, whose primary role is to remove DNA replication errors preventing them from manifesting as mutations, thereby increasing the overall genome stability. Defects in MMR are associated with increased cancer risk in humans and other...... organisms. Here, we characterize the interaction between MMR and a proofreading-deficient allele of the human replicative DNA polymerase delta, PolδD316A;E318A, which has a higher capacity for strand displacement DNA synthesis than wild type Polδ. Human cell lines overexpressing PolδD316A;E318A display...

Loop Transfer Matrix and Loop Quantum Mechanics

International Nuclear Information System (INIS)

Savvidy, George K.

2000-01-01

The gonihedric model of random surfaces on a 3d Euclidean lattice has equivalent representation in terms of transfer matrix K(Q i ,Q f ), which describes the propagation of loops Q. We extend the previous construction of the loop transfer matrix to the case of nonzero self-intersection coupling constant κ. We introduce the loop generalization of Fourier transformation which allows to diagonalize transfer matrices, that depend on symmetric difference of loops only and express all eigenvalues of 3d loop transfer matrix through the correlation functions of the corresponding 2d statistical system. The loop Fourier transformation allows to carry out the analogy with quantum mechanics of point particles, to introduce conjugate loop momentum P and to define loop quantum mechanics. We also consider transfer matrix on 4d lattice which describes propagation of memebranes. This transfer matrix can also be diagonalized by using the generalized Fourier transformation, and all its eigenvalues are equal to the correlation functions of the corresponding 3d statistical system. In particular the free energy of the 4d membrane system is equal to the free energy of 3d gonihedric system of loops and is equal to the free energy of 2d Ising model. (author)

Structural and Molecular Basis for Coordination in a Viral DNA Packaging Motor

Directory of Open Access Journals (Sweden)

Huzhang Mao

2016-03-01

Full Text Available Ring NTPases are a class of ubiquitous molecular motors involved in basic biological partitioning processes. dsDNA viruses encode ring ATPases that translocate their genomes to near-crystalline densities within pre-assembled viral capsids. Here, X-ray crystallography, cryoEM, and biochemical analyses of the dsDNA packaging motor in bacteriophage phi29 show how individual subunits are arranged in a pentameric ATPase ring and suggest how their activities are coordinated to translocate dsDNA. The resulting pseudo-atomic structure of the motor and accompanying functional analyses show how ATP is bound in the ATPase active site; identify two DNA contacts, including a potential DNA translocating loop; demonstrate that a trans-acting arginine finger is involved in coordinating hydrolysis around the ring; and suggest a functional coupling between the arginine finger and the DNA translocating loop. The ability to visualize the motor in action illuminates how the different motor components interact with each other and with their DNA substrate.

A Molecular Staple: D-Loops in the I Domain of Bacteriophage P22 Coat Protein Make Important Intercapsomer Contacts Required for Procapsid Assembly

Science.gov (United States)

D'Lima, Nadia G.

2015-01-01

ABSTRACT Bacteriophage P22, a double-stranded DNA (dsDNA) virus, has a nonconserved 124-amino-acid accessory domain inserted into its coat protein, which has the canonical HK97 protein fold. This I domain is involved in virus capsid size determination and stability, as well as protein folding. The nuclear magnetic resonance (NMR) solution structure of the I domain revealed the presence of a D-loop, which was hypothesized to make important intersubunit contacts between coat proteins in adjacent capsomers. Here we show that amino acid substitutions of residues near the tip of the D-loop result in aberrant assembly products, including tubes and broken particles, highlighting the significance of the D-loops in proper procapsid assembly. Using disulfide cross-linking, we showed that the tips of the D-loops are positioned directly across from each other both in the procapsid and the mature virion, suggesting their importance in both states. Our results indicate that D-loop interactions act as “molecular staples” at the icosahedral 2-fold symmetry axis and significantly contribute to stabilizing the P22 capsid for DNA packaging. IMPORTANCE Many dsDNA viruses have morphogenic pathways utilizing an intermediate capsid, known as a procapsid. These procapsids are assembled from a coat protein having the HK97 fold in a reaction driven by scaffolding proteins or delta domains. Maturation of the capsid occurs during DNA packaging. Bacteriophage HK97 uniquely stabilizes its capsid during maturation by intercapsomer cross-linking, but most virus capsids are stabilized by alternate means. Here we show that the I domain that is inserted into the coat protein of bacteriophage P22 is important in the process of proper procapsid assembly. Specifically, the I domain allows for stabilizing interactions across the capsid 2-fold axis of symmetry via a D-loop. When amino acid residues at the tip of the D-loop are mutated, aberrant assembly products, including tubes, are formed instead

Detection of circular telomeric DNA without 2D gel electrophoresis.

Science.gov (United States)

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

2008-09-01

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

DNA Length Modulates the Affinity of Fragments of Genomic DNA for the Nuclear Matrix In Vitro.

Science.gov (United States)

García-Vilchis, David; Aranda-Anzaldo, Armando

2017-12-01

Classical observations have shown that during the interphase the chromosomal DNA of metazoans is organized in supercoiled loops attached to a compartment known as the nuclear matrix (NM). Fragments of chromosomal DNA able to bind the isolated NM in vitro are known as matrix associated/attachment/addressed regions or MARs. No specific consensus sequence or motif has been found that may constitute a universal, defining feature of MARs. On the other hand, high-salt resistant DNA-NM interactions in situ define true DNA loop anchorage regions or LARs, that might correspond to a subset of the potential MARs but are not necessarily identical to MARs characterized in vitro, since there are several examples of MARs able to bind the NM in vitro but which are not actually bound to the NM in situ. In the present work we assayed the capacity of two LARs, as well as of shorter fragments within such LARs, for binding to the NM in vitro. Paradoxically the isolated (≈2 kb) LARs cannot bind to the NM in vitro while their shorter (≈300 pb) sub-fragments and other non-related but equally short DNA fragments, bind to the NM in a high-salt resistant fashion. Our results suggest that the ability of a given DNA fragment for binding to the NM in vitro primarily depends on the length of the fragment, suggesting that binding to the NM is modulated by the local topology of the DNA fragment in suspension that it is known to depend on the DNA length. J. Cell. Biochem. 118: 4487-4497, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

BLM has early and late functions in homologous recombination repair in mouse embryonic stem cells

DEFF Research Database (Denmark)

Chu, W K; Hanada, K; Kanaar, R

2010-01-01

function of BLM remains unclear. Multiple roles have been proposed for BLM in the homologous recombination (HR) repair pathway, including 'early' functions, such as the stimulation of resection of DNA double-strand break ends or displacement of the invading strand of DNA displacement loops, and 'late......' roles, such as dissolution of double Holliday junctions. However, most of the evidence for these putative roles comes from in vitro biochemical data. In this study, we report the characterization of mouse embryonic stem cells with disruption of Blm and/or Rad54 genes. We show that Blm has roles both...

Displacement ventilation

DEFF Research Database (Denmark)

Kosonen, Risto; Melikov, Arsen Krikor; Mundt, Elisabeth

The aim of this Guidebook is to give the state-of-the art knowledge of the displacement ventilation technology, and to simplify and improve the practical design procedure. The Guidebook discusses methods of total volume ventilation by mixing ventilation and displacement ventilation and it gives...... insights of the performance of the displacement ventilation. It also shows practical case studies in some typical applications and the latest research findings to create good local micro-climatic conditions....

Displacement cascades and defects annealing in tungsten, Part I: Defect database from molecular dynamics simulations

Energy Technology Data Exchange (ETDEWEB)

Setyawan, Wahyu [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Nandipati, Giridhar [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Roche, Kenneth J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Univ. of Washington, Seattle, WA (United States); Heinisch, Howard L. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Wirth, Brian D. [Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab., Oak Ridge, TN (United States); Kurtz, Richard J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

2015-07-01

Molecular dynamics simulations have been used to generate a comprehensive database of surviving defects due to displacement cascades in bulk tungsten. Twenty-one data points of primary knock-on atom (PKA) energies ranging from 100 eV (sub-threshold energy) to 100 keV (~780^×_Ed, where _Ed = 128 eV is the average displacement threshold energy) have been completed at 300 K, 1025 K and 2050 K. Within this range of PKA energies, two regimes of power-law energy-dependence of the defect production are observed. A distinct power-law exponent characterizes the number of Frenkel pairs produced within each regime. The two regimes intersect at a transition energy which occurs at approximately 250^×_Ed. The transition energy also marks the onset of the formation of large self-interstitial atom (SIA) clusters (size 14 or more). The observed defect clustering behavior is asymmetric, with SIA clustering increasing with temperature, while the vacancy clustering decreases. This asymmetry increases with temperature such that at 2050 K (~0.5_Tm) practically no large vacancy clusters are formed, meanwhile large SIA clusters appear in all simulations. The implication of such asymmetry on the long-term defect survival and damage accumulation is discussed. In addition, <100> {110} SIA loops are observed to form directly in the highest energy cascades, while vacancy <100> loops are observed to form at the lowest temperature and highest PKA energies, although the appearance of both the vacancy and SIA loops with Burgers vector of <100> type is relatively rare.

The Affinity of the S9.6 Antibody for Double-Stranded RNAs Impacts the Accurate Mapping of R-Loops in Fission Yeast.

Science.gov (United States)

Hartono, Stella R; Malapert, Amélie; Legros, Pénélope; Bernard, Pascal; Chédin, Frédéric; Vanoosthuyse, Vincent

2018-02-02

R-loops, which result from the formation of stable DNA:RNA hybrids, can both threaten genome integrity and act as physiological regulators of gene expression and chromatin patterning. To characterize R-loops in fission yeast, we used the S9.6 antibody-based DRIPc-seq method to sequence the RNA strand of R-loops and obtain strand-specific R-loop maps at near nucleotide resolution. Surprisingly, preliminary DRIPc-seq experiments identified mostly RNase H-resistant but exosome-sensitive RNAs that mapped to both DNA strands and resembled RNA:RNA hybrids (dsRNAs), suggesting that dsRNAs form widely in fission yeast. We confirmed in vitro that S9.6 can immuno-precipitate dsRNAs and provide evidence that dsRNAs can interfere with its binding to R-loops. dsRNA elimination by RNase III treatment prior to DRIPc-seq allowed the genome-wide and strand-specific identification of genuine R-loops that responded in vivo to RNase H levels and displayed classical features associated with R-loop formation. We also found that most transcripts whose levels were altered by in vivo manipulation of RNase H levels did not form detectable R-loops, suggesting that prolonged manipulation of R-loop levels could indirectly alter the transcriptome. We discuss the implications of our work in the design of experimental strategies to probe R-loop functions. Copyright © 2017 Elsevier Ltd. All rights reserved.

A ratiometric electrochemical biosensor for the exosomal microRNAs detection based on bipedal DNA walkers propelled by locked nucleic acid modified toehold mediate strand displacement reaction.

Science.gov (United States)

Zhang, Jing; Wang, Liang-Liang; Hou, Mei-Feng; Xia, Yao-Kun; He, Wen-Hui; Yan, An; Weng, Yun-Ping; Zeng, Lu-Peng; Chen, Jing-Hua

2018-04-15

Sensitive and selective detection of microRNAs (miRNAs) in cancer cells derived exosomes have attracted rapidly growing interest owing to their potential in diagnostic and prognostic applications. Here, we design a ratiometric electrochemical biosensor based on bipedal DNA walkers for the attomolar detection of exosomal miR-21. In the presence of miR-21, DNA walkers are activated to walk continuously along DNA tracks, resulting in conformational changes as well as considerable increases of the signal ratio produced by target-respond and target-independent reporters. With the signal cascade amplification of DNA walkers, the biosensor exhibits ultrahigh sensitivity with the limit of detection (LOD) down to 67 aM. Furthermore, owing to the background-correcting function of target-independent reporters termed as reference reporters, the biosensor is robust and stable enough to be applied in the detection of exosomal miR-21 extracted from breast cancer cell lines and serums. In addition, because locked nucleic acid (LNA) modified toehold mediate strand displacement reaction (TMSDR) has extraordinary discriminative ability, the biosensor displays excellent selectivity even against the single-base-mismatched target. It is worth mentioning that our sensor is regenerative and stable for at least 5 cycles without diminution in sensitivity. In brief, the high sensitivity, selectivity and reproducibility, together with cheap, make the proposed biosensor a promising approach for exosomal miRNAs detection, in conjunction with early point-of-care testing (POCT) of cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of single-nucleotide polymorphisms using an ON-OFF switching of regenerated biosensor based on a locked nucleic acid-integrated and toehold-mediated strand displacement reaction.

Science.gov (United States)

Gao, Zhong Feng; Ling, Yu; Lu, Lu; Chen, Ning Yu; Luo, Hong Qun; Li, Nian Bing

2014-03-04

Although various strategies have been reported for single-nucleotide polymorphisms (SNPs) detection, development of a time-saving, specific, and regenerated electrochemical sensing platform still remains a realistic goal. In this study, an ON-OFF switching of a regenerated biosensor based on a locked nucleic acid (LNA)-integrated and toehold-mediated strand displacement reaction technique is constructed for detection of SNPs. The LNA-integrated and methylene blue-labeled capture probe with an external toehold is designed to switch on the sensing system. The mutant-type DNA probe completes complementary with the capture probe to trigger the strand displacement reaction, which switches off the sensing system. However, when the single-base mismatched wild-type DNA probe is presented, the strand displacement reaction cannot be achieved; therefore, the sensing system still keeps the ON state. This DNA sensor is stable over five reuses. We further testify that the LNA-integrated sequence has better recognition ability for SNPs detection compared to the DNA-integrated sequence. Moreover, this DNA senor exhibits a remarkable discrimination capability of SNPs among abundant wild-type targets and 6000-fold (m/m) excess of genomic DNA. In addition, it is selective enough in complex and contaminant-ridden samples, such as human urine, soil, saliva, and beer. Overall, these results demonstrate that this reliable DNA sensor is easy to be fabricated, simple to operate, and stable enough to be readily regenerated.

Possible role(s) of nuclear matrix and DNA loop organization in fixation or repair of DNA double-strand breaks

International Nuclear Information System (INIS)

Malyapa, R.S.; Wright, W.D.; Roti Roti, J.L.

1995-01-01

DNA double-strand breaks produced by ionizing radiation are considered to be a critical radiation-induced lesion responsible, in part, for cell killing. However, the manner in which structures within the nucleus involving DNA organization contribute to the balance between fixation or repair of these critical lesions remains largely obscure. The repair process requires both functional enzymes and substrate availability, i.e., access to and orientation of damage sites. Therefore, the ability to repair damaged DNA could be influenced not only by DNA integrity but also by the spatial organization of DNA. Therefore, the authors investigated the possibility that radiation-induced DNA damage differentially affects DNA supercoiling ability in cells of differing radiosensitivities using radioresistant and radiosensitive mutants of different origins. This study was also designed to determine if differences in the composition of the nuclear matrix exist between cell lines of each origin. Results from these studies indicate that differences in the composition of the nuclear matrix proteins and DNA stability might be related to intrinsic radiation resistance

Structure determination of uracil-DNA N-glycosylase from Deinococcus radiodurans in complex with DNA.

Science.gov (United States)

Pedersen, Hege Lynum; Johnson, Kenneth A; McVey, Colin E; Leiros, Ingar; Moe, Elin

2015-10-01

Uracil-DNA N-glycosylase (UNG) is a DNA-repair enzyme in the base-excision repair (BER) pathway which removes uracil from DNA. Here, the crystal structure of UNG from the extremophilic bacterium Deinococcus radiodurans (DrUNG) in complex with DNA is reported at a resolution of 1.35 Å. Prior to the crystallization experiments, the affinity between DrUNG and different DNA oligonucleotides was tested by electrophoretic mobility shift assays (EMSAs). As a result of this analysis, two 16 nt double-stranded DNAs were chosen for the co-crystallization experiments, one of which (16 nt AU) resulted in well diffracting crystals. The DNA in the co-crystal structure contained an abasic site (substrate product) flipped into the active site of the enzyme, with no uracil in the active-site pocket. Despite the high resolution, it was not possible to fit all of the terminal nucleotides of the DNA complex into electron density owing to disorder caused by a lack of stabilizing interactions. However, the DNA which was in contact with the enzyme, close to the active site, was well ordered and allowed detailed analysis of the enzyme-DNA interaction. The complex revealed that the interaction between DrUNG and DNA is similar to that in the previously determined crystal structure of human UNG (hUNG) in complex with DNA [Slupphaug et al. (1996). Nature (London), 384, 87-92]. Substitutions in a (here defined) variable part of the leucine loop result in a shorter loop (eight residues instead of nine) in DrUNG compared with hUNG; regardless of this, it seems to fulfil its role and generate a stabilizing force with the minor groove upon flipping out of the damaged base into the active site. The structure also provides a rationale for the previously observed high catalytic efficiency of DrUNG caused by high substrate affinity by demonstrating an increased number of long-range electrostatic interactions between the enzyme and the DNA. Interestingly, specific interactions between residues

Loop kinematics

International Nuclear Information System (INIS)

Migdal, A.A.

1982-01-01

Basic operators acting in the loop space are introduced. The topology of this space and properties of the Stokes type loop functionals are discussed. The parametrically invariant loop calculus developed here is used in the loop dynamics

Replicating animal mitochondrial DNA

Directory of Open Access Journals (Sweden)

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.

Job Displacement and Crime

DEFF Research Database (Denmark)

Bennett, Patrick; Ouazad, Amine

We use a detailed employer-employee data set matched with detailed crime information (timing of crime, fines, convictions, crime type) to estimate the impact of job loss on an individual's probability to commit crime. We focus on job losses due to displacement, i.e. job losses in firms losing...... a substantial share of their workers, for workers with at least three years of tenure. Displaced workers are more likely to commit offenses leading to conviction (probation, prison terms) for property crimes and for alcohol-related traffic violations in the two years following displacement. We find no evidence...... that displaced workers' propensity to commit crime is higher than non-displaced workers before the displacement event; but it is significantly higher afterwards. Displacement impacts crime over and above what is explained by earnings losses and weeks of unemployment following displacement....

Genome-Derived Cytosolic DNA Mediates Type I Interferon-Dependent Rejection of B Cell Lymphoma Cells

Directory of Open Access Journals (Sweden)

Yu J. Shen

2015-04-01

Full Text Available The DNA damage response (DDR induces the expression of type I interferons (IFNs, but the underlying mechanisms are poorly understood. Here, we show the presence of cytosolic DNA in different mouse and human tumor cells. Treatment of cells with genotoxic agents increased the levels of cytosolic DNA in a DDR-dependent manner. Cloning of cytosolic DNA molecules from mouse lymphoma cells suggests that cytosolic DNA is derived from unique genomic loci and has the potential to form non-B DNA structures, including R-loops. Overexpression of Rnaseh1, which resolves R-loops, reduced the levels of cytosolic DNA, type I Ifn transcripts, and type I IFN-dependent rejection of lymphoma cells. Live-cell imaging showed a dynamic contact of cytosolic DNA with mitochondria, an important organelle for innate immune recognition of cytosolic nucleotides. In summary, we found that cytosolic DNA is present in many tumor cells and contributes to the immunogenicity of tumor cells.

Structural and Molecular Basis for Coordination in a Viral DNA Packaging Motor.

Science.gov (United States)

Mao, Huzhang; Saha, Mitul; Reyes-Aldrete, Emilio; Sherman, Michael B; Woodson, Michael; Atz, Rockney; Grimes, Shelley; Jardine, Paul J; Morais, Marc C

2016-03-01

Ring NTPases are a class of ubiquitous molecular motors involved in basic biological partitioning processes. dsDNA viruses encode ring ATPases that translocate their genomes to near-crystalline densities within pre-assembled viral capsids. Here, X-ray crystallography, cryoEM, and biochemical analyses of the dsDNA packaging motor in bacteriophage phi29 show how individual subunits are arranged in a pentameric ATPase ring and suggest how their activities are coordinated to translocate dsDNA. The resulting pseudo-atomic structure of the motor and accompanying functional analyses show how ATP is bound in the ATPase active site; identify two DNA contacts, including a potential DNA translocating loop; demonstrate that a trans-acting arginine finger is involved in coordinating hydrolysis around the ring; and suggest a functional coupling between the arginine finger and the DNA translocating loop. The ability to visualize the motor in action illuminates how the different motor components interact with each other and with their DNA substrate. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

DNA2—An Important Player in DNA Damage Response or Just Another DNA Maintenance Protein?

Directory of Open Access Journals (Sweden)

Elzbieta Pawłowska

2017-07-01

Full Text Available The human DNA2 (DNA replication helicase/nuclease 2 protein is expressed in both the nucleus and mitochondria, where it displays ATPase-dependent nuclease and helicase activities. DNA2 plays an important role in the removing of long flaps in DNA replication and long-patch base excision repair (LP-BER, interacting with the replication protein A (RPA and the flap endonuclease 1 (FEN1. DNA2 can promote the restart of arrested replication fork along with Werner syndrome ATP-dependent helicase (WRN and Bloom syndrome protein (BLM. In mitochondria, DNA2 can facilitate primer removal during strand-displacement replication. DNA2 is involved in DNA double strand (DSB repair, in which it is complexed with BLM, RPA and MRN for DNA strand resection required for homologous recombination repair. DNA2 can be a major protein involved in the repair of complex DNA damage containing a DSB and a 5′ adduct resulting from a chemical group bound to DNA 5′ ends, created by ionizing radiation and several anticancer drugs, including etoposide, mitoxantrone and some anthracyclines. The role of DNA2 in telomere end maintenance and cell cycle regulation suggests its more general role in keeping genomic stability, which is impaired in cancer. Therefore DNA2 can be an attractive target in cancer therapy. This is supported by enhanced expression of DNA2 in many cancer cell lines with oncogene activation and premalignant cells. Therefore, DNA2 can be considered as a potential marker, useful in cancer therapy. DNA2, along with PARP1 inhibition, may be considered as a potential target for inducing synthetic lethality, a concept of killing tumor cells by targeting two essential genes.

Displacement Ventilation

DEFF Research Database (Denmark)

Nielsen, Peter Vilhelm

Displacement ventilation is an interesting new type of air distribution principle which should be considered in connection with design of comfort ventilation in both smal1 and large spaces. Research activities on displacement ventilation are large all over the world and new knowledge of design...... methods appears continuously. This book gives an easy introduction to the basis of displacement ventilation and the chapters are written in the order which is used in a design procedure. The main text is extended by five appendices which show some of the new research activities taking place at Aalborg...

A Biological Micro Actuator: Graded and Closed-Loop Control of Insect Leg Motion by Electrical Stimulation of Muscles

Science.gov (United States)

Cao, Feng; Zhang, Chao; Vo Doan, Tat Thang; Li, Yao; Sangi, Daniyal Haider; Koh, Jie Sheng; Huynh, Ngoc Anh; Aziz, Mohamed Fareez Bin; Choo, Hao Yu; Ikeda, Kazuo; Abbeel, Pieter; Maharbiz, Michel M.; Sato, Hirotaka

2014-01-01

In this study, a biological microactuator was demonstrated by closed-loop motion control of the front leg of an insect (Mecynorrhina torquata, beetle) via electrical stimulation of the leg muscles. The three antagonistic pairs of muscle groups in the front leg enabled the actuator to have three degrees of freedom: protraction/retraction, levation/depression, and extension/flexion. We observed that the threshold amplitude (voltage) required to elicit leg motions was approximately 1.0 V; thus, we fixed the stimulation amplitude at 1.5 V to ensure a muscle response. The leg motions were finely graded by alternation of the stimulation frequencies: higher stimulation frequencies elicited larger leg angular displacement. A closed-loop control system was then developed, where the stimulation frequency was the manipulated variable for leg-muscle stimulation (output from the final control element to the leg muscle) and the angular displacement of the leg motion was the system response. This closed-loop control system, with an optimized proportional gain and update time, regulated the leg to set at predetermined angular positions. The average electrical stimulation power consumption per muscle group was 148 µW. These findings related to and demonstrations of the leg motion control offer promise for the future development of a reliable, low-power, biological legged machine (i.e., an insect–machine hybrid legged robot). PMID:25140875

A biological micro actuator: graded and closed-loop control of insect leg motion by electrical stimulation of muscles.

Directory of Open Access Journals (Sweden)

Feng Cao

Full Text Available In this study, a biological microactuator was demonstrated by closed-loop motion control of the front leg of an insect (Mecynorrhina torquata, beetle via electrical stimulation of the leg muscles. The three antagonistic pairs of muscle groups in the front leg enabled the actuator to have three degrees of freedom: protraction/retraction, levation/depression, and extension/flexion. We observed that the threshold amplitude (voltage required to elicit leg motions was approximately 1.0 V; thus, we fixed the stimulation amplitude at 1.5 V to ensure a muscle response. The leg motions were finely graded by alternation of the stimulation frequencies: higher stimulation frequencies elicited larger leg angular displacement. A closed-loop control system was then developed, where the stimulation frequency was the manipulated variable for leg-muscle stimulation (output from the final control element to the leg muscle and the angular displacement of the leg motion was the system response. This closed-loop control system, with an optimized proportional gain and update time, regulated the leg to set at predetermined angular positions. The average electrical stimulation power consumption per muscle group was 148 µW. These findings related to and demonstrations of the leg motion control offer promise for the future development of a reliable, low-power, biological legged machine (i.e., an insect-machine hybrid legged robot.

Structure of uracil-DNA glycosylase from Mycobacterium tuberculosis: insights into interactions with ligands

International Nuclear Information System (INIS)

Kaushal, Prem Singh; Talawar, Ramappa K.; Varshney, Umesh; Vijayan, M.

2010-01-01

The molecule of uracil-DNA glycosylase from M. tuberculosis exhibits domain motion on binding to DNA or a proteinaceous inhibitor. The highly conserved DNA-binding region interacts with a citrate ion in the structure. Uracil N-glycosylase (Ung) is the most thoroughly studied of the group of uracil DNA-glycosylase (UDG) enzymes that catalyse the first step in the uracil excision-repair pathway. The overall structure of the enzyme from Mycobacterium tuberculosis is essentially the same as that of the enzyme from other sources. However, differences exist in the N- and C-terminal stretches and some catalytic loops. Comparison with appropriate structures indicate that the two-domain enzyme closes slightly when binding to DNA, while it opens slightly when binding to the proteinaceous inhibitor Ugi. The structural changes in the catalytic loops on complexation reflect the special features of their structure in the mycobacterial protein. A comparative analysis of available sequences of the enzyme from different sources indicates high conservation of amino-acid residues in the catalytic loops. The uracil-binding pocket in the structure is occupied by a citrate ion. The interactions of the citrate ion with the protein mimic those of uracil, in addition to providing insights into other possible interactions that inhibitors could be involved in

Soft Pneumatic Bending Actuator with Integrated Carbon Nanotube Displacement Sensor

Directory of Open Access Journals (Sweden)

Tim Giffney

2016-02-01

Full Text Available The excellent compliance and large range of motion of soft actuators controlled by fluid pressure has lead to strong interest in applying devices of this type for biomimetic and human-robot interaction applications. However, in contrast to soft actuators fabricated from stretchable silicone materials, conventional technologies for position sensing are typically rigid or bulky and are not ideal for integration into soft robotic devices. Therefore, in order to facilitate the use of soft pneumatic actuators in applications where position sensing or closed loop control is required, a soft pneumatic bending actuator with an integrated carbon nanotube position sensor has been developed. The integrated carbon nanotube position sensor presented in this work is flexible and well suited to measuring the large displacements frequently encountered in soft robotics. The sensor is produced by a simple soft lithography process during the fabrication of the soft pneumatic actuator, with a greater than 30% resistance change between the relaxed state and the maximum displacement position. It is anticipated that integrated resistive position sensors using a similar design will be useful in a wide range of soft robotic systems.

Simple force balance accelerometer/seismometer based on a tuning fork displacement sensor

International Nuclear Information System (INIS)

Stuart-Watson, D.; Tapson, J.

2004-01-01

Seismometers and microelectromechanical system accelerometers use the force-balance principle to obtain measurements. In these instruments the displacement of a mass object by an unknown force is sensed using a very high-resolution displacement sensor. The position of the object is then stabilized by applying an equal and opposite force to it. The magnitude of the stabilizing force is easily measured, and is assumed to be equivalent to the unknown force. These systems are critically dependent on the displacement sensor. In this article we use a resonant quartz tuning fork as the sensor. The tuning fork is operated so that its oscillation is lightly damped by the proximity of the movable mass object. Changes in the position of the mass object cause changes in the phase of the fork's resonance; this is used as the feedback variable in controlling the mass position. We have developed an acceleration sensor using this principle. The mass object is a piezoelectric bimorph diaphragm which is anchored around its perimeter, allowing direct electronic control of the displacement of its center. The tuning fork is brought very close to the diaphragm center, and is connected into a self-oscillating feedback circuit which has phase and amplitude as outputs. The diaphragm position is adjusted by a feedback loop, using phase as the feedback variable, to keep it in a constant position with respect to the tuning fork. The measured noise for this sensor is approximately 10.0 mg in a bandwidth of 100 Hz, which is substantially better than commercial systems of equivalent cost and size

3D and 1D calculation of hysteresis loops and energy products for anisotropic nanocomposite films with perpendicular anisotropy

International Nuclear Information System (INIS)

Yuan, X.H.; Zhao, G.P.; Yue, Ming; Ye, L.N.; Xia, J.; Zhang, X.C.; Chang, J.

2013-01-01

In this paper, the magnetic reversal process, hysteresis loops and energy products for exchange-coupled Nd 2 Fe 14 B/α-Fe bilayers are studied systematically by a three-dimensional (3D) model. The 3D calculations are numerically solved using the finite difference method, where the results are carefully compared with those calculated by one-dimensional (1D) model. It is found that the calculated hysteresis loops and energy products based on the two methods are consistent with each other. Both nucleation fields and coercivities decrease monotonically as the soft layer thickness L s increases. In addition, the calculated spatial distributions of magnetization orientations in the thickness direction at various applied fields based on both methods signify a three-step magnetic reversal process, which are nucleation, growth and displacement of the domain wall. The calculated magnetic orientations within the film plane, however, are totally different according to the two methods. The 3D calculation exhibits a process of vortex formation and annihilation. On the other hand, the 1D calculation gives a quasi-coherent one, where magnetization orientation is coherent in the film plane and varies in the thickness direction. This new reversal mechanism displayed in the film plane has a systematic influence on the nucleation fields, coercivity and energy products. - Highlights: • Consistent hysteresis loops and energy products for 3D and 1D calculation. • Domain wall formation, evolution and displacement perpendicular to the film plane. • Vortex formation, annihilation and better loop squareness in 3D calculation. • Larger nucleation fields, remanence and smaller coercivity in 3D calculation

Preparation of genomic DNA from a single species of uncultured magnetotactic bacterium by multiple-displacement amplification.

Science.gov (United States)

Arakaki, Atsushi; Shibusawa, Mie; Hosokawa, Masahito; Matsunaga, Tadashi

2010-03-01

Magnetotactic bacteria comprise a phylogenetically diverse group that is capable of synthesizing intracellular magnetic particles. Although various morphotypes of magnetotactic bacteria have been observed in the environment, bacterial strains available in pure culture are currently limited to a few genera due to difficulties in their enrichment and cultivation. In order to obtain genetic information from uncultured magnetotactic bacteria, a genome preparation method that involves magnetic separation of cells, flow cytometry, and multiple displacement amplification (MDA) using phi29 polymerase was used in this study. The conditions for the MDA reaction using samples containing 1 to 100 cells were evaluated using a pure-culture magnetotactic bacterium, "Magnetospirillum magneticum AMB-1," whose complete genome sequence is available. Uniform gene amplification was confirmed by quantitative PCR (Q-PCR) when 100 cells were used as a template. This method was then applied for genome preparation of uncultured magnetotactic bacteria from complex bacterial communities in an aquatic environment. A sample containing 100 cells of the uncultured magnetotactic coccus was prepared by magnetic cell separation and flow cytometry and used as an MDA template. 16S rRNA sequence analysis of the MDA product from these 100 cells revealed that the amplified genomic DNA was from a single species of magnetotactic bacterium that was phylogenetically affiliated with magnetotactic cocci in the Alphaproteobacteria. The combined use of magnetic separation, flow cytometry, and MDA provides a new strategy to access individual genetic information from magnetotactic bacteria in environmental samples.

Two-loop polygon Wilson loops in N = 4 SYM

International Nuclear Information System (INIS)

Anastasiou, C.; Brandhuber, A.; Heslop, P.; Spence, B.; Travaglini, G.; Khoze, V.V.

2009-01-01

We compute for the first time the two-loop corrections to arbitrary n-gon lightlike Wilson loops in N = 4 supersymmetric Yang-Mills theory, using efficient numerical methods. The calculation is motivated by the remarkable agreement between the finite part of planar six-point MHV amplitudes and hexagon Wilson loops which has been observed at two loops. At n = 6 we confirm that the ABDK/BDS ansatz must be corrected by adding a remainder function, which depends only on conformally invariant ratios of kinematic variables. We numerically compute remainder functions for n = 7,8 and verify dual conformal invariance. Furthermore, we study simple and multiple collinear limits of the Wilson loop remainder functions and demonstrate that they have precisely the form required by the collinear factorisation of the corresponding two-loop n-point amplitudes. The number of distinct diagram topologies contributing to the n-gon Wilson loops does not increase with n, and there is a fixed number of 'master integrals', which we have computed. Thus we have essentially computed general polygon Wilson loops, and if the correspondence with amplitudes continues to hold, all planar n-point two-loop MHV amplitudes in the N = 4 theory.

Alternative loop rings

CERN Document Server

Goodaire, EG; Polcino Milies, C

1996-01-01

For the past ten years, alternative loop rings have intrigued mathematicians from a wide cross-section of modern algebra. As a consequence, the theory of alternative loop rings has grown tremendously. One of the main developments is the complete characterization of loops which have an alternative but not associative, loop ring. Furthermore, there is a very close relationship between the algebraic structures of loop rings and of group rings over 2-groups. Another major topic of research is the study of the unit loop of the integral loop ring. Here the interaction between loop rings and group ri

Signal-on electrochemiluminescence biosensor for microRNA-319a detection based on two-stage isothermal strand-displacement polymerase reaction.

Science.gov (United States)

Wang, Minghui; Zhou, Yunlei; Yin, Huanshun; Jiang, Wenjing; Wang, Haiyan; Ai, Shiyun

2018-06-01

MicroRNAs play crucial role in regulating gene expression in organism, thus it is very necessary to exploit an efficient method for the sensitive and specific detection of microRNA. Herein, a signal-on electrochemiluminescence biosensor was fabricated for microRNA-319a detection based on two-stage isothermal strand-displacement polymerase reaction (ISDPR). In the presence of target microRNA, amounts of trigger DNA could be generated by the first ISDPR. Then, the trigger DNA and the primer hybridized simultaneously with the hairpin probe to open the stem of the probe, and then the ECL signal will be emitted. In the presence of phi29 DNA polymerase and dNTPs, the trigger DNA could be displaced to initiate a new cycle which was the second ISDPR. Due to the two-stage amplification, this method presented excellent detection sensitivity with a low detection limit of 0.14 fM. Moreover, the applicability of the developed method was demonstrated by detecting the change of microRNA-319a content in the leaves of rice seedlings after the rice seeds were incubated with chemical mutagen of ethyl methanesulfonate. Copyright © 2018 Elsevier B.V. All rights reserved.

The rolling-circle melting-pot model for porcine circovirus DNA replication

Science.gov (United States)

A stem-loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication (Ori) among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. Porcine circo...

Displacement characteristics of a piezoactuator-based prototype microactuator with a hydraulic displacement amplification system

Energy Technology Data Exchange (ETDEWEB)

Muralidhara [NMAMIT, Nitte (India); Rao, Rathnamala [NITK, Surathkal (India)

2015-11-15

In this study, a new piezoactuator-based prototype microactuator is proposed with a hydraulic displacement amplification system. A piezoactuator is used to deflect a diaphragm which displaces a certain volume of hydraulic fluid into a smaller-diameter piston chamber, thereby amplifying the displacement at the other end of the piston. An electro-mechanical model is implemented to estimate the displacement of a multilayer piezoelectric actuator for the applied input voltage considering the hysteresis behavior. The displacement characteristics of the proposed microactuator are studied for triangular actuation voltage signal. Results of the experiments and simulation of the displacement behavior of the stacked piezoactuator and the amplified displacement of the prototype actuator were compared. Experimental results suggest that the mathematical model developed for the new piezoactuator-based prototype actuator is capable of estimating its displacement behavior accurately, within an error of 1.2%.

Displacement characteristics of a piezoactuator-based prototype microactuator with a hydraulic displacement amplification system

International Nuclear Information System (INIS)

Muralidhara; Rao, Rathnamala

2015-01-01

In this study, a new piezoactuator-based prototype microactuator is proposed with a hydraulic displacement amplification system. A piezoactuator is used to deflect a diaphragm which displaces a certain volume of hydraulic fluid into a smaller-diameter piston chamber, thereby amplifying the displacement at the other end of the piston. An electro-mechanical model is implemented to estimate the displacement of a multilayer piezoelectric actuator for the applied input voltage considering the hysteresis behavior. The displacement characteristics of the proposed microactuator are studied for triangular actuation voltage signal. Results of the experiments and simulation of the displacement behavior of the stacked piezoactuator and the amplified displacement of the prototype actuator were compared. Experimental results suggest that the mathematical model developed for the new piezoactuator-based prototype actuator is capable of estimating its displacement behavior accurately, within an error of 1.2%.

Measuring vulnerability to disaster displacement

Science.gov (United States)

Brink, Susan A.; Khazai, Bijan; Power, Christopher; Wenzel, Friedemann

2015-04-01

Large scale disasters can cause devastating impacts in terms of population displacement. Between 2008 and 2013, on average 27 million people were displaced annually by disasters (Yonetani 2014). After large events such as hurricane Katrina or the Port-au-Prince earthquake, images of inadequate public shelter and concerns about large scale and often inequitable migration have been broadcast around the world. Population displacement can often be one of the most devastating and visible impacts of a natural disaster. Despite the importance of population displacement in disaster events, measures to understand the socio-economic vulnerability of a community often use broad metrics to estimate the total socio-economic risk of an event rather than focusing on the specific impacts that a community faces in a disaster. Population displacement is complex and multi-causal with the physical impact of a disaster interacting with vulnerability arising from the response, environmental issues (e.g., weather), cultural concerns (e.g., expectations of adequate shelter), and many individual factors (e.g., mobility, risk perception). In addition to the complexity of the causes, population displacement is difficult to measure because of the wide variety of different terms and definitions and its multi-dimensional nature. When we speak of severe population displacement, we may refer to a large number of displaced people, an extended length of displacement or associated difficulties such as poor shelter quality, risk of violence and crime in shelter communities, discrimination in aid, a lack of access to employment or other difficulties that can be associated with large scale population displacement. We have completed a thorough review of the literature on disaster population displacement. Research has been conducted on historic events to understand the types of negative impacts associated with population displacement and also the vulnerability of different groups to these impacts. We

An evaluation of multiple annealing and looping based genome amplification using a synthetic bacterial community

KAUST Repository

Wang, Yong; Gao, Zhaoming; Xu, Ying; Li, Guangyu; He, Lisheng; Qian, Peiyuan

2016-01-01

-generation-sequencing technology. Using a synthetic bacterial community, the amplification efficiency of the Multiple Annealing and Looping Based Amplification Cycles (MALBAC) kit that is originally developed to amplify the single-cell genomic DNA of mammalian organisms

The inhibition of anti-DNA binding to DNA by nucleic acid binding polymers.

Directory of Open Access Journals (Sweden)

Nancy A Stearns

Full Text Available Antibodies to DNA (anti-DNA are the serological hallmark of systemic lupus erythematosus (SLE and can mediate disease pathogenesis by the formation of immune complexes. Since blocking immune complex formation can attenuate disease manifestations, the effects of nucleic acid binding polymers (NABPs on anti-DNA binding in vitro were investigated. The compounds tested included polyamidoamine dendrimer, 1,4-diaminobutane core, generation 3.0 (PAMAM-G3, hexadimethrine bromide, and a β-cylodextrin-containing polycation. As shown with plasma from patients with SLE, NABPs can inhibit anti-DNA antibody binding in ELISA assays. The inhibition was specific since the NABPs did not affect binding to tetanus toxoid or the Sm protein, another lupus autoantigen. Furthermore, the polymers could displace antibody from preformed complexes. Together, these results indicate that NABPs can inhibit the formation of immune complexes and may represent a new approach to treatment.

Study of mitochondria D-loop gene to detect the heterogeneity of gemak in Turnicidae family

Science.gov (United States)

Setiati, N.; Partaya

2018-03-01

As a part of life biodiversity, birds in Turnicidae family should be preserved from the extinction and its type heterogeneity decline. One effort for giving the strategic base of plasma nutfah conservation is through genetic heterogeneity study. The aim of the research is to analyze D-loop gen from DNA mitochondria of gemak bird in Turnicidae family molecularly. From the result of the analysis, it may be known the genetic heterogeneity of gemak bird based on the sequence of D-loop gen. The collection of both types of gemak of Turnicidae family is still easy since we can find them in ricefield area after harvest particularly for Gemakloreng (Turnix sylvatica), it means while gemak tegalan (Turnixsusciator) is getting difficult to find. Based on the above DNA quantification standard, the blood sample of Gemak in this research is mostly grouped into pure blood (ranges from 1,63 – 1,90), and it deserves to be used for PCR analysis. The sequencing analysis has not detected the sequence of nucleotide completely. However, it indicates sequence polymorphism of base as the arranger of D-loop gen. D-loop gen may identify genetic heterogeneity of gemak bird of Turnicidae family, but it is necessary to perform further sequencing analysis with PCR-RFLP technique. This complete nucleotide sequence is obtained and easy to detect after being cut restriction enzyme.

Random walk loop soups and conformal loop ensembles

NARCIS (Netherlands)

van de Brug, T.; Camia, F.; Lis, M.

2016-01-01

The random walk loop soup is a Poissonian ensemble of lattice loops; it has been extensively studied because of its connections to the discrete Gaussian free field, but was originally introduced by Lawler and Trujillo Ferreras as a discrete version of the Brownian loop soup of Lawler and Werner, a

Effects of respiration on target and critical structure positions during treatment assessed with movie-loop electronic portal imaging

International Nuclear Information System (INIS)

Herman, Michael G.; Khadivi, Kevin O.; Kleinberg, Lawrence; Gage, Irene; Abrams, Ross A.

1997-01-01

Purpose: To determine the extent of organ and target motion due to patient respiration during chest radiotherapy using electronic portal imaging, to examine these effects on treatment volumes and to show that simulation and treatment port films do not reflect this range of motion. Materials and Methods: Twenty four patients consisting of 17 tangential breast and 7 AP-PA lung field arrangements were imaged during daily radiation treatment. Eight to 10 sequential movie-loop images were acquired during each field of each fraction with a liquid ion chamber electronic portal imaging device (EPID). Motion relative to the reference image was assessed orthogonally to the central axis of the beam. In tangential breast images, cranial, caudad and lateral lung-chest wall landmarks were used; for AP-PA lung, visible tumor, mediastinum and bronchus. Inter and intra-fractional landmark displacements were determined through off-line analysis. Intra-fractional displacements, determined from multiple images within one fraction, indicate motion of the landmark during treatment. Inter-fractional data represents motion between treatment fractions as seen in routine portal film imaging. The effects on treatment volumes were assessed for the largest displacements using the EPID data together with CT reconstruction. Results: The mean, maximum and standard deviation (σ) for observed respiration induced displacements in the cranio-caudad (CC) and lateral directions relative to the beam are summarized both within (intra) and between (inter) fractions: These data indicate that while the mean displacements are small, the standard deviations are significant and the maximum motion observed during a fraction due to respiration may exceed 3 cm in certain cases. In addition, the intra-fractional displacements significantly exceed the inter-fractional displacements, which suggests that anatomical motion is not fully quantified in routine portal imaging. In lung treatments where the largest

Primer-Independent DNA Synthesis by a Family B DNA Polymerase from Self-Replicating Mobile Genetic Elements

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Modesto Redrejo-Rodríguez

2017-11-01

Full Text Available Family B DNA polymerases (PolBs play a central role during replication of viral and cellular chromosomes. Here, we report the discovery of a third major group of PolBs, which we denote primer-independent PolB (piPolB, that might be a link between the previously known protein-primed and RNA/DNA-primed PolBs. PiPolBs are encoded by highly diverse mobile genetic elements, pipolins, integrated in the genomes of diverse bacteria and also present as circular plasmids in mitochondria. Biochemical characterization showed that piPolB displays efficient DNA polymerization activity that can use undamaged and damaged templates and is endowed with proofreading and strand displacement capacities. Remarkably, the protein is also capable of template-dependent de novo DNA synthesis, i.e., DNA-priming activity, thereby breaking the long-standing dogma that replicative DNA polymerases require a pre-existing primer for DNA synthesis. We suggest that piPolBs are involved in self-replication of pipolins and may also contribute to bacterial DNA damage tolerance.

Droplet Microfluidics Approach for Single-DNA Molecule Amplification and Condensation into DNA-Magnesium-Pyrophosphate Particles

Directory of Open Access Journals (Sweden)

Greta Zubaite

2017-02-01

Full Text Available Protein expression in vitro has broad applications in directed evolution, synthetic biology, proteomics and drug screening. However, most of the in vitro expression systems rely on relatively high DNA template concentrations to obtain sufficient amounts of proteins, making it harder to perform in vitro screens on gene libraries. Here, we report a technique for the generation of condensed DNA particles that can serve as efficient templates for in vitro gene expression. We apply droplet microfluidics to encapsulate single-DNA molecules in 3-picoliter (pL volume droplets and convert them into 1 μm-sized DNA particles by the multiple displacement amplification reaction driven by phi29 DNA polymerase. In the presence of magnesium ions and inorganic pyrophosphate, the amplified DNA condensed into the crystalline-like particles, making it possible to purify them from the reaction mix by simple centrifugation. Using purified DNA particles, we performed an in vitro transcription-translation reaction and successfully expressed complex enzyme β-galactosidase in droplets and in the 384-well format. The yield of protein obtained from DNA particles was significantly higher than from the corresponding amount of free DNA templates, thus opening new possibilities for high throughput screening applications.

Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis

NARCIS (Netherlands)

Hamdan, Samir M.; Loparo, Joseph J.; Takahashi, Masateru; Richardson, Charles C.; Oijen, Antoine M. van

2009-01-01

In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to

Resonant neutron-induced atomic displacements

Energy Technology Data Exchange (ETDEWEB)

Elmaghraby, Elsayed K., E-mail: e.m.k.elmaghraby@gmail.com

2017-05-01

Highlights: • Neutron induced atomic displacements was investigated based on scattering of energy of neutron. • Model for cascade function (multiplication of displacements with increasing energy transfer) was proposed and justified. • Parameterizations for the dpa induced in all elements were performed. • Table containing all necessary parameters to calculate the displacement density induced by neutron is given. • Contribution of non resonance displacement and resonant-neutron induced displacements are distinguished. - Abstract: A model for displacement cascade function was modified to account for the continuous variation of displacement density in the material in response to neutron exposure. The model is based on the Gaussian distribution of displacement energies of atoms in a material. Analytical treatment for moderated epithermal neutron field was given in which the displacement density was divided into two terms, discrete-resonance term and continuum term. Calculation are done for all isotopes using ENDF/B VII.1 data files and temperature dependent cross section library. Weighted elemental values were reported a fitting was performed to obtain energy-dependent formula of displacement density and reduce the number of parameters. Results relevant the present specification of the cascade function are tabulated for each element to enable calculation of displacement density at any value of displacement energy in the between 5 eV and 55 eV.

Renormalization of loop functions for all loops

International Nuclear Information System (INIS)

Brandt, R.A.; Neri, F.; Sato, M.

1981-01-01

It is shown that the vacuum expectation values W(C 1 ,xxx, C/sub n/) of products of the traces of the path-ordered phase factors P exp[igcontour-integral/sub C/iA/sub μ/(x)dx/sup μ/] are multiplicatively renormalizable in all orders of perturbation theory. Here A/sub μ/(x) are the vector gauge field matrices in the non-Abelian gauge theory with gauge group U(N) or SU(N), and C/sub i/ are loops (closed paths). When the loops are smooth (i.e., differentiable) and simple (i.e., non-self-intersecting), it has been shown that the generally divergent loop functions W become finite functions W when expressed in terms of the renormalized coupling constant and multiplied by the factors e/sup -K/L(C/sub i/), where K is linearly divergent and L(C/sub i/) is the length of C/sub i/. It is proved here that the loop functions remain multiplicatively renormalizable even if the curves have any finite number of cusps (points of nondifferentiability) or cross points (points of self-intersection). If C/sub γ/ is a loop which is smooth and simple except for a single cusp of angle γ, then W/sub R/(C/sub γ/) = Z(γ)W(C/sub γ/) is finite for a suitable renormalization factor Z(γ) which depends on γ but on no other characteristic of C/sub γ/. This statement is made precise by introducing a regularization, or via a loop-integrand subtraction scheme specified by a normalization condition W/sub R/(C-bar/sub γ/) = 1 for an arbitrary but fixed loop C-bar/sub γ/. Next, if C/sub β/ is a loop which is smooth and simple except for a cross point of angles β, then W(C/sub β/) must be renormalized together with the loop functions of associated sets S/sup i//sub β/ = ]C/sup i/ 1 ,xxx, C/sup i//sub p/i] (i = 2,xxx,I) of loops C/sup i//sub q/ which coincide with certain parts of C/sub β/equivalentC 1 1 . Then W/sub R/(S/sup i//sub β/) = Z/sup i/j(β)W(S/sup j//sub β/) is finite for a suitable matrix Z/sup i/j

Surface-attached molecular beacons light the way for DNA sequencing

Czech Academy of Sciences Publication Activity Database

Paleček, Emil

2004-01-01

Roč. 22, č. 2 (2004), s. 55-58 ISSN 0167-7799 R&D Projects: GA AV ČR IBS5004355; GA ČR GA204/03/0566 Institutional research plan: CEZ:AV0Z5004920 Keywords : molecular beacon * DNA stem-loop structure * DNA sensors Subject RIV: BO - Biophysics Impact factor: 8.606, year: 2004

Studies on the effects of persistent RNA priming on DNA replication and genomic stability

OpenAIRE

Stuckey, Ruth

2014-01-01

[EN]: DNA replication and transcription take place on the same DNA template, and the correct interplay between these processes ensures faithful genome duplication. DNA replication must be highly coordinated with other cell cycle events, such as segregation of fully replicated DNA in order to maintain genomic integrity. Transcription generates RNA:DNA hybrids, transient intermediate structures that are degraded by the ribonuclease H (RNaseH) class of enzymes. RNA:DNA hybrids can form R-loops, ...

Rapid and Simple Detection of Hot Spot Point Mutations of Epidermal Growth Factor Receptor, BRAF, and NRAS in Cancers Using the Loop-Hybrid Mobility Shift Assay

Science.gov (United States)

Matsukuma, Shoichi; Yoshihara, Mitsuyo; Kasai, Fumio; Kato, Akinori; Yoshida, Akira; Akaike, Makoto; Kobayashi, Osamu; Nakayama, Haruhiko; Sakuma, Yuji; Yoshida, Tsutomu; Kameda, Yoichi; Tsuchiya, Eiju; Miyagi, Yohei

2006-01-01

A simple and rapid method to detect the epidermal growth factor receptor hot spot mutation L858R in lung adenocarcinoma was developed based on principles similar to the universal heteroduplex generator technology. A single-stranded oligonucleotide with an internal deletion was used to generate heteroduplexes (loop-hybrids) bearing a loop in the complementary strand derived from the polymerase chain reaction product of the normal or mutant allele. By placing deletion in the oligonucleotide adjacent to the mutational site, difference in electrophoretic mobility between loop-hybrids with normal and mutated DNA was distinguishable in a native polyacrylamide gel. The method was also modified to detect in-frame deletion mutations of epidermal growth factor receptor in lung adenocarcinomas. In addition, the method was adapted to detect hot spot mutations in the B-type Raf kinase (BRAF) at V600 and in a Ras-oncogene (NRAS) at Q61, the mutations commonly found in thyroid carcinomas. Our mutation detection system, designated the loop-hybrid mobility shift assay was sensitive enough to detect mutant DNA comprising 7.5% of the total DNA. As a simple and straightforward mutation detection technique, loop-hybrid mobility shift assay may be useful for the molecular diagnosis of certain types of clinical cancers. Other applications are also discussed. PMID:16931592

A lateral flow biosensor for detection of single nucleotide polymorphism by circular strand displacement reaction.

Science.gov (United States)

Xiao, Zhuo; Lie, Puchang; Fang, Zhiyuan; Yu, Luxin; Chen, Junhua; Liu, Jie; Ge, Chenchen; Zhou, Xuemeng; Zeng, Lingwen

2012-09-04

A lateral flow biosensor for detection of single nucleotide polymorphism based on circular strand displacement reaction (CSDPR) has been developed. Taking advantage of high fidelity of T4 DNA ligase, signal amplification by CSDPR, and the optical properties of gold nanoparticles, this assay has reached a detection limit of 0.01 fM.

Job Displacement and Crime

DEFF Research Database (Denmark)

Bennett, Patrick; Ouazad, Amine

theory of crime. Marital dissolution is more likely post-displacement, and we find small intra-family externalities of adult displacement on younger family members’ crime. The impact of displacement on crime is stronger in municipalities with higher capital and labor income inequalities....

Hierarchically assembled DNA origami tubules with reconfigurable chirality

International Nuclear Information System (INIS)

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

2013-01-01

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

Mitochondrial DNA: An Endogenous Trigger for Immune Paralysis.

Science.gov (United States)

Schäfer, Simon T; Franken, Lars; Adamzik, Michael; Schumak, Beatrix; Scherag, André; Engler, Andrea; Schönborn, Niels; Walden, Jennifer; Koch, Susanne; Baba, Hideo A; Steinmann, Jörg; Westendorf, Astrid M; Fandrey, Joachim; Bieber, Thomas; Kurts, Christian; Frede, Stilla; Peters, Jürgen; Limmer, Andreas

2016-04-01

Critically ill patients are at high risk to suffer from sepsis, even in the absence of an initial infectious source, but the molecular mechanisms for their increased sepsis susceptibility, including a suppressed immune system, remain unclear. Although microbes and pathogen-associated molecular pattern are accepted inducers of sepsis and septic immunosuppression, the role of endogenous Toll-like receptor (TLR) ligands, such as mitochondrial DNA (mtDNA), in altering the immune response is unknown. Mitochondrial DNA serum concentrations of the mitochondrial genes D-Loop and adenosine triphosphatase 6 were determined (quantitative polymerase chain reaction) in 165 septic patients and 50 healthy volunteers. Furthermore, cytotoxic T-cell activity was analyzed in wild-type and TLR9 knockout mice, with/without previous mtDNA administration, followed by injection of an ovalbumin-expressing adenoviral vector. Mitochondrial DNA serum concentrations were increased in septic patients (adenosine triphosphatase 6, 123-fold; D-Loop, 76-fold, P < 0.0001) compared with volunteers. Furthermore, a single mtDNA injection caused profound, TLR9-dependent immunosuppression of adaptive T-cell cytotoxicity in wild-type but not in TLR9 knockout mice and evoked various immunosuppressive mechanisms including the destruction of the splenic microstructure, deletion of cross-presenting dendritic cells, and up-regulation of programmed cell death ligand 1 and indoleamine 2,3-dioxygenase. Several of these findings in mice were mirrored in septic patients, and mtDNA concentrations were associated with an increased 30-day mortality. The findings of this study imply that mtDNA, an endogenous danger associated molecular pattern, is a hitherto unknown inducer of septic immunoparalysis and one possible link between initial inflammation and subsequent immunosuppression in critically ill patients.

Base excision repair of chemotherapeutically-induced alkylated DNA damage predominantly causes contractions of expanded GAA repeats associated with Friedreich's ataxia.

Directory of Open Access Journals (Sweden)

Yanhao Lai

Full Text Available Expansion of GAA·TTC repeats within the first intron of the frataxin gene is the cause of Friedreich's ataxia (FRDA, an autosomal recessive neurodegenerative disorder. However, no effective treatment for the disease has been developed as yet. In this study, we explored a possibility of shortening expanded GAA repeats associated with FRDA through chemotherapeutically-induced DNA base lesions and subsequent base excision repair (BER. We provide the first evidence that alkylated DNA damage induced by temozolomide, a chemotherapeutic DNA damaging agent can induce massive GAA repeat contractions/deletions, but only limited expansions in FRDA patient lymphoblasts. We showed that temozolomide-induced GAA repeat instability was mediated by BER. Further characterization of BER of an abasic site in the context of (GAA20 repeats indicates that the lesion mainly resulted in a large deletion of 8 repeats along with small expansions. This was because temozolomide-induced single-stranded breaks initially led to DNA slippage and the formation of a small GAA repeat loop in the upstream region of the damaged strand and a small TTC loop on the template strand. This allowed limited pol β DNA synthesis and the formation of a short 5'-GAA repeat flap that was cleaved by FEN1, thereby leading to small repeat expansions. At a later stage of BER, the small template loop expanded into a large template loop that resulted in the formation of a long 5'-GAA repeat flap. Pol β then performed limited DNA synthesis to bypass the loop, and FEN1 removed the long repeat flap ultimately causing a large repeat deletion. Our study indicates that chemotherapeutically-induced alkylated DNA damage can induce large contractions/deletions of expanded GAA repeats through BER in FRDA patient cells. This further suggests the potential of developing chemotherapeutic alkylating agents to shorten expanded GAA repeats for treatment of FRDA.

Single Molecule Study of DNA Organization and Recombination

Science.gov (United States)

Xiao, Botao

We have studied five projects related to DNA organization and recombination using mainly single molecule force-spectroscopy and statistical tools. First, HU is one of the most abundant DNA-organizing proteins in bacterial chromosomes and participates in gene regulation. We report experiments that study the dependence of DNA condensation by HU on force, salt and HU concentration. A first important result is that at physiological salt levels, HU only bends DNA, resolving a previous paradox of why a chromosome-compacting protein should have a DNA-stiffening function. A second major result is quantitative demonstration of strong dependencies of HU-DNA dissociation on both salt concentration and force. Second, we have used a thermodynamic Maxwell relation to count proteins driven off large DNAs by tension, an effect important to understanding DNA organization. Our results compare well with estimates of numbers of proteins HU and Fis in previous studies. We have also shown that a semi-flexible polymer model describes our HU experimental data well. The force-dependent binding suggests mechano-chemical mechanisms for gene regulation. Third, the elusive role of protein H1 in chromatin has been clarified with purified H1 and Xenopus extracts. We find that H1 compacts DNA by both bending and looping. Addition of H1 enhances chromatin formation and maintains the plasticity of the chromatin. Fourth, the topology and mechanics of DNA twisting are critical to DNA organization and recombination. We have systematically measured DNA extension as a function of linking number density from 0.08 to -2 with holding forces from 0.2 to 2.4 pN. Unlike previous proposals, the DNA extension decreases with negative linking number. Finally, DNA recombination is a dynamic process starting from enzyme-DNA binding. We report that the Int-DBD domain of lambda integrase binds to DNA without compaction at low Int-DBD concentration. High concentration of Int-DBD loops DNA below a threshold force

Development of Capillary Loop Convective Polymerase Chain Reaction Platform with Real-Time Fluorescence Detection

Directory of Open Access Journals (Sweden)

Wen-Pin Chou

2017-02-01

Full Text Available Polymerase chain reaction (PCR has been one of the principal techniques of molecular biology and diagnosis for decades. Conventional PCR platforms, which work by rapidly heating and cooling the whole vessel, need complicated hardware designs, and cause energy waste and high cost. On the other hand, partial heating on the various locations of vessels to induce convective solution flows by buoyancy have been used for DNA amplification in recent years. In this research, we develop a new convective PCR platform, capillary loop convective polymerase chain reaction (clcPCR, which can generate one direction flow and make the PCR reaction more stable. The U-shaped loop capillaries with 1.6 mm inner diameter are designed as PCR reagent containers. The clcPCR platform utilizes one isothermal heater for heating the bottom of the loop capillary and a CCD device for detecting real-time amplifying fluorescence signals. The stable flow was generated in the U-shaped container and the amplification process could be finished in 25 min. Our experiments with different initial concentrations of DNA templates demonstrate that clcPCR can be applied for precise quantification. Multiple sample testing and real-time quantification will be achieved in future studies.

An amplified graphene oxide-based fluorescence aptasensor based on target-triggered aptamer hairpin switch and strand-displacement polymerization recycling for bioassays.

Science.gov (United States)

Hu, Kun; Liu, Jinwen; Chen, Jia; Huang, Yong; Zhao, Shulin; Tian, Jianniao; Zhang, Guohai

2013-04-15

An amplified graphene oxide (GO) based fluorescence aptasensor based on target-triggered aptamer hairpin switch and strand-displacement polymerization recycling is developed for bioassays. The dye-labeled single-strand DNA (aptamer hairpin) was adsorbed on the surface of GO, which result in the fluorescence quenching of dye, and exhibiting minimal background fluorescence. Upon the target, primer and polymerase, the stem of the aptamer hairpin was opened, and binds with the primer to triggers the circular target strand-displacement polymerization reaction, which produces huge amounts of duplex helixes DNA and lead to strong fluorescence emission due to shielding of nucelobases within its double-helix structure. During the polymerization reaction, the primer was extended, and target was displaced. And the displaced target recognizes and hybridizes with another hairpin probe, triggering the next round of polymerization reaction, and the circle process induces fluorescence signal amplification for the detection of analyte. To test the feasibility of the aptasensor systems, interferon-gamma (IFN-γ) was employed as a model analyte. A detection limit as low as 1.5 fM is obtained based on the GO aptasensor with a linear range of three orders of magnitude. The present method was successfully applied for the detection of IFN-γ in human plasma. Copyright © 2012 Elsevier B.V. All rights reserved.

Dynamic and Progressive Control of DNA Origami Conformation by Modulating DNA Helicity with Chemical Adducts.

Science.gov (United States)

Chen, Haorong; Zhang, Hanyu; Pan, Jing; Cha, Tae-Gon; Li, Shiming; Andréasson, Joakim; Choi, Jong Hyun

2016-05-24

DNA origami has received enormous attention for its ability to program complex nanostructures with a few nanometer precision. Dynamic origami structures that change conformation in response to environmental cues or external signals hold great promises in sensing and actuation at the nanoscale. The reconfiguration mechanism of existing dynamic origami structures is mostly limited to single-stranded hinges and relies almost exclusively on DNA hybridization or strand displacement. Here, we show an alternative approach by demonstrating on-demand conformation changes with DNA-binding molecules, which intercalate between base pairs and unwind DNA double helices. The unwinding effect modulates the helicity mismatch in DNA origami, which significantly influences the internal stress and the global conformation of the origami structure. We demonstrate the switching of a polymerized origami nanoribbon between different twisting states and a well-constrained torsional deformation in a monomeric origami shaft. The structural transformation is shown to be reversible, and binding isotherms confirm the reconfiguration mechanism. This approach provides a rapid and reversible means to change DNA origami conformation, which can be used for dynamic and progressive control at the nanoscale.

Mining protein loops using a structural alphabet and statistical exceptionality

Directory of Open Access Journals (Sweden)

Martin Juliette

2010-02-01

recurrent motifs exhibit a significant level of amino-acid conservation with at least four significant positions and 87% of long loops contain at least one such word. We complement our analysis with the detection of statistically over-represented patterns of structural letters as in conventional DNA sequence analysis. About 30% (930 of structural words are over-represented, and cover about 40% of loop lengths. Interestingly, these words exhibit lower structural variability and higher sequential specificity, suggesting structural or functional constraints. Conclusions We developed a method to systematically decompose and study protein loops using recurrent structural motifs. This method is based on the structural alphabet HMM-SA and not on structural alignment and geometrical parameters. We extracted meaningful structural motifs that are found in both short and long loops. To our knowledge, it is the first time that pattern mining helps to increase the signal-to-noise ratio in protein loops. This finding helps to better describe protein loops and might permit to decrease the complexity of long-loop analysis. Detailed results are available at http://www.mti.univ-paris-diderot.fr/publication/supplementary/2009/ACCLoop/.

Mining protein loops using a structural alphabet and statistical exceptionality.

Science.gov (United States)

Regad, Leslie; Martin, Juliette; Nuel, Gregory; Camproux, Anne-Claude

2010-02-04

amino-acid conservation with at least four significant positions and 87% of long loops contain at least one such word. We complement our analysis with the detection of statistically over-represented patterns of structural letters as in conventional DNA sequence analysis. About 30% (930) of structural words are over-represented, and cover about 40% of loop lengths. Interestingly, these words exhibit lower structural variability and higher sequential specificity, suggesting structural or functional constraints. We developed a method to systematically decompose and study protein loops using recurrent structural motifs. This method is based on the structural alphabet HMM-SA and not on structural alignment and geometrical parameters. We extracted meaningful structural motifs that are found in both short and long loops. To our knowledge, it is the first time that pattern mining helps to increase the signal-to-noise ratio in protein loops. This finding helps to better describe protein loops and might permit to decrease the complexity of long-loop analysis. Detailed results are available at http://www.mti.univ-paris-diderot.fr/publication/supplementary/2009/ACCLoop/.

Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1.

Science.gov (United States)

Gloor, Jason W; Balakrishnan, Lata; Campbell, Judith L; Bambara, Robert A

2012-08-01

In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ∼ 30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation.

Like-charge attraction and opposite-charge decomplexation between polymers and DNA molecules

OpenAIRE

Buyukdagli, Sahin

2016-01-01

We scrutinize the effect of polyvalent ions on polymer-DNA interactions. We extend a recently developed test charge theory to the case of a stiff polymer interacting with a DNA molecule in an electrolyte mixture. The theory accounts for one-loop level electrostatic correlation effects such as the ionic cloud deformation around the strongly charged DNA molecule as well as image-charge forces induced by the low DNA permittivity. Our model can reproduce and explain various characteristics of the...

Intersegmental interactions in supercoiled DNA: atomic force microscope study

Energy Technology Data Exchange (ETDEWEB)

Shlyakhtenko, Luda S.; Miloseska, Lela; Potaman, Vladimir N.; Sinden, Richard R.; Lyubchenko, Yuri L

2003-10-15

Intersegmental interactions in DNA facilitated by the neutralization of electrostatic repulsion was studied as a function of salt concentration and DNA supercoiling. DNA samples with defined superhelical densities were deposited onto aminopropyl mica at different ionic conditions and imaged in air after drying of the samples. Similar to hydrodynamic data, we did not observe a collapse of supercoiled DNA, as proposed earlier by cryo-EM studies. Instead, the formation of the contacts between DNA helices within supercoiled loops with no visible space between the duplexes was observed. The length of such close contacts increased upon increasing NaCl concentration. DNA supercoiling was a critical factor for the stabilization of intersegmental contacts. Implications of the observed effect for understanding DNA compaction in the cell and for regulation DNA transactions via interaction of distantly separated DNA regions are discussed.

Colorimetric tests for diagnosis of filarial infection and vector surveillance using non-instrumented nucleic acid loop-mediated isothermal amplification (NINA-LAMP.

Directory of Open Access Journals (Sweden)

Catherine B Poole

Full Text Available Accurate detection of filarial parasites in humans is essential for the implementation and evaluation of mass drug administration programs to control onchocerciasis and lymphatic filariasis. Determining the infection levels in vector populations is also important for assessing transmission, deciding when drug treatments may be terminated and for monitoring recrudescence. Immunological methods to detect infection in humans are available, however, cross-reactivity issues have been reported. Nucleic acid-based molecular assays offer high levels of specificity and sensitivity, and can be used to detect infection in both humans and vectors. In this study we developed loop-mediated isothermal amplification (LAMP tests to detect three different filarial DNAs in human and insect samples using pH sensitive dyes for enhanced visual detection of amplification. Furthermore, reactions were performed in a portable, non-instrumented nucleic acid amplification (NINA device that provides a stable heat source for LAMP. The efficacy of several strand displacing DNA polymerases were evaluated in combination with neutral red or phenol red dyes. Colorimetric NINA-LAMP assays targeting Brugia Hha I repeat, Onchocerca volvulus GST1a and Wuchereria bancrofti LDR each exhibit species-specificity and are also highly sensitive, detecting DNA equivalent to 1/10-1/5000th of one microfilaria. Reaction times varied depending on whether a single copy gene (70 minutes, O. volvulus or repetitive DNA (40 min, B. malayi and W. bancrofti was employed as a biomarker. The NINA heater can be used to detect multiple infections simultaneously. The accuracy, simplicity and versatility of the technology suggests that colorimetric NINA-LAMP assays are ideally suited for monitoring the success of filariasis control programs.

Rapid, highly sensitive and highly specific gene detection by combining enzymatic amplification and DNA chip detection simultaneously

Directory of Open Access Journals (Sweden)

Koji Hashimoto

2016-05-01

Full Text Available We have developed a novel gene detection method based on the loop-mediated isothermal amplification (LAMP reaction and the DNA dissociation reaction on the same DNA chip surface to achieve a lower detection limit, broader dynamic range and faster detection time than are attainable with a conventional DNA chip. Both FAM- and thiol-labeled DNA probe bound to the complementary sequence accompanying Dabcyl was immobilized on the gold surface via Au/thiol bond. The LAMP reaction was carried out on the DNA probe fixed gold surface. At first, Dabcyl molecules quenched the FAM fluorescence. According to the LAMP reaction, the complementary sequence with Dabcyl was competitively reacted with the amplified targeted sequence. As a result, the FAM fluorescence increased owing to dissociation of the complementary sequence from the DNA probe. The simultaneous reaction of LAMP and DNA chip detection was achieved, and 103 copies of the targeted gene were detected within an hour by measuring fluorescence intensity of the DNA probe. Keywords: Biosensor, DNA chip, Loop-mediated isothermal amplification (LAMP, Fluorescence detection, Gold substrate, Au/thiol bond

Closed loop kinesthetic feedback for postural control rehabilitation.

Science.gov (United States)

Vérité, Fabien; Bachta, Wael; Morel, Guillaume

2014-01-01

Postural control rehabilitation may benefit from the use of smart devices providing biofeedback. This approach consists of increasing the patients perception of their postural state. Namely, postural state is monitored and fed back in real time to the patients through one or more sensory channels. This allows implementing rehabilitation exercises where the patients control their posture with the help of additional sensory inputs. In this paper, a closed loop control of the Center-Of-Pressure (CoP) based on kinesthetic feedback is proposed as a new form of biofeedback. The motion of a one Degree of Freedom (DoF) translational device, lightly touched by the patient's forefinger, is servoed to the patient's CoP position extracted from the measurements of a force plate on which he/she stands. As a result, the patient's CoP can be controllably displaced. A first set of experiments is used to prove the feasibility of this closed-loop control under ideal conditions favoring the perception of the kinesthetic feedback, while the subject is totally unaware of the context. A second set of experiments is then proposed to evaluate the robustness of this approach under experimental conditions that are more realistic with regards to the clinical context of a rehabilitation program involving biofeedback-based exercises.

Two-loop hard-thermal-loop thermodynamics with quarks

International Nuclear Information System (INIS)

Andersen, Jens O.; Petitgirard, Emmanuel; Strickland, Michael

2004-01-01

We calculate the quark contribution to the free energy of a hot quark-gluon plasma to two-loop order using hard-thermal-loop (HTL) perturbation theory. All ultraviolet divergences can be absorbed into renormalizations of the vacuum energy and the HTL quark and gluon mass parameters. The quark and gluon HTL mass parameters are determined self-consistently by a variational prescription. Combining the quark contribution with the two-loop HTL perturbation theory free energy for pure glue we obtain the total two-loop QCD free energy. Comparisons are made with lattice estimates of the free energy for N f =2 and with exact numerical results obtained in the large-N f limit

DNA supercoiling: changes during cellular differentiation and activation of chromatin transcription

International Nuclear Information System (INIS)

Luchnik, A.N.; Bakayev, V.V.; Glaser, V.M.; Moscow State Univ., USSR)

1983-01-01

In this paper it is reported that elastic DNA torsional tension has been observed in a fraction of isolated SV40 minichromosomes, which are shown to be transcriptionally active, and that the number of DNA topological (titratable superhelical) turns in closed superhelical loops of nuclear DNA decreases during cellular differentiation, which, we propose, may be responsible for the coordinate switch in transcription of genes controlling cellular proliferation. 37 references, 6 figures, 2 tables

The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences.

Directory of Open Access Journals (Sweden)

Fabian König

Full Text Available The monoclonal antibody S9.6 is a widely-used tool to purify, analyse and quantify R-loop structures in cells. A previous study using the surface plasmon resonance technology and a single-chain variable fragment (scFv of S9.6 showed high affinity (0.6 nM for DNA-RNA and also a high affinity (2.7 nM for RNA-RNA hybrids. We used the microscale thermophoresis method allowing surface independent interaction studies and electromobility shift assays to evaluate additional RNA-DNA hybrid sequences and to quantify the binding affinities of the S9.6 antibody with respect to distinct sequences and their GC-content. Our results confirm high affinity binding to previously analysed sequences, but reveals that binding affinities are highly sequence specific. Our study presents R-loop sequences that independent of GC-content and in different sequence variations exhibit either no binding, binding affinities in the micromolar range and as well high affinity binding in the nanomolar range. Our study questions the usefulness of the S9.6 antibody in the quantitative analysis of R-loop sequences in vivo.

A study of the effect of helium concentration and displacement damage on the microstructure of helium ion irradiated tungsten

Science.gov (United States)

Harrison, R. W.; Greaves, G.; Hinks, J. A.; Donnelly, S. E.

2017-11-01

Transmission electron microscopy (TEM) with in-situ He ion irradiation has been used to examine the damage microstructure of W when varying the helium concentration to displacement damage ratio, irradiation temperature and total dose. Irradiations employed 15, 60 or 85 keV He ions, at temperatures between 500 and 1000 °C up to doses of ∼3.0 DPA. Once nucleated and grown to an observable size in the TEM, bubble diameter as a function of irradiation dose did not measurably increase at irradiation temperatures of 500 °C between 1.0 and 3.0 DPA; this is attributed to the low mobility of vacancies and He/vacancy complexes at these temperatures. Bubble diameter increased slightly for irradiation temperatures of 750 °C and rapidly increased when irradiated at 1000 °C. Dislocation loops were observed at irradiation temperatures of 500 and 750 °C and no loops were observed at 1000 °C. Burgers vectors of the dislocations were determined to be b = ±½ type only and both vacancy and interstitial loops were observed. The proportion of interstitial loops increased with He-appm/DPA ratio and this is attributed to the concomitant increase in bubble areal density, which reduces the vacancy flux for both the growth of vacancy-type loops and the annihilation of interstitial clusters.

Internal Displacement: Livelihood saving responses

OpenAIRE

Deborah Hines

2001-01-01

Deborah Hines explores how to assist the internally displaced and those prone to displacement. She considers the major causes of internal displacement, making the case for a more comprehensive set of policy and operational actions in response to situations of internal displacement. Development (2001) 44, 34–39. doi:10.1057/palgrave.development.1110289

Replication slippage of the thermophilic DNA polymerases B and D from the Euryarchaeota Pyrococcus abyssi

Directory of Open Access Journals (Sweden)

Melissa G. eCastillo-Lizardo

2014-08-01

Full Text Available Replication slippage or slipped-strand mispairing involves the misalignment of DNA strands during the replication of repeated DNA sequences, and can lead to genetic rearrangements such as microsatellite instability. Here, we show that PolB and PolD replicative DNA polymerases from the archaeal model Pyrococcus abyssi (Pab slip in vitro during replication of a single-stranded DNA template carrying a hairpin structure and short direct repeats. We find that this occurs in both their wild-type (exo+ and exonuclease deficient (exo- forms. The slippage behavior of PabPolB and PabPolD, probably due to limited strand displacement activity, resembles that observed for the high fidelity Pyrococcus furiosus (Pfu DNA polymerase. The presence of PabPCNA inhibited PabPolB and PabPolD slippage. We propose a model whereby PabPCNA stimulates strand displacement activity and polymerase progression through the hairpin, thus permitting the error-free replication of repetitive sequences.

LoopIng: a template-based tool for predicting the structure of protein loops.

KAUST Repository

Messih, Mario Abdel; Lepore, Rosalba; Tramontano, Anna

2015-01-01

) and significant enhancements for long loops (11-20 residues). The quality of the predictions is robust to errors that unavoidably affect the stem regions when these are modeled. The method returns a confidence score for the predicted template loops and has

Time Course of Detection of Human Male DNA from Stained Blood Sample on Various Surfaces by Loop Mediated Isothermal Amplification and Polymerase Chain Reaction

Directory of Open Access Journals (Sweden)

Panan Kanchanaphum

2018-01-01

Full Text Available This study explores determining the sex of humans from blood stains taken from different surfaces and compares the time course of detection with the conventional PCR, Conventional Loop Mediated Isothermal Amplification (LAMP, and LAMP-Lateral Flow Dipstick (LFD. For the DNA templates, 7 male and 7 female blood stained samples were extracted and added to LAMP and PCR reaction solution to amplify the SRY gene. The DNA samples were extracted from the following blood stained materials: cloth, wood, clay, and tile. Then, the samples were stored at room temperature for 1, 7, 30, and 60 day(s. After the DNA amplification, the gel electrophoresis process was applied to detect LAMP product. The LFD was combined with the LAMP to detect LAMP product on the male cloth samples. For the male samples, the time course of detection on the first and seventh days indicated positive for both LAMP and PCR products on all the surfaces while no DNA amplification was found on any of the female samples. On day 30, positive LAMP product was still found on all the male samples. However, it had faded on the tiles. Moreover, all the male samples, which had tested positive for PCR product, were blurred and unclear. On day 60, LAMP product was still found on all the male samples. Conversely, the PCR method resulted in no bands showing for any of the male samples. However, the LAMP-LFD method detected product on all the male samples of cloth. The results show that the LAMP is an effective, practical, and reliable molecular-biological method. Moreover, the LFD can increase the efficiency and sensitivity of the LAMP, making it more suitable for field studies because gel electrophoresis apparatus is not required.

Inheritance of mitochondrial DNA in serially recloned pigs by somatic cell nuclear transfer (SCNT)

Energy Technology Data Exchange (ETDEWEB)

Do, Minhwa; Jang, Won-Gu; Hwang, Jeong Hee; Jang, Hoon; Kim, Eun-Jung; Jeong, Eun-Jeong [Regenerative Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305 806 (Korea, Republic of); Shim, Hosup [Department of Physiology, Dankook University School of Medicine, Cheonan 330 714 (Korea, Republic of); Hwang, Sung Soo; Oh, Keon Bong; Byun, Sung June [Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Suwon (Korea, Republic of); Kim, Jin-Hoi [Department of Animal Biotechnology, Konkuk University, Seoul 143 701 (Korea, Republic of); Lee, Jeong Woong, E-mail: jwlee@kribb.re.kr [Regenerative Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305 806 (Korea, Republic of)

2012-08-10

Highlights: Black-Right-Pointing-Pointer We success serial SCNT through the third generation using pig fibroblasts. Black-Right-Pointing-Pointer Donor-specific mtDNA in the recloned pigs was detected. Black-Right-Pointing-Pointer SCNT affect mtDNA mounts. -- Abstract: Somatic cell nuclear transfer (SCNT) has been established for the transmission of specific nuclear DNA. However, the fate of donor mitochondrial DNA (mtDNA) remains unclear. Here, we examined the fate of donor mtDNA in recloned pigs through third generations. Fibroblasts of recloned pigs were obtained from offspring of each generation produced by fusion of cultured fibroblasts from a Minnesota miniature pig (MMP) into enucleated oocytes of a Landrace pig. The D-loop regions from the mtDNA of donor and recipient differ at nucleotide sequence positions 16050 (A{yields}T), 16062 (T{yields}C), and 16135 (G{yields}A). In order to determine the fate of donor mtDNA in recloned pigs, we analyzed the D-loop region of the donor's mtDNA by allele-specific PCR (AS-PCR) and real-time PCR. Donor mtDNA was successfully detected in all recloned offspring (F1, F2, and F3). These results indicate that heteroplasmy that originate from donor and recipient mtDNA is maintained in recloned pigs, resulting from SCNT, unlike natural reproduction.

Nucleic Acid Analogue Induced Transcription of Double Stranded DNA

DEFF Research Database (Denmark)

1998-01-01

RNA is transcribed from a double stranded DNA template by forming a complex by hybridizing to the template at a desired transcription initiation site one or more oligonucleic acid analogues of the PNA type capable of forming a transcription initiation site with the DNA and exposing the complex...... to the action of a DNA dependant RNA polymerase in the presence of nucleoside triphosphates. Equal length transcripts may be obtained by placing a block to transcription downstream from the initiation site or by cutting the template at such a selected location. The initiation site is formed by displacement...... of one strand of the DNA locally by the PNA hybridization....

A label-free ultrasensitive fluorescence detection of viable Salmonella enteritidis using enzyme-induced cascade two-stage toehold strand-displacement-driven assembly of G-quadruplex DNA.

Science.gov (United States)

Zhang, Peng; Liu, Hui; Ma, Suzhen; Men, Shuai; Li, Qingzhou; Yang, Xin; Wang, Hongning; Zhang, Anyun

2016-06-15

The harm of Salmonella enteritidis (S. enteritidis ) to public health mainly by contaminating fresh food and water emphasizes the urgent need for rapid detection techniques to help control the spread of the pathogen. In this assay, an newly designed capture probe complex that contained specific S. enteritidis-aptamer and hybridized signal target sequence was used for viable S. enteritidis recognition directly. In the presence of the target S. enteritidis, single-stranded target sequences were liberated and initiated the replication-cleavage reaction, producing numerous G-quadruplex structures with a linker on the 3'-end. And then, the sensing system took innovative advantage of quadratic linker-induced strand-displacement for the first time to release target sequence in succession, leading to the cyclic reuse of the target sequences and cascade signal amplification, thereby achieving the successive production of G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binded to these G-quadruplex structures and generated significantly enhanced fluorescent signals to achieve highly sensitive detection of S. enteritidis down to 60 CFU/mL with a linear range from 10(2) to 10(7)CFU/mL. By coupling the cascade two-stage target sequences-recyclable toehold strand-displacement with aptamer-based target recognition successfully, it is the first report on a novel non-label, modification-free and DNA extraction-free ultrasensitive fluorescence biosensor for detecting viable S. enteritidis directly, which can discriminate from dead S. enteritidis. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel mode for transcription inhibition mediated by PNA-induced R-loops with a model in vitro system.

Science.gov (United States)

D'Souza, Alicia D; Belotserkovskii, Boris P; Hanawalt, Philip C

2018-02-01

The selective inhibition of transcription of a chosen gene by an artificial agent has numerous applications. Usually, these agents are designed to bind a specific nucleotide sequence in the promoter or within the transcribed region of the chosen gene. However, since optimal binding sites might not exist within the gene, it is of interest to explore the possibility of transcription inhibition when the agent is designed to bind at other locations. One of these possibilities arises when an additional transcription initiation site (e.g. secondary promoter) is present upstream from the primary promoter of the target gene. In this case, transcription inhibition might be achieved by inducing the formation of an RNA-DNA hybrid (R-loop) upon transcription from the secondary promoter. The R-loop could extend into the region of the primary promoter, to interfere with promoter recognition by RNA polymerase and thereby inhibit transcription. As a sequence-specific R-loop-inducing agent, a peptide nucleic acid (PNA) could be designed to facilitate R-loop formation by sequestering the non-template DNA strand. To investigate this mode for transcription inhibition, we have employed a model system in which a PNA binding site is localized between the T3 and T7 phage RNA polymerase promoters, which respectively assume the roles of primary and secondary promoters. In accord with our model, we have demonstrated that with PNA-bound DNA substrates, transcription from the T7 promoter reduces transcription from the T3 promoter by 30-fold, while in the absence of PNA binding there is no significant effect of T7 transcription upon T3 transcription. Copyright © 2018 Elsevier B.V. All rights reserved.

Gold nanoparticle enhanced fluorescence anisotropy for the assay of single nucleotide polymorphisms (SNPs) based on toehold-mediated strand-displacement reaction.

Science.gov (United States)

Wang, Xinyi; Zou, Mingjian; Huang, Hongduan; Ren, Yuqian; Li, Limei; Yang, Xiaoda; Li, Na

2013-03-15

We developed a highly differentiating, homogeneous gold nanoparticle (AuNP) enhanced fluorescence anisotropic method for single nucleotide polymorphism (SNP) detection at nanomolar level using toehold-mediated strand-displacement reaction. The template strand, containing a toehold domain with an allele-specific site, was immobilized on the surface of AuNPs, and the solution fluorescence anisotropy was markedly enhanced when the fluorescein-labeled blocking DNA was attached to the AuNP via hybridization. Strand-displacement by the target ssDNA strand resulted in detachment of fluorescein-labeled DNA from AuNPs, and thus decreased fluorescence anisotropy. The drastic kinetic difference in strand-displacement from toehold design was used to distinguish between the perfectly matched and the single-base mismatched strands. Free energy changes were calculated to elucidate the dependence of the differentiation ability on the mutation site in the toehold region. A solid negative signal change can be obtained for single-base mismatched strand in the dynamic range of the calibration curve, and a more than 10-fold signal difference can still be observed in a mixed solution containing 100 times the single-base mismatched strand, indicating the good specificity of the method. This proposed method can be performed with a standard spectrofluorimeter in a homogeneous and cost-effective manner, and has the potential to be extended to the application of fluorescence anisotropy method of SNP detection. Copyright © 2012 Elsevier B.V. All rights reserved.

Structure and Variability of the Loop Current along the Yucatan Slope and Shelf Break.

Science.gov (United States)

Sheinbaum, J.; Athie, G.; Candela, J.; Ochoa, J.; Romero, A.

2016-02-01

Yucatan Current and Loop Current variability is investigated using data from an array of moorings that was deployed during 2006-2011 at the western Yucatan Channel and two other strategic cross-sections further north over the Campeche Bank, where the core and western edge of the currents are usually located. Measurements show the cores of the Yucatan Current and Loop Current have a more offshore (onshore) position in summer (winter-spring) suggesting seasonality and a relation to transport variations. Some eastward displacements of the currents are associated with periods of positive horizontal shear (cyclonic vorticity anomalies) propagating northward from the Caribbean coast of Mexico into the Gulf. Ten of the thirteen Loop Current eddies released between 2006 and 2011 were found to be clearly related to these propagating cyclonic anomalies that after crossing the Yucatan Channel produce intense pulses of eddy kinetic energy in the mooring sections downstream. Current structure and variability above and below 1000 m depth have very different characteristics. Diffferences are also found between western and eastern mooring measurements at similar depths. Wind forcing, coastally trapped waves and small scale frontal eddies appear to be the source of this east-west asymmetry.

How to open the treasure chest? Optimising DNA extraction from herbarium specimens.

Science.gov (United States)

Särkinen, Tiina; Staats, Martijn; Richardson, James E; Cowan, Robyn S; Bakker, Freek T

2012-01-01

Herbarium collections are potentially an enormous resource for DNA studies, but the use of herbarium specimens in molecular studies has thus far been slowed down by difficulty in obtaining amplifiable DNA. Here we compare a set of commercially available DNA extraction protocols and their performance in terms of DNA purity and yield, and PCR amplification success as measured by using three differentially sized markers, the rbcL barcoding marker (cpDNA), the LEAFY exon 3 (nrDNA), and the trnL((UAA)) P6 loop (cpDNA). Results reveal large differences between extraction methods, where DNA purity rather than yield is shown to be strongly correlated with PCR success. Amplicon size shows similarly strong correlation with PCR success, with the shortest fragment showing the highest success rate (78%, P6 loop, 10-143 base pairs (bp)) and the largest fragment the lowest success (10%, rbcL, 670 bp). The effect of specimen preparation method on PCR success was also tested. Results show that drying method strongly affects PCR success, especially the availability of fragments longer than 250 bp, where longer fragments are more available for PCR amplification in air dried material compared to alcohol dried specimens. Results from our study indicate that projects relying on poor-quality starting material such as herbarium or scat samples should focus on extracting pure DNA and aim to amplify short target regions (herbarium samples available into barcoding initiatives and other molecular studies.

Nanoliter reactors improve multiple displacement amplification of genomes from single cells.

Directory of Open Access Journals (Sweden)

Yann Marcy

2007-09-01

Full Text Available Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-microl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.

Neocortical electrical stimulation for epilepsy : Closed-loop versus open-loop

NARCIS (Netherlands)

Vassileva, Albena; van Blooijs, Dorien; Leijten, Frans; Huiskamp, Geertjan

2018-01-01

The aim of this review is to evaluate whether open-loop or closed-loop neocortical electrical stimulation should be the preferred approach to manage seizures in intractable epilepsy. Twenty cases of open-loop neocortical stimulation with an implanted device have been reported, in 5 case studies.

Signal replication in a DNA nanostructure

Science.gov (United States)

Mendoza, Oscar; Houmadi, Said; Aimé, Jean-Pierre; Elezgaray, Juan

2017-01-01

Logic circuits based on DNA strand displacement reaction are the basic building blocks of future nanorobotic systems. The circuits tethered to DNA origami platforms present several advantages over solution-phase versions where couplings are always diffusion-limited. Here we consider a possible implementation of one of the basic operations needed in the design of these circuits, namely, signal replication. We show that with an appropriate preparation of the initial state, signal replication performs in a reproducible way. We also show the existence of side effects concomitant to the high effective concentrations in tethered circuits, such as slow leaky reactions and cross-activation.

A universal molecular translator for non-nucleic acid targets that enables dynamic DNA assemblies and logic operations.

Science.gov (United States)

Tang, Wei; Hu, Shichao; Wang, Huaming; Zhao, Yan; Li, Na; Liu, Feng

2014-11-28

A universal molecular translator based on the target-triggered DNA strand displacement was developed, which was able to convert various kinds of non-nucleic acid targets into a unique output DNA. This translation strategy was successfully applied in directing dynamic DNA assemblies and in realizing three-input logic gate operations.

Highly sensitive chemiluminescent point mutation detection by circular strand-displacement amplification reaction.

Science.gov (United States)

Shi, Chao; Ge, Yujie; Gu, Hongxi; Ma, Cuiping

2011-08-15

Single nucleotide polymorphism (SNP) genotyping is attracting extensive attentions owing to its direct connections with human diseases including cancers. Here, we have developed a highly sensitive chemiluminescence biosensor based on circular strand-displacement amplification and the separation by magnetic beads reducing the background signal for point mutation detection at room temperature. This method took advantage of both the T4 DNA ligase recognizing single-base mismatch with high selectivity and the strand-displacement reaction of polymerase to perform signal amplification. The detection limit of this method was 1.3 × 10(-16)M, which showed better sensitivity than that of most of those reported detection methods of SNP. Additionally, the magnetic beads as carrier of immobility was not only to reduce the background signal, but also may have potential apply in high through-put screening of SNP detection in human genome. Copyright © 2011 Elsevier B.V. All rights reserved.

Active Site Sharing and Subterminal Hairpin Recognition in a New Class of DNA Transposases

Energy Technology Data Exchange (ETDEWEB)

Ronning, Donald R.; Guynet, Catherine; Ton-Hoang, Bao; Perez, Zhanita N.; Ghirlando, Rodolfo; Chandler, Michael; Dyda, Fred (Centre Nat); (NIH)

2010-07-20

Many bacteria harbor simple transposable elements termed insertion sequences (IS). In Helicobacter pylori, the chimeric IS605 family elements are particularly interesting due to their proximity to genes encoding gastric epithelial invasion factors. Protein sequences of IS605 transposases do not bear the hallmarks of other well-characterized transposases. We have solved the crystal structure of full-length transposase (TnpA) of a representative member, ISHp608. Structurally, TnpA does not resemble any characterized transposase; rather, it is related to rolling circle replication (RCR) proteins. Consistent with RCR, Mg{sup 2+} and a conserved tyrosine, Tyr127, are essential for DNA nicking and the formation of a covalent intermediate between TnpA and DNA. TnpA is dimeric, contains two shared active sites, and binds two DNA stem loops representing the conserved inverted repeats near each end of ISHp608. The cocrystal structure with stem-loop DNA illustrates how this family of transposases specifically recognizes and pairs ends, necessary steps during transposition.

A multiple-pass ring oscillator based dual-loop phase-locked loop

International Nuclear Information System (INIS)

Chen Danfeng; Ren Junyan; Deng Jingjing; Li Wei; Li Ning

2009-01-01

A dual-loop phase-locked loop (PLL) for wideband operation is proposed. The dual-loop architecture combines a coarse-tuning loop with a fine-tuning one, enabling a wide tuning range and low voltage-controlled oscillator (VCO) gain without poisoning phase noise and reference spur suppression performance. An analysis of the phase noise and reference spur of the dual-loop PLL is emphasized. A novel multiple-pass ring VCO is designed for the dual-loop application. It utilizes both voltage-control and current-control simultaneously in the delay cell. The PLL is fabricated in Jazz 0.18-μm RF CMOS technology. The measured tuning range is from 4.2 to 5.9 GHz. It achieves a low phase noise of -99 dBc/Hz - 1 MHz offset from a 5.5 GHz carrier.

A multiple-pass ring oscillator based dual-loop phase-locked loop

Energy Technology Data Exchange (ETDEWEB)

Chen Danfeng; Ren Junyan; Deng Jingjing; Li Wei; Li Ning, E-mail: dfchen@fudan.edu.c [State Key Laboratory of ASIC and System, Fudan University, Shanghai 201203 (China)

2009-10-15

A dual-loop phase-locked loop (PLL) for wideband operation is proposed. The dual-loop architecture combines a coarse-tuning loop with a fine-tuning one, enabling a wide tuning range and low voltage-controlled oscillator (VCO) gain without poisoning phase noise and reference spur suppression performance. An analysis of the phase noise and reference spur of the dual-loop PLL is emphasized. A novel multiple-pass ring VCO is designed for the dual-loop application. It utilizes both voltage-control and current-control simultaneously in the delay cell. The PLL is fabricated in Jazz 0.18-{mu}m RF CMOS technology. The measured tuning range is from 4.2 to 5.9 GHz. It achieves a low phase noise of -99 dBc/Hz - 1 MHz offset from a 5.5 GHz carrier.

Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3'-to-5' DNA translocase and helicase that prefers to unwind 3'-tailed RNA:DNA hybrids.

Science.gov (United States)

Ordonez, Heather; Shuman, Stewart

2013-05-17

We are interested in the distinctive roster of helicases of Mycobacterium, a genus of the phylum Actinobacteria that includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis Lhr as the exemplar of a novel clade of superfamily II helicases, by virtue of its biochemical specificities and signature domain organization. Lhr is a 1507-amino acid monomeric nucleic acid-dependent ATPase that uses the energy of ATP hydrolysis to drive unidirectional 3'-to-5' translocation along single strand DNA and to unwind duplexes en route. The ATPase is more active in the presence of calcium than magnesium. ATP hydrolysis is triggered by either single strand DNA or single strand RNA, yet the apparent affinity for a DNA activator is 11-fold higher than for an RNA strand of identical size and nucleobase sequence. Lhr is 8-fold better at unwinding an RNA:DNA hybrid than it is at displacing a DNA:DNA duplex of identical nucleobase sequence. The truncated derivative Lhr-(1-856) is an autonomous ATPase, 3'-to-5' translocase, and RNA:DNA helicase. Lhr-(1-856) is 100-fold better RNA:DNA helicase than DNA:DNA helicase. Lhr homologs are found in bacteria representing eight different phyla, being especially prevalent in Actinobacteria (including M. tuberculosis) and Proteobacteria (including Escherichia coli).

Equilibrious Strand Exchange Promoted by DNA Conformational Switching

Science.gov (United States)

Wu, Zhiguo; Xie, Xiao; Li, Puzhen; Zhao, Jiayi; Huang, Lili; Zhou, Xiang

2013-01-01

Most of DNA strand exchange reactions in vitro are based on toehold strategy which is generally nonequilibrium, and intracellular strand exchange mediated by proteins shows little sequence specificity. Herein, a new strand exchange promoted by equilibrious DNA conformational switching is verified. Duplexes containing c-myc sequence which is potentially converted into G-quadruplex are designed in this strategy. The dynamic equilibrium between duplex and G4-DNA is response to the specific exchange of homologous single-stranded DNA (ssDNA). The SER is enzyme free and sequence specific. No ATP is needed and the displaced ssDNAs are identical to the homologous ssDNAs. The SER products and exchange kenetics are analyzed by PAGE and the RecA mediated SER is performed as the contrast. This SER is a new feature of G4-DNAs and a novel strategy to utilize the dynamic equilibrium of DNA conformations.

Sequence-structure relationships in RNA loops: establishing the basis for loop homology modeling.

Science.gov (United States)

Schudoma, Christian; May, Patrick; Nikiforova, Viktoria; Walther, Dirk

2010-01-01

The specific function of RNA molecules frequently resides in their seemingly unstructured loop regions. We performed a systematic analysis of RNA loops extracted from experimentally determined three-dimensional structures of RNA molecules. A comprehensive loop-structure data set was created and organized into distinct clusters based on structural and sequence similarity. We detected clear evidence of the hallmark of homology present in the sequence-structure relationships in loops. Loops differing by structures. Thus, our results support the application of homology modeling for RNA loop model building. We established a threshold that may guide the sequence divergence-based selection of template structures for RNA loop homology modeling. Of all possible sequences that are, under the assumption of isosteric relationships, theoretically compatible with actual sequences observed in RNA structures, only a small fraction is contained in the Rfam database of RNA sequences and classes implying that the actual RNA loop space may consist of a limited number of unique loop structures and conserved sequences. The loop-structure data sets are made available via an online database, RLooM. RLooM also offers functionalities for the modeling of RNA loop structures in support of RNA engineering and design efforts.

Biomimetic Molecular Signaling using DNA Walkers on Microparticles.

Science.gov (United States)

Damase, Tulsi Ram; Spencer, Adam; Samuel, Bamidele; Allen, Peter B

2017-06-22

We report the release of catalytic DNA walkers from hydrogel microparticles and the detection of those walkers by substrate-coated microparticles. This might be considered a synthetic biology analog of molecular signal release and reception. One type of particles was coated with components of a DNA one-step strand displacement (OSD) reaction to release the walker. A second type of particle was coated with substrate (or "track") for the molecular walker. We distinguish these particle types using fluorescence barcoding: we synthesized and distinguished multiple particle types with multicolor fluorescence microscopy and automated image analysis software. This represents a step toward amplified, multiplex, and microscopically localized detection based on DNA nanotechnology.

Localization of ultraviolet-induced excision repair in the nucleus and the distribution of repair events in higher order chromatin loops in mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Mullenders, L.H.F.; Zeeland, A.A. van; Natarajan, A.T.

1987-01-01

Several lines of evidence indicate that eukaryotic DNA is arranged in highly supercoiled domains or loops, and that the repeating loops are constrained by attachment to a nuclear skeletal structure termed the nuclear matrix. We have investigated whether the repair of DNA damage occurs in the nuclear matrix compartment. Normal human fibroblasts, ultraviolet (u.v.)-irradiated with 30 J m/sup -2/ and post-u.v. incubated in the presence of hydroxyurea, did not show any evidence for the occurrence of repair synthesis at the nuclear matrix. 5 J m/sup -2/ repair synthesis seems to initiate at the nuclear matrix, although only part of the total repair could be localized there. In u.v.-irradiated (30 J m/sup -2/) normal human fibroblast post-u.v. incubated in the presence of hydroxyurea and arabinsosylcytosine for 2h, multiple single-stranded regions are generated in a DNA loop as a result of the inhibition of the excision repair process. Preferential repair of certain domains in the chromatin was shown to occur in xeroderma pigmentosum cells of complementation group C (XP-C) in contrast to XP-D cells and Syrian hamster embryonic cells.

Magnetic Actuation of Self-Assembled DNA Hinges

Science.gov (United States)

Lauback, S.; Mattioli, K.; Armstrong, M.; Miller, C.; Pease, C.; Castro, C.; Sooryakumar, R.

DNA nanotechnology offers a broad range of applications spanning from the creation of nanoscale devices, motors and nanoparticle templates to the development of precise drug delivery systems. Central to advancing this technology is the ability to actuate or reconfigure structures in real time, which is currently achieved primarily by DNA strand displacement yielding slow actuation times (about 1-10min). Here we exploit superparamagnetic beads to magnetically actuate DNA structures which also provides a system to measure forces associated with molecular interactions. DNA nanodevices are folded using DNA origami, whereby a long single-stranded DNA is folded into a precise compact geometry using hundreds of short oligonucleotides. Our DNA nanodevice is a nanohinge from which rod shaped DNA nanostructures are polymerized into micron-scale filaments forming handles for actuation. By functionalizing one arm of the hinge and the filament ends, the hinge can be attached to a surface while still allowing an arm to rotate and the filaments can be labeled with magnetic beads enabling the hinge to be actuated almost instantaneously by external magnetic fields. These results lay the groundwork to establish real-time manipulation and direct force application of DNA constructs.

Stress-specific response of the p53-Mdm2 feedback loop

Directory of Open Access Journals (Sweden)

Jensen Mogens H

2010-07-01

Full Text Available Abstract Background The p53 signalling pathway has hundreds of inputs and outputs. It can trigger cellular senescence, cell-cycle arrest and apoptosis in response to diverse stress conditions, including DNA damage, hypoxia and nutrient deprivation. Signals from all these inputs are channeled through a single node, the transcription factor p53. Yet, the pathway is flexible enough to produce different downstream gene expression patterns in response to different stresses. Results We construct a mathematical model of the negative feedback loop involving p53 and its inhibitor, Mdm2, at the core of this pathway, and use it to examine the effect of different stresses that trigger p53. In response to DNA damage, hypoxia, etc., the model exhibits a wide variety of specific output behaviour - steady states with low or high levels of p53 and Mdm2, as well as spiky oscillations with low or high average p53 levels. Conclusions We show that even a simple negative feedback loop is capable of exhibiting the kind of flexible stress-specific response observed in the p53 system. Further, our model provides a framework for predicting the differences in p53 response to different stresses and single nucleotide polymorphisms.

Overproduction of single-stranded-DNA-binding protein specifically inhibits recombination of UV-irradiated bacteriophage DNA in Escherichia coli

International Nuclear Information System (INIS)

Moreau, P.L.

1988-01-01

Overproduction of single-stranded DNA (ssDNA)-binding protein (SSB) in uvr Escherichia coli mutants results in a wide range of altered phenotypes. (i) Cell survival after UV irradiation is decreased; (ii) expression of the recA-lexA regulon is slightly reduced after UV irradiation, whereas it is increased without irradiation; and (iii) recombination of UV-damaged lambda DNA is inhibited, whereas recombination of nonirradiated DNA is unaffected. These results are consistent with the idea that in UV-damaged bacteria, SSB is first required to allow the formation of short complexes of RecA protein and ssDNA that mediate cleavage of the LexA protein. However, in a second stage, SSB should be displaced from ssDNA to permit the production of longer RecA-ssDNA nucleoprotein filaments that are required for strand pairing and, hence, recombinational repair. Since bacteria overproducing SSB appear identical in physiological respects to recF mutant bacteria, it is suggested that the RecF protein (alone or with other proteins of the RecF pathway) may help RecA protein to release SSB from ssDNA

A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily.

Science.gov (United States)

Sang, Pau Biak; Srinath, Thiruneelakantan; Patil, Aravind Goud; Woo, Eui-Jeon; Varshney, Umesh

2015-09-30

Uracil DNA glycosylases (UDGs) are an important group of DNA repair enzymes, which pioneer the base excision repair pathway by recognizing and excising uracil from DNA. Based on two short conserved sequences (motifs A and B), UDGs have been classified into six families. Here we report a novel UDG, UdgX, from Mycobacterium smegmatis and other organisms. UdgX specifically recognizes uracil in DNA, forms a tight complex stable to sodium dodecyl sulphate, 2-mercaptoethanol, urea and heat treatment, and shows no detectable uracil excision. UdgX shares highest homology to family 4 UDGs possessing Fe-S cluster. UdgX possesses a conserved sequence, KRRIH, which forms a flexible loop playing an important role in its activity. Mutations of H in the KRRIH sequence to S, G, A or Q lead to gain of uracil excision activity in MsmUdgX, establishing it as a novel member of the UDG superfamily. Our observations suggest that UdgX marks the uracil-DNA for its repair by a RecA dependent process. Finally, we observed that the tight binding activity of UdgX is useful in detecting uracils in the genomes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The single-strand DNA binding activity of human PC4 preventsmutagenesis and killing by oxidative DNA damage

Energy Technology Data Exchange (ETDEWEB)

Wang, Jen-Yeu; Sarker, Altaf Hossain; Cooper, Priscilla K.; Volkert, Michael R.

2004-02-01

Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Yeast mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub l{Delta} mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair is suggested by the demonstration that Sub1 acts in a peroxide-resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show XPG recruits PC4 to a bubble-containing DNA substrate with resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate.

The use of a displacement device negatively affects the performance of dogs (Canis familiaris) in visible object displacement tasks.

Science.gov (United States)

Müller, Corsin A; Riemer, Stefanie; Range, Friederike; Huber, Ludwig

2014-08-01

Visible and invisible displacement tasks have been used widely for comparative studies of animals' understanding of object permanence, with evidence accumulating that some species can solve invisible displacement tasks and, thus, reach Piagetian stage 6 of object permanence. In contrast, dogs appear to rely on associative cues, such as the location of the displacement device, during invisible displacement tasks. It remains unclear, however, whether dogs, and other species that failed in invisible displacement tasks, do so because of their inability to form a mental representation of the target object, or simply because of the involvement of a more salient but potentially misleading associative cue, the displacement device. Here we show that the use of a displacement device impairs the performance of dogs also in visible displacement tasks: their search accuracy was significantly lower when a visible displacement was performed with a displacement device, and only two of initially 42 dogs passed the sham-baiting control conditions. The negative influence of the displacement device in visible displacement tasks may be explained by strong associative cues overriding explicit information about the target object's location, reminiscent of an overshadowing effect, and/or object individuation errors as the target object is placed within the displacement device and moves along a spatiotemporally identical trajectory. Our data suggest that a comprehensive appraisal of a species' performance in object permanence tasks should include visible displacement tasks with the same displacement device used in invisible displacements, which typically has not been done in the past.

LOOP CALCULUS AND BELIEF PROPAGATION FOR Q-ARY ALPHABET: LOOP TOWER

Energy Technology Data Exchange (ETDEWEB)

CHERTKOV, MICHAEL [Los Alamos National Laboratory; CHERNYAK, VLADIMIR [Los Alamos National Laboratory

2007-01-10

Loop calculus introduced in [1], [2] constitutes a new theoretical tool that explicitly expresses symbol Maximum-A-Posteriori (MAP) solution of a general statistical inference problem via a solution of the Belief Propagation (BP) equations. This finding brought a new significance to the BP concept, which in the past was thought of as just a loop-free approximation. In this paper they continue a discussion of the Loop Calculus, partitioning the results into three Sections. In Section 1 they introduce a new formulation of the Loop Calculus in terms of a set of transformations (gauges) that keeping the partition function of the problem invariant. The full expression contains two terms referred to as the 'ground state' and 'excited states' contributions. The BP equations are interpreted as a special (BP) gauge fixing condition that emerges as a special orthogonality constraint between the ground state and excited states, which also selects loop contributions as the only surviving ones among the excited states. In Section 2 they demonstrate how the invariant interpretation of the Loop Calculus, introduced in Section 1, allows a natural extension to the case of a general q-ary alphabet, this is achieved via a loop tower sequential construction. The ground level in the tower is exactly equivalent to assigning one color (out of q available) to the 'ground state' and considering all 'excited' states colored in the remaining (q-1) colors, according to the loop calculus rule. Sequentially, the second level in the tower corresponds to selecting a loop from the previous step, colored in (q-1) colors, and repeating the same ground vs excited states splitting procedure into one and (q-2) colors respectively. The construction proceeds till the full (q-1)-levels deep loop tower (and the corresponding contributions to the partition function) are established. In Section 3 they discuss an ultimate relation between the loop calculus and the Bethe

Beam stability in synchrotrons with digital filters in the feedback loop of a transverse damper

International Nuclear Information System (INIS)

Zhabitskij, V.M.

2009-01-01

The stability of an ion beam in synchrotrons with digital filters in the feedback loop of a transverse damper is treated. Solving the characteristic equation allows one to calculate the achievable damping rates as a function of instability growth rate, feedback gain and parameters of the signal processing. A transverse feedback system (TFS) is required in synchrotrons to stabilize the high intensity ion beams against transverse instabilities and to damp the beam injection errors. The TFS damper kicker (DK) corrects the transverse momentum of a bunch in proportion to its displacement from the closed orbit at the location of the beam position monitor (BPM). The digital signal processing unit in the feedback loop between BPM and DK ensures a condition to achieve optimal damping. Damping rates of the feedback systems with digital notch, Hilbert and all-pass filters are analyzed in comparison with those in an ideal feedback system

Displacement data assimilation

Energy Technology Data Exchange (ETDEWEB)

Rosenthal, W. Steven [Pacific Northwest Laboratory, Richland, WA 99354 (United States); Venkataramani, Shankar [Department of Mathematics and Program in Applied Mathematics, University of Arizona, Tucson, AZ 85721 (United States); Mariano, Arthur J. [Rosenstiel School of Marine & Atmospheric Science, University of Miami, Miami, FL 33149 (United States); Restrepo, Juan M., E-mail: restrepo@math.oregonstate.edu [Department of Mathematics, Oregon State University, Corvallis, OR 97331 (United States)

2017-02-01

We show that modifying a Bayesian data assimilation scheme by incorporating kinematically-consistent displacement corrections produces a scheme that is demonstrably better at estimating partially observed state vectors in a setting where feature information is important. While the displacement transformation is generic, here we implement it within an ensemble Kalman Filter framework and demonstrate its effectiveness in tracking stochastically perturbed vortices.

Urine sample used for detection of toxoplasma gondii infection by loop-mediated isothermal amplification (LAMP).

Science.gov (United States)

Hu, Xin; Pan, Chang-Wang; Li, Ya-Fei; Wang, Han; Tan, Feng

2012-02-01

In this study, a loop-mediated isothermal amplification (LAMP) assay was established to detect Toxoplasma gondii DNA in mice infected with T. gondii PRU strain. This LAMP assay was based on the sequence of highly repetitive B1 gene. The detection limit of T. gondii LAMP assay was 1 pg of T. gondii DNA, which was evaluated using 10-fold serially diluted DNA of cultured parasites. The LAMP assay was also highly specific for T. gondii and able to detect T. gondii DNA in urine of mice treated with dexamethasone at 90 day post infection (p.i.), although this assay could not detect the DNA in mice urine 2-6 days p.i. These results demonstrated that LAMP is effective for evaluation of therapy effectiveness for T. gondii infection. The established LAMP assay may represent a useful and practical tool for the routine diagnosis and therapeutic evaluation of human toxoplasmosis.

Mixed DNA/Oligo(ethylene glycol) Functionalized Gold Surface Improve DNA Hybridization in Complex Media

International Nuclear Information System (INIS)

Lee, C.; Gamble, L.; Grainger, D.; Castner, D.

2006-01-01

Reliable, direct 'sample-to-answer' capture of nucleic acid targets from complex media would greatly improve existing capabilities of DNA microarrays and biosensors. This goal has proven elusive for many current nucleic acid detection technologies attempting to produce assay results directly from complex real-world samples, including food, tissue, and environmental materials. In this study, we have investigated mixed self-assembled thiolated single-strand DNA (ssDNA) monolayers containing a short thiolated oligo(ethylene glycol) (OEG) surface diluent on gold surfaces to improve the specific capture of DNA targets from complex media. Both surface composition and orientation of these mixed DNA monolayers were characterized with x-ray photoelectron spectroscopy (XPS) and near-edge x-ray absorption fine structure (NEXAFS). XPS results from sequentially adsorbed ssDNA/OEG monolayers on gold indicate that thiolated OEG diluent molecules first incorporate into the thiolated ssDNA monolayer and, upon longer OEG exposures, competitively displace adsorbed ssDNA molecules from the gold surface. NEXAFS polarization dependence results (followed by monitoring the N 1s→π* transition) indicate that adsorbed thiolated ssDNA nucleotide base-ring structures in the mixed ssDNA monolayers are oriented more parallel to the gold surface compared to DNA bases in pure ssDNA monolayers. This supports ssDNA oligomer reorientation towards a more upright position upon OEG mixed adlayer incorporation. DNA target hybridization on mixed ssDNA probe/OEG monolayers was monitored by surface plasmon resonance (SPR). Improvements in specific target capture for these ssDNA probe surfaces due to incorporation of the OEG diluent were demonstrated using two model biosensing assays, DNA target capture from complete bovine serum and from salmon genomic DNA mixtures. SPR results demonstrate that OEG incorporation into the ssDNA adlayer improves surface resistance to both nonspecific DNA and protein

The localization of ultraviolet-induced excision repair in the nucleus and the distribution of repair events in higher order chromatin loops in mammalian cells

International Nuclear Information System (INIS)

Mullenders, L.H.F.; Zeeland, A.A. van; Natarajan, A.T.

1987-01-01

Several lines of evidence indicate that eukaryotic DNA is arranged in highly supercoiled domains or loops, and that the repeating loops are constrained by attachment to a nuclear skeletal structure termed the nuclear matrix. We have investigated whether the repair of DNA damage occurs in the nuclear matrix compartment. Normal human fibroblasts, ultraviolet (u.v.)-irradiated with 30 J m -2 and post-u.v. incubated in the presence of hydroxyurea, did not show any evidence for the occurrence of repair synthesis at the nuclear matrix. 5 J m -2 repair synthesis seems to initiate at the nuclear matrix, although only part of the total repair could be localized there. In u.v.-irradiated (30 J m -2 ) normal human fibroblast post-u.v. incubated in the presence of hydroxyurea and arabinsosylcytosine for 2h, multiple single-stranded regions are generated in a DNA loop as a result of the inhibition of the excision repair process. Preferential repair of certain domains in the chromatin was shown to occur in xeroderma pigmentosum cells of complementation group C (XP-C) in contrast to XP-D cells and Syrian hamster embryonic cells. (author)

Introduction to Loop Heat Pipes

Science.gov (United States)

Ku, Jentung

2015-01-01

This is the presentation file for the short course Introduction to Loop Heat Pipes, to be conducted at the 2015 Thermal Fluids and Analysis Workshop, August 3-7, 2015, Silver Spring, Maryland. This course will discuss operating principles and performance characteristics of a loop heat pipe. Topics include: 1) pressure profiles in the loop; 2) loop operating temperature; 3) operating temperature control; 4) loop startup; 4) loop shutdown; 5) loop transient behaviors; 6) sizing of loop components and determination of fluid inventory; 7) analytical modeling; 8) examples of flight applications; and 9) recent LHP developments.

Displacement cascades in diatomic materials

International Nuclear Information System (INIS)

Parkin, D.M.; Coulter, C.A.

1981-01-01

A new function, the specified-projectile displacement function p/sub ijk/ (E), is introduced to describe displacement cascades in polyatomic materials. This function describes the specific collision events that produce displacements and hence adds new information not previously available. Calculations of p/sub ijk/ (E) for MgO, Al 2 O 3 and TaO are presented and discussed. Results show that the parameters that have the largest effect on displacement collision events are the PKA energy and the mass ratio of the atom types in the material. It is further shown that the microscopic nature of the displacement events changes over the entire recoil energy range relevant to fusion neutron spectra and that these changes are different in materials whose mass ratio is near one than in those where it is far from one

Reactor loops at Chalk River

International Nuclear Information System (INIS)

Sochaski, R.O.

1962-07-01

This report describes broadly the nine in-reactor loops, and their components, located in and around the NRX and NRU reactors at Chalk River. First an introduction and general description is given of the loops and their function, supplemented with a table outlining some loop specifications and nine simplified flow sheets, one for each individual loop. The report then proceeds to classify each loop into two categories, the 'main loop circuit' and the 'auxiliary circuit', and descriptions are given of each circuit's components in turn. These components, in part, are comprised of the main loop pumps, the test section, loop heaters, loop coolers, delayed-neutron monitors, surge tank, Dowtherm coolers, loop piping. Here again photographs, drawings and tables are included to provide a clearer understanding of the descriptive literature and to include, in tables, some specifications of the more important components in each loop. (author)

LNA effects on DNA binding and conformation

DEFF Research Database (Denmark)

Pabon-Martinez, Y Vladimir; Xu, You; Villa, Alessandra

2017-01-01

-substitution in the duplex pyrimidine strand alters the double helix structure, affecting x-displacement, slide and twist favoring triplex formation through enhanced TFO major groove accommodation. Collectively, these findings should facilitate the design of potent anti-gene ONs.......The anti-gene strategy is based on sequence-specific recognition of double-strand DNA by triplex forming (TFOs) or DNA strand invading oligonucleotides to modulate gene expression. To be efficient, the oligonucleotides (ONs) should target DNA selectively, with high affinity. Here we combined...... hybridization analysis and electrophoretic mobility shift assay with molecular dynamics (MD) simulations to better understand the underlying structural features of modified ONs in stabilizing duplex- and triplex structures. Particularly, we investigated the role played by the position and number of locked...

Biomolecular and Structural Analyses of Cauliflower-like DNAs by Ultraviolet, Circular Dichroism, and Fluorescence Spectroscopies in Comparison with Natural DNA

OpenAIRE

Gill, Pooria; Ranjbar, Bijan; Saber, Reza; Khajeh, Khosro; Mohammadian, Mehdi

2011-01-01

Cauliflower-like DNAs are stem-loop DNAs that are fabricated periodically in inverted repetitions from deoxyribonucleic acid phosphates (dNTPs) by loop-mediated isothermal amplification (LAMP). Cauliflower-like DNAs have ladder-shape behaviors on gel electrophoresis, and increasing the time of LAMP leads to multiplying the repetitions, stem-loops, and electrophoretic bands. Cauliflower-like DNAs were fabricated via LAMP using two loop primers, two bumper primers, dNTPs, a λ-phage DNA template...

Development of an optical displacement transducer for CO2 laser auto-focusing

International Nuclear Information System (INIS)

Brown, D.P.D.

1986-01-01

The PCMWP has partially funded the development of an optical displacement transducer by UKAEA Culham Laboratory (Laser Applications Group). This report covers all work which was done up to the end of the 1984/5 financial year. The purpose of the transducer is to sense automatically the standoff gap between the workpiece and a laser cutting head and subsequently to control an auto-focusing head for the CO 2 laser cutting process. Development of the transducer has reached a stage where it can be mounted on an industrial robot and incorporated into a closed loop servo control system so that standoff gap can be closely controlled whilst traversing variable geometry workpieces. The transducer has been shown to be insensitive to angular displacement of the workpiece (within limits) and to workpiece type or surface finish with the exception of transparent materials. Separate trials have shown it to be unaffected by CO 2 laser light and it has been used, with a motorised laser head, to control standoff whilst the laser cuts a range of contoured materials. The scientific principle of the transducer has thus been proved and a laboratory system has been successfully tested. Its performance has matched the specification with the exception of its ability to sense transparent materials. (author)

Combined Discrete Space Voltage Vector with Direct Torque Control for Bearingless Brushless DC Motor and Closed-Loop Suspended Force Control

Directory of Open Access Journals (Sweden)

Weiran Wang

2013-06-01

Full Text Available In order to improve the performance of bearingless brushless DC motor, a closed-loop suspended force controller combining the discrete space voltage vector modulation is applied and the direct torque control is presented in this paper. Firstly, we increase the number of the control vector to reduce the torque ripple. Then, the suspending equation is constructed which is spired by the direct torque control algorithm. As a result, the closed-loop suspended force controller is built. The simulated and experimental results evaluate the performance of the proposed method. The more advantage is that the proposed algorithm can achieve the fast torque response, reduce the torque ripple, and follow ideal stator flux track. Furthermore, the motor which implants the closed-loop suspended force controller cannot onlyobtain the dynamic response rapidly and displacement control accurately, but also has the characteristics of bearingless brushless DC motor (such as simple structure, high energy efficiency, small volume and low failure rate.

Computational prediction of CTCF/cohesin-based intra-TAD loops that insulate chromatin contacts and gene expression in mouse liver.

Science.gov (United States)

Matthews, Bryan J; Waxman, David J

2018-05-14

CTCF and cohesin are key drivers of 3D-nuclear organization, anchoring the megabase-scale Topologically Associating Domains (TADs) that segment the genome. Here, we present and validate a computational method to predict cohesin-and-CTCF binding sites that form intra-TAD DNA loops. The intra-TAD loop anchors identified are structurally indistinguishable from TAD anchors regarding binding partners, sequence conservation, and resistance to cohesin knockdown; further, the intra-TAD loops retain key functional features of TADs, including chromatin contact insulation, blockage of repressive histone mark spread, and ubiquity across tissues. We propose that intra-TAD loops form by the same loop extrusion mechanism as the larger TAD loops, and that their shorter length enables finer regulatory control in restricting enhancer-promoter interactions, which enables selective, high-level expression of gene targets of super-enhancers and genes located within repressive nuclear compartments. These findings elucidate the role of intra-TAD cohesin-and-CTCF binding in nuclear organization associated with widespread insulation of distal enhancer activity. © 2018, Matthews et al.

Distribution of sizes of erased loops for loop-erased random walks

OpenAIRE

Dhar, Deepak; Dhar, Abhishek

1997-01-01

We study the distribution of sizes of erased loops for loop-erased random walks on regular and fractal lattices. We show that for arbitrary graphs the probability $P(l)$ of generating a loop of perimeter $l$ is expressible in terms of the probability $P_{st}(l)$ of forming a loop of perimeter $l$ when a bond is added to a random spanning tree on the same graph by the simple relation $P(l)=P_{st}(l)/l$. On $d$-dimensional hypercubical lattices, $P(l)$ varies as $l^{-\\sigma}$ for large $l$, whe...

Modelling toehold-mediated RNA strand displacement.

Science.gov (United States)

Šulc, Petr; Ouldridge, Thomas E; Romano, Flavio; Doye, Jonathan P K; Louis, Ard A

2015-03-10

We study the thermodynamics and kinetics of an RNA toehold-mediated strand displacement reaction with a recently developed coarse-grained model of RNA. Strand displacement, during which a single strand displaces a different strand previously bound to a complementary substrate strand, is an essential mechanism in active nucleic acid nanotechnology and has also been hypothesized to occur in vivo. We study the rate of displacement reactions as a function of the length of the toehold and temperature and make two experimentally testable predictions: that the displacement is faster if the toehold is placed at the 5' end of the substrate; and that the displacement slows down with increasing temperature for longer toeholds. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

An Extended Surface Loop on Toxoplasma gondii Apical Membrane Antigen 1 (AMA1 Governs Ligand Binding Selectivity.

Directory of Open Access Journals (Sweden)

Michelle L Parker

Full Text Available Apicomplexan parasites are the causative agents of globally prevalent diseases including malaria and toxoplasmosis. These obligate intracellular pathogens have evolved a sophisticated host cell invasion strategy that relies on a parasite-host cell junction anchored by interactions between apical membrane antigens (AMAs on the parasite surface and rhoptry neck 2 (RON2 proteins discharged from the parasite and embedded in the host cell membrane. Key to formation of the AMA1-RON2 complex is displacement of an extended surface loop on AMA1 called the DII loop. While conformational flexibility of the DII loop is required to expose the mature RON2 binding groove, a definitive role of this substructure has not been elucidated. To establish a role of the DII loop in Toxoplasma gondii AMA1, we engineered a form of the protein where the mobile portion of the loop was replaced with a short Gly-Ser linker (TgAMA1ΔDIIloop. Isothermal titration calorimetry measurements with a panel of RON2 peptides revealed an influential role for the DII loop in governing selectivity. Most notably, an Eimeria tenella RON2 (EtRON2 peptide that showed only weak binding to TgAMA1 bound with high affinity to TgAMA1ΔDIIloop. To define the molecular basis for the differential binding, we determined the crystal structure of TgAMA1ΔDIIloop in complex with the EtRON2 peptide. When analyzed in the context of existing AMA1-RON2 structures, spatially distinct anchor points in the AMA1 groove were identified that, when engaged, appear to provide the necessary traction to outcompete the DII loop. Collectively, these data support a model where the AMA1 DII loop serves as a structural gatekeeper to selectively filter out ligands otherwise capable of binding with high affinity in the AMA1 apical groove. These data also highlight the importance of considering the functional implications of the DII loop in the ongoing development of therapeutic intervention strategies targeting the AMA1-RON

Mitochondrial DNA haplogroup phylogeny of the dog: Proposal for a cladistic nomenclature.

Science.gov (United States)

Fregel, Rosa; Suárez, Nicolás M; Betancor, Eva; González, Ana M; Cabrera, Vicente M; Pestano, José

2015-05-01

Canis lupus familiaris mitochondrial DNA analysis has increased in recent years, not only for the purpose of deciphering dog domestication but also for forensic genetic studies or breed characterization. The resultant accumulation of data has increased the need for a normalized and phylogenetic-based nomenclature like those provided for human maternal lineages. Although a standardized classification has been proposed, haplotype names within clades have been assigned gradually without considering the evolutionary history of dog mtDNA. Moreover, this classification is based only on the D-loop region, proven to be insufficient for phylogenetic purposes due to its high number of recurrent mutations and the lack of relevant information present in the coding region. In this study, we design 1) a refined mtDNA cladistic nomenclature from a phylogenetic tree based on complete sequences, classifying dog maternal lineages into haplogroups defined by specific diagnostic mutations, and 2) a coding region SNP analysis that allows a more accurate classification into haplogroups when combined with D-loop sequencing, thus improving the phylogenetic information obtained in dog mitochondrial DNA studies. Copyright © 2015 Elsevier B.V. All rights reserved.

PHOTOSPHERIC PROPERTIES OF WARM EUV LOOPS AND HOT X-RAY LOOPS

Energy Technology Data Exchange (ETDEWEB)

Kano, R. [National Astronomical Observatory of Japan, 2-21-1 Osawa, Mitaka, Tokyo 181-8588 (Japan); Ueda, K. [Department of Astronomy, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Tsuneta, S., E-mail: ryouhei.kano@nao.ac.jp [Institute of Space and Astronautical Science, Japan Aerospace Exploration Agency, 3-1-1 Yoshinodai, Chuo, Sagamihara, Kanagawa 252-5210 (Japan)

2014-02-20

We investigate the photospheric properties (vector magnetic fields and horizontal velocity) of a well-developed active region, NOAA AR 10978, using the Hinode Solar Optical Telescope specifically to determine what gives rise to the temperature difference between ''warm loops'' (1-2 MK), which are coronal loops observed in EUV wavelengths, and ''hot loops'' (>3 MK), coronal loops observed in X-rays. We found that outside sunspots, the magnetic filling factor in the solar network varies with location and is anti-correlated with the horizontal random velocity. If we accept that the observed magnetic features consist of unresolved magnetic flux tubes, this anti-correlation can be explained by the ensemble average of flux-tube motion driven by small-scale random flows. The observed data are consistent with a flux tube width of ∼77 km and horizontal flow at ∼2.6 km s{sup –1} with a spatial scale of ∼120 km. We also found that outside sunspots, there is no significant difference between warm and hot loops either in the magnetic properties (except for the inclination) or in the horizontal random velocity at their footpoints, which are identified with the Hinode X-Ray Telescope and the Transition Region and Coronal Explorer. The energy flux injected into the coronal loops by the observed photospheric motion of the magnetic fields is estimated to be 2 × 10{sup 6} erg s{sup –1} cm{sup –2}, which is the same for both warm and hot loops. This suggests that coronal properties (e.g., loop length) play a more important role in giving rise to temperature differences of active-region coronal loops than photospheric parameters.

Plasmid DNA Delivery: Nanotopography Matters.

Science.gov (United States)

Song, Hao; Yu, Meihua; Lu, Yao; Gu, Zhengying; Yang, Yannan; Zhang, Min; Fu, Jianye; Yu, Chengzhong

2017-12-20

Plasmid DNA molecules with unique loop structures have widespread bioapplications, in many cases relying heavily on delivery vehicles to introduce them into cells and achieve their functions. Herein, we demonstrate that control over delicate nanotopography of silica nanoparticles as plasmid DNA vectors has significant impact on the transfection efficacy. For silica nanoparticles with rambutan-, raspberry-, and flower-like morphologies composed of spike-, hemisphere-, and bowl-type subunit nanotopographies, respectively, the rambutan-like nanoparticles with spiky surfaces demonstrate the highest plasmid DNA binding capability and transfection efficacy of 88%, higher than those reported for silica-based nanovectors. Moreover, it is shown that the surface spikes of rambutan nanoparticles provide a continuous open space to bind DNA chains via multivalent interactions and protect the gene molecules sheltered in the spiky layer against nuclease degradation, exhibiting no significant transfection decay. This unique protection feature is in great contrast to a commercial transfection agent with similar transfection performance but poor protection capability against enzymatic cleavage. Our study provides new understandings in the rational design of nonviral vectors for efficient gene delivery.

Pre-steady-state fluorescence analysis of damaged DNA transfer from human DNA glycosylases to AP endonuclease APE1.

Science.gov (United States)

Kuznetsova, Alexandra A; Kuznetsov, Nikita A; Ishchenko, Alexander A; Saparbaev, Murat K; Fedorova, Olga S

2014-10-01

DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (apurinic/apyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-endonucleases. Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4(cat) and UNG from different structural superfamilies were used. We found that all DNA glycosylases may utilise direct protein-protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1. We hypothesize a fast "flip-flop" exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4(cat), AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions. Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway. Copyright © 2014 Elsevier B.V. All rights reserved.

MRI of displaced meniscal fragments

International Nuclear Information System (INIS)

Dunoski, Brian; Zbojniewicz, Andrew M.; Laor, Tal

2012-01-01

A torn meniscus frequently requires surgical fixation or debridement as definitive treatment. Meniscal tears with associated fragment displacement, such as bucket handle and flap tears, can be difficult to recognize and accurately describe on MRI, and displaced fragments can be challenging to identify at surgery. A displaced meniscal fragment can be obscured by synovium or be in a location not usually evaluated at arthroscopy. We present a pictorial essay of meniscal tears with displaced fragments in patients referred to a pediatric hospital in order to increase recognition and accurate interpretation by the radiologist, who in turn can help assist the surgeon in planning appropriate therapy. (orig.)

MRI of displaced meniscal fragments

Energy Technology Data Exchange (ETDEWEB)

Dunoski, Brian [University of Cincinnati College of Medicine, Department of Radiology, Cincinnati Children' s Hospital Medical Center, Cincinnati, OH (United States); Children' s Hospital of Michigan, Department of Radiology, Detroit, MI (United States); Zbojniewicz, Andrew M.; Laor, Tal [University of Cincinnati College of Medicine, Department of Radiology, Cincinnati Children' s Hospital Medical Center, Cincinnati, OH (United States)

2012-01-15

A torn meniscus frequently requires surgical fixation or debridement as definitive treatment. Meniscal tears with associated fragment displacement, such as bucket handle and flap tears, can be difficult to recognize and accurately describe on MRI, and displaced fragments can be challenging to identify at surgery. A displaced meniscal fragment can be obscured by synovium or be in a location not usually evaluated at arthroscopy. We present a pictorial essay of meniscal tears with displaced fragments in patients referred to a pediatric hospital in order to increase recognition and accurate interpretation by the radiologist, who in turn can help assist the surgeon in planning appropriate therapy. (orig.)

Strip biosensor for amplified detection of nerve growth factor-beta based on a molecular translator and catalytic DNA circuit.

Science.gov (United States)

Liu, Jun; Lai, Ting; Mu, Kejie; Zhou, Zheng

2014-10-07