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Sample records for dna direct chemical

  1. DNA adducts-chemical addons.

    PubMed

    Rajalakshmi, T R; AravindhaBabu, N; Shanmugam, K T; Masthan, K M K

    2015-04-01

    DNA adduct is a piece of DNA covalently bond to a chemical (safrole, benzopyrenediol epoxide, acetaldehyde). This process could be the start of a cancerous cell. When a chemical binds to DNA, it gets damaged resulting in abnormal replication. This could be the start of a mutation and without proper DNA repair, this can lead to cancer. It is this chemical that binds with the DNA is our prime area of concern. Instead of performing the whole body analysis for diagnosing cancer, this test could be carried out for early detection of cancer. When scanning tunneling microscope is used, the DNA results can be obtained earlier. DNA adducts in scientific experiments are used as biomarkers. PMID:26015708

  2. DNA adducts-chemical addons

    PubMed Central

    Rajalakshmi, T. R.; AravindhaBabu, N.; Shanmugam, K. T.; Masthan, K. M. K.

    2015-01-01

    DNA adduct is a piece of DNA covalently bond to a chemical (safrole, benzopyrenediol epoxide, acetaldehyde). This process could be the start of a cancerous cell. When a chemical binds to DNA, it gets damaged resulting in abnormal replication. This could be the start of a mutation and without proper DNA repair, this can lead to cancer. It is this chemical that binds with the DNA is our prime area of concern. Instead of performing the whole body analysis for diagnosing cancer, this test could be carried out for early detection of cancer. When scanning tunneling microscope is used, the DNA results can be obtained earlier. DNA adducts in scientific experiments are used as biomarkers. PMID:26015708

  3. Synthesis of chemically modified DNA.

    PubMed

    Shivalingam, Arun; Brown, Tom

    2016-06-15

    Naturally occurring DNA is encoded by the four nucleobases adenine, cytosine, guanine and thymine. Yet minor chemical modifications to these bases, such as methylation, can significantly alter DNA function, and more drastic changes, such as replacement with unnatural base pairs, could expand its function. In order to realize the full potential of DNA in therapeutic and synthetic biology applications, our ability to 'write' long modified DNA in a controlled manner must be improved. This review highlights methods currently used for the synthesis of moderately long chemically modified nucleic acids (up to 1000 bp), their limitations and areas for future expansion. PMID:27284032

  4. DNA sequencing: chemical methods

    SciTech Connect

    Ambrose, B.J.B.; Pless, R.C.

    1987-01-01

    Limited base-specific or base-selective cleavage of a defined DNA fragment yields polynucleotide products, the length of which correlates with the positions of the particular base (or bases) in the original fragment. Sverdlov and co-workers recognized the possibility of using this principle for the determination of DNA sequences. In 1977 a fully elaborated method was introduced based on this principle, which allowed routine analysis of DNA sequences over distances greater than 100 nucleotide unite from a defined, radiolabeled terminus. Six procedures for partial cleavage were described. Simultaneous parallel resolution of an appropriate set of partial cleavage mixtures by polyacrylamide gel electrophoresis, followed by visualization of the radioactive bands by autoradiography, allows the deduction of nucleotide sequence.

  5. Direct ionization of DNA in solution

    SciTech Connect

    Miller, J.H.; Wilson, W.E.; Ritchie, R.H.

    1993-07-01

    Most of the energy absorbed in the cell nucleus from a radiation field goes into the aqueous medium that surrounds macromolecules, like DNA, which are critical to the normal function of cells. This part of the energy deposition produces numerous reactive species that can diffuse to DNA sequences and induce chemical changes. The average diffusion distance of the free radicals that mediate this indirect mode of DNA damage is only a few nanometers because the cellular medium contains a high concentration of molecules that rapidly scavenge the radiation-induced species. Under these conditions, direct interaction of the radiation field with DNA can not be neglected as a potential mode of damage induction. Two aspects of the direct effect are discussed in this paper: (1) screening of the interaction between DNA and charged particles by the dielectric response of the aqueous medium and (2) the impact-parameter dependence of these interactions.

  6. Recognition and repair of chemically heterogeneous structures at DNA ends.

    PubMed

    Andres, Sara N; Schellenberg, Matthew J; Wallace, Bret D; Tumbale, Percy; Williams, R Scott

    2015-01-01

    Exposure to environmental toxicants and stressors, radiation, pharmaceutical drugs, inflammation, cellular respiration, and routine DNA metabolism all lead to the production of cytotoxic DNA strand breaks. Akin to splintered wood, DNA breaks are not "clean." Rather, DNA breaks typically lack DNA 5'-phosphate and 3'-hydroxyl moieties required for DNA synthesis and DNA ligation. Failure to resolve damage at DNA ends can lead to abnormal DNA replication and repair, and is associated with genomic instability, mutagenesis, neurological disease, ageing and carcinogenesis. An array of chemically heterogeneous DNA termini arises from spontaneously generated DNA single-strand and double-strand breaks (SSBs and DSBs), and also from normal and/or inappropriate DNA metabolism by DNA polymerases, DNA ligases and topoisomerases. As a front line of defense to these genotoxic insults, eukaryotic cells have accrued an arsenal of enzymatic first responders that bind and protect damaged DNA termini, and enzymatically tailor DNA ends for DNA repair synthesis and ligation. These nucleic acid transactions employ direct damage reversal enzymes including Aprataxin (APTX), Polynucleotide kinase phosphatase (PNK), the tyrosyl DNA phosphodiesterases (TDP1 and TDP2), the Ku70/80 complex and DNA polymerase β (POLβ). Nucleolytic processing enzymes such as the MRE11/RAD50/NBS1/CtIP complex, Flap endonuclease (FEN1) and the apurinic endonucleases (APE1 and APE2) also act in the chemical "cleansing" of DNA breaks to prevent genomic instability and disease, and promote progression of DNA- and RNA-DNA damage response (DDR and RDDR) pathways. Here, we provide an overview of cellular first responders dedicated to the detection and repair of abnormal DNA termini. PMID:25111769

  7. Recognition and repair of chemically heterogeneous structures at DNA ends

    PubMed Central

    Andres, Sara N.; Schellenberg, Matthew J.; Wallace, Bret D.; Tumbale, Percy; Williams, R. Scott

    2014-01-01

    Exposure to environmental toxicants and stressors, radiation, pharmaceutical drugs, inflammation, cellular respiration, and routine DNA metabolism all lead to the production of cytotoxic DNA strand breaks. Akin to splintered wood, DNA breaks are not “clean”. Rather, DNA breaks typically lack DNA 5'-phosphate and 3'-hydroxyl moieties required for DNA synthesis and DNA ligation. Failure to resolve damage at DNA ends can lead to abnormal DNA replication and repair, and is associated with genomic instability, mutagenesis, neurological disease, ageing and carcinogenesis. An array of chemically heterogeneous DNA termini arises from spontaneously generated DNA single-strand and double-strand breaks (SSBs and DSBs), and also from normal and/or inappropriate DNA metabolism by DNA polymerases, DNA ligases and topoisomerases. As a front line of defense to these genotoxic insults, eukaryotic cells have accrued an arsenal of enzymatic first responders that bind and protect damaged DNA termini, and enzymatically tailor DNA ends for DNA repair synthesis and ligation. These nucleic acid transactions employ direct damage reversal enzymes including Aprataxin (APTX), Polynucleotide kinase phosphatase (PNK), the tyrosyl DNA phosphodiesterases (TDP1 and TDP2), the Ku70/80 complex and DNA polymerase β (POLβ). Nucleolytic processing enzymes such as the MRE11/RAD50/NBS1/CtIP complex, Flap endonuclease (FEN1) and the apurinic endonucleases (APE1 and APE2) also act in the chemical "cleansing" of DNA breaks to prevent genomic instability and disease, and promote progression of DNA- and RNA-DNA damage response (DDR and RDDR) pathways. Here, we provide an overview of cellular first responders dedicated to the detection and repair of abnormal DNA termini. PMID:25111769

  8. Chemical approaches to DNA nanotechnology.

    PubMed

    Endo, Masayuki; Sugiyama, Hiroshi

    2009-10-12

    Due to its self-assembling nature, DNA is undoubtedly an excellent molecule for the creation of various multidimensional nanostructures and the placement of functional molecules and materials. DNA molecules behave according to the programs of their sequences. Mixtures of numbers of DNA molecules can be placed precisely and organized into single structures to form nanoarchitectures. Once the appropriate sequences for the target nanostructure are established, the predesigned structure can be built up by self-assembly of the designed DNA strands. DNA nanotechnology has already reached the stage at which the organization of desired functional molecules and nanomaterials can be programmed on a defined DNA scaffold. In this review, we will focus on DNA nanotechnology and describe the potential of synthetic chemistry to contribute to the further development of DNA nanomaterials. PMID:19714700

  9. RNA-directed DNA methylation in plants.

    PubMed

    Movahedi, Ali; Sun, Weibu; Zhang, Jiaxin; Wu, Xiaolong; Mousavi, Mohaddesseh; Mohammadi, Kourosh; Yin, Tongming; Zhuge, Qiang

    2015-11-01

    In plants, many small interfering RNAs (siRNAs) direct de novo methylation by DNA methyltransferase. DNA methylation typically occurs by RNA-directed DNA methylation (RdDM), which directs transcriptional gene silencing of transposons and endogenous transgenes. RdDM is driven by non-coding RNAs (ncRNAs) produced by DNA-dependent RNA polymerases IV and V (PolIV and PolV). The production of siRNAs is initiated by PolIV and ncRNAs produced by PolIV are precursors of 24-nucleotide siRNAs. In contrast, ncRNAs produced by PolV are involved in scaffolding RNAs. In this review, we summarize recent studies of RdDM. In particular, we focus on the mechanisms involved in chromatin remodeling by PolIV and PolV. PMID:26183954

  10. The structure of DNA by direct imaging

    PubMed Central

    Marini, Monica; Falqui, Andrea; Moretti, Manola; Limongi, Tania; Allione, Marco; Genovese, Alessandro; Lopatin, Sergei; Tirinato, Luca; Das, Gobind; Torre, Bruno; Giugni, Andrea; Gentile, Francesco; Candeloro, Patrizio; Di Fabrizio, Enzo

    2015-01-01

    The structure of DNA was determined in 1953 by x-ray fiber diffraction. Several attempts have been made to obtain a direct image of DNA with alternative techniques. The direct image is intended to allow a quantitative evaluation of all relevant characteristic lengths present in a molecule. A direct image of DNA, which is different from diffraction in the reciprocal space, is difficult to obtain for two main reasons: the intrinsic very low contrast of the elements that form the molecule and the difficulty of preparing the sample while preserving its pristine shape and size. We show that through a preparation procedure compatible with the DNA physiological conditions, a direct image of a single suspended DNA molecule can be obtained. In the image, all relevant lengths of A-form DNA are measurable. A high-resolution transmission electron microscope that operates at 80 keV with an ultimate resolution of 1.5 Å was used for this experiment. Direct imaging of a single molecule can be used as a method to address biological problems that require knowledge at the single-molecule level, given that the average information obtained by x-ray diffraction of crystals or fibers is not sufficient for detailed structure determination, or when crystals cannot be obtained from biological molecules or are not sufficient in understanding multiple protein configurations. PMID:26601243

  11. The structure of DNA by direct imaging.

    PubMed

    Marini, Monica; Falqui, Andrea; Moretti, Manola; Limongi, Tania; Allione, Marco; Genovese, Alessandro; Lopatin, Sergei; Tirinato, Luca; Das, Gobind; Torre, Bruno; Giugni, Andrea; Gentile, Francesco; Candeloro, Patrizio; Di Fabrizio, Enzo

    2015-08-01

    The structure of DNA was determined in 1953 by x-ray fiber diffraction. Several attempts have been made to obtain a direct image of DNA with alternative techniques. The direct image is intended to allow a quantitative evaluation of all relevant characteristic lengths present in a molecule. A direct image of DNA, which is different from diffraction in the reciprocal space, is difficult to obtain for two main reasons: the intrinsic very low contrast of the elements that form the molecule and the difficulty of preparing the sample while preserving its pristine shape and size. We show that through a preparation procedure compatible with the DNA physiological conditions, a direct image of a single suspended DNA molecule can be obtained. In the image, all relevant lengths of A-form DNA are measurable. A high-resolution transmission electron microscope that operates at 80 keV with an ultimate resolution of 1.5 Å was used for this experiment. Direct imaging of a single molecule can be used as a method to address biological problems that require knowledge at the single-molecule level, given that the average information obtained by x-ray diffraction of crystals or fibers is not sufficient for detailed structure determination, or when crystals cannot be obtained from biological molecules or are not sufficient in understanding multiple protein configurations. PMID:26601243

  12. DNA-Encoded Dynamic Combinatorial Chemical Libraries.

    PubMed

    Reddavide, Francesco V; Lin, Weilin; Lehnert, Sarah; Zhang, Yixin

    2015-06-26

    Dynamic combinatorial chemistry (DCC) explores the thermodynamic equilibrium of reversible reactions. Its application in the discovery of protein binders is largely limited by difficulties in the analysis of complex reaction mixtures. DNA-encoded chemical library (DECL) technology allows the selection of binders from a mixture of up to billions of different compounds; however, experimental results often show low a signal-to-noise ratio and poor correlation between enrichment factor and binding affinity. Herein we describe the design and application of DNA-encoded dynamic combinatorial chemical libraries (EDCCLs). Our experiments have shown that the EDCCL approach can be used not only to convert monovalent binders into high-affinity bivalent binders, but also to cause remarkably enhanced enrichment of potent bivalent binders by driving their in situ synthesis. We also demonstrate the application of EDCCLs in DNA-templated chemical reactions. PMID:26014116

  13. DNA Damage, Homology-Directed Repair, and DNA Methylation

    PubMed Central

    Angrisano, Tiziana; Morano, Annalisa; Lee, Bongyong; Pardo, Alba Di; Messina, Samantha; Iuliano, Rodolfo; Fusco, Alfredo; Santillo, Maria R; Muller, Mark T; Chiariotti, Lorenzo; Gottesman, Max E; Avvedimento, Enrico V

    2007-01-01

    To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments. PMID:17616978

  14. T7 replisome directly overcomes DNA damage

    PubMed Central

    Sun, Bo; Pandey, Manjula; Inman, James T.; Yang, Yi; Kashlev, Mikhail; Patel, Smita S.; Wang, Michelle D.

    2015-01-01

    Cells and viruses possess several known ‘restart' pathways to overcome lesions during DNA replication. However, these ‘bypass' pathways leave a gap in replicated DNA or require recruitment of accessory proteins, resulting in significant delays to fork movement or even cell division arrest. Using single-molecule and ensemble methods, we demonstrate that the bacteriophage T7 replisome is able to directly replicate through a leading-strand cyclobutane pyrimidine dimer (CPD) lesion. We show that when a replisome encounters the lesion, a substantial fraction of DNA polymerase (DNAP) and helicase stay together at the lesion, the replisome does not dissociate and the helicase does not move forward on its own. The DNAP is able to directly replicate through the lesion by working in conjunction with helicase through specific helicase–DNAP interactions. These observations suggest that the T7 replisome is fundamentally permissive of DNA lesions via pathways that do not require fork adjustment or replisome reassembly. PMID:26675048

  15. T7 replisome directly overcomes DNA damage

    NASA Astrophysics Data System (ADS)

    Sun, Bo; Pandey, Manjula; Inman, James T.; Yang, Yi; Kashlev, Mikhail; Patel, Smita S.; Wang, Michelle D.

    2015-12-01

    Cells and viruses possess several known `restart' pathways to overcome lesions during DNA replication. However, these `bypass' pathways leave a gap in replicated DNA or require recruitment of accessory proteins, resulting in significant delays to fork movement or even cell division arrest. Using single-molecule and ensemble methods, we demonstrate that the bacteriophage T7 replisome is able to directly replicate through a leading-strand cyclobutane pyrimidine dimer (CPD) lesion. We show that when a replisome encounters the lesion, a substantial fraction of DNA polymerase (DNAP) and helicase stay together at the lesion, the replisome does not dissociate and the helicase does not move forward on its own. The DNAP is able to directly replicate through the lesion by working in conjunction with helicase through specific helicase-DNAP interactions. These observations suggest that the T7 replisome is fundamentally permissive of DNA lesions via pathways that do not require fork adjustment or replisome reassembly.

  16. Chemical Information: Print. Directed Study.

    ERIC Educational Resources Information Center

    Stone, Catherine C.

    This report provides a survey and evaluation of chemical information literature. Contained in this survey are an overview of the chemical literature field, comments on obtaining access to this literature and annotated bibliographies of primary, secondary, and tertiary sources as well as special topics. Primary sources include journals, patents,…

  17. Programmable chemical controllers made from DNA.

    PubMed

    Chen, Yuan-Jyue; Dalchau, Neil; Srinivas, Niranjan; Phillips, Andrew; Cardelli, Luca; Soloveichik, David; Seelig, Georg

    2013-10-01

    Biological organisms use complex molecular networks to navigate their environment and regulate their internal state. The development of synthetic systems with similar capabilities could lead to applications such as smart therapeutics or fabrication methods based on self-organization. To achieve this, molecular control circuits need to be engineered to perform integrated sensing, computation and actuation. Here we report a DNA-based technology for implementing the computational core of such controllers. We use the formalism of chemical reaction networks as a 'programming language' and our DNA architecture can, in principle, implement any behaviour that can be mathematically expressed as such. Unlike logic circuits, our formulation naturally allows complex signal processing of intrinsically analogue biological and chemical inputs. Controller components can be derived from biologically synthesized (plasmid) DNA, which reduces errors associated with chemically synthesized DNA. We implement several building-block reaction types and then combine them into a network that realizes, at the molecular level, an algorithm used in distributed control systems for achieving consensus between multiple agents. PMID:24077029

  18. Programmable chemical controllers made from DNA

    NASA Astrophysics Data System (ADS)

    Chen, Yuan-Jyue; Dalchau, Neil; Srinivas, Niranjan; Phillips, Andrew; Cardelli, Luca; Soloveichik, David; Seelig, Georg

    2013-10-01

    Biological organisms use complex molecular networks to navigate their environment and regulate their internal state. The development of synthetic systems with similar capabilities could lead to applications such as smart therapeutics or fabrication methods based on self-organization. To achieve this, molecular control circuits need to be engineered to perform integrated sensing, computation and actuation. Here we report a DNA-based technology for implementing the computational core of such controllers. We use the formalism of chemical reaction networks as a 'programming language' and our DNA architecture can, in principle, implement any behaviour that can be mathematically expressed as such. Unlike logic circuits, our formulation naturally allows complex signal processing of intrinsically analogue biological and chemical inputs. Controller components can be derived from biologically synthesized (plasmid) DNA, which reduces errors associated with chemically synthesized DNA. We implement several building-block reaction types and then combine them into a network that realizes, at the molecular level, an algorithm used in distributed control systems for achieving consensus between multiple agents.

  19. Programmable chemical controllers made from DNA

    PubMed Central

    Chen, Yuan-Jyue; Dalchau, Neil; Srinivas, Niranjan; Phillips, Andrew; Cardelli, Luca; Soloveichik, David; Seelig, Georg

    2014-01-01

    Biological organisms use complex molecular networks to navigate their environment and regulate their internal state. The development of synthetic systems with similar capabilities could lead to applications such as smart therapeutics or fabrication methods based on self-organization. To achieve this, molecular control circuits need to be engineered to perform integrated sensing, computation and actuation. Here we report a DNA-based technology for implementing the computational core of such controllers. We use the formalism of chemical reaction networks as a 'programming language', and our DNA architecture can, in principle, implement any behaviour that can be mathematically expressed as such. Unlike logic circuits, our formulation naturally allows complex signal processing of intrinsically analogue biological and chemical inputs. Controller components can be derived from biologically synthesized (plasmid) DNA, which reduces errors associated with chemically synthesized DNA. We implement several building-block reaction types and then combine them into a network that realizes, at the molecular level, an algorithm used in distributed control systems for achieving consensus between multiple agents. PMID:24077029

  20. Robust 3D DNA FISH using directly labeled probes.

    PubMed

    Bolland, Daniel J; King, Michelle R; Reik, Wolf; Corcoran, Anne E; Krueger, Christel

    2013-01-01

    3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions. PMID:23978815

  1. Robust 3D DNA FISH Using Directly Labeled Probes

    PubMed Central

    Bolland, Daniel J.; King, Michelle R.; Reik, Wolf; Corcoran, Anne E.; Krueger, Christel

    2013-01-01

    3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions. PMID:23978815

  2. Determining orientation and direction of DNA sequences

    DOEpatents

    Goodwin, Edwin H.; Meyne, Julianne

    2000-01-01

    Determining orientation and direction of DNA sequences. A method by which fluorescence in situ hybridization can be made strand specific is described. Cell cultures are grown in a medium containing a halogenated nucleotide. The analog is partially incorporated in one DNA strand of each chromatid. This substitution takes place in opposite strands of the two sister chromatids. After staining with the fluorescent DNA-binding dye Hoechst 33258, cells are exposed to long-wavelength ultraviolet light which results in numerous strand nicks. These nicks enable the substituted strand to be denatured and solubilized by heat, treatment with high or low pH aqueous solutions, or by immersing the strands in 2.times.SSC (0.3M NaCl+0.03M sodium citrate), to name three procedures. It is unnecessary to enzymatically digest the strands using Exo III or another exonuclease in order to excise and solubilize nucleotides starting at the sites of the nicks. The denaturing/solubilizing process removes most of the substituted strand while leaving the prereplication strand largely intact. Hybridization of a single-stranded probe of a tandem repeat arranged in a head-to-tail orientation will result in hybridization only to the chromatid with the complementary strand present.

  3. The fate of the chemical warfare agent during DNA extraction.

    PubMed

    Wilkinson, Della A; Hulst, Albert G; de Reuver, Leo P J; van Krimpen, Simon H; van Baar, Ben M L

    2007-11-01

    Forensic laboratories do not have the infrastructure to process or store contaminated DNA samples that have been recovered from a crime scene contaminated with chemical or biological warfare agents. Previous research has shown that DNA profiles can be recovered from blood exposed to several chemical warfare agents after the agent has been removed. The fate of four toxic agents, sulfur mustard, sodium 2-fluoroacetate, sarin, and diazinon, in a lysis buffer used in Promega DNA IQ extraction protocol was studied to determine if extraction would render the samples safe. Two independent analytical methods were used per agent, selected from GC-MS, 1H NMR, 19F NMR, (31)P NMR, or LC-ES MS. The methods were validated before use. Determinations were carried out in a semi-quantitative way, by direct comparison to standards. Agent levels in the elution buffer were found to be below the detectable limits for mustard, sarin, sodium 2-fluoroacetate or low (<0.02 mg/mL) for diazinon. Therefore, once extracted these DNA samples could be safely processed in a forensic laboratory. PMID:18093062

  4. Comparisons of direct extraction methods of microbial DNA from different paddy soils.

    PubMed

    Islam, Md Rashedul; Sultana, Tahera; Melvin Joe, M; Cho, Jang-Cheon; Sa, Tongmin

    2012-07-01

    Molecular analyses for the study of soil microbial communities often depend on the direct extraction of DNA from soils. The present work compares the effectiveness of three different methods of extracting microbial DNA from seven different paddy soils. Comparison among different DNA extraction methods against different paddy soil samples revealed a marked variation in DNA yields from 3.18-20.17 μg DNA/g of dry soil. However, irrespective of the soil samples and extraction methods the DNA fragment size was >10 kb. Among the methods evaluated, method-C (chemical-enzymatic-mechanical) had better cell lysis efficiency and DNA yield. After purification of crude DNA by Purification Kit, A260/A230 and A260/A280 ratios of the DNA obtained by method-C reached up to 2.27 and 1.89, respectively, sustaining the efficacy of this technique in removing humic acid, protein and other contaminants. Results of the comprehensive evaluation of DNA extraction methods suggest that method-C is superior to other two methods (chemical-enzymatic and chemical-mechanical), and was the best choice for extraction of total DNA from soil samples. Since soil type and microbial community characteristics influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods according to experimental goals. PMID:23961194

  5. Direct Detection and Sequencing of Damaged DNA Bases

    PubMed Central

    2011-01-01

    Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template - as a by-product of the sequencing method - through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications. PMID:22185597

  6. How Can Plant DNA Viruses Evade siRNA-Directed DNA Methylation and Silencing?

    PubMed Central

    Pooggin, Mikhail M.

    2013-01-01

    Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short interfering RNAs (siRNAs), which can potentially direct viral DNA methylation and transcriptional silencing. However, growing evidence indicates that the circular double-stranded DNA accumulating in the nucleus for Pol II-mediated transcription of viral genes is not methylated. Hence, DNA viruses most likely evade or suppress RNA-directed DNA methylation. This review describes the specialized mechanisms of replication and silencing evasion evolved by geminiviruses and pararetoviruses, which rescue viral DNA from repressive methylation and interfere with transcriptional and post-transcriptional silencing of viral genes. PMID:23887650

  7. Enhancing DNA Crystal Durability through Chemical Crosslinking.

    PubMed

    Zhang, Diana; Paukstelis, Paul J

    2016-06-16

    Three-dimensional (3D) DNA crystals have been envisioned as a powerful tool for the positional control of biological and non-biological arrays on the nanoscale. However, most DNA crystals contain short duplex regions that can result in low thermal stability. Additionally, because DNA is a polyanion, DNA crystals often require high cation concentrations to maintain their integrity. Here, we demonstrate that a DNA alkylating mustard, bis(2-chloroethyl)amine, can form interstrand crosslinks within a model 3D DNA crystal. The crosslinking procedure did not alter crystal X-ray diffraction properties, but it did significantly improve the overall stability of the crystals under a variety of conditions. Crosslinked crystals showed enhanced stability at elevated temperature and were stable at Mg(2+) concentrations as low as 1 mm. Remarkably, the crosslinked crystals showed significant resistance to DNase I treatment, while also having improved longevity in tissue culture mediums. Characterization of the crosslinked species suggest that there are multiple crosslinking sites, but that the most prevalent interstrand crosslink involves an unpaired 3'-terminal guanosine residue. The improved stability of these DNA crystals suggests that simple treatment with alkylating reagents might be sufficient to stabilize crystals and other DNA constructs for improved functionality in biological and non-biological applications. PMID:27108768

  8. Label-free and selective photoelectrochemical detection of chemical DNA methylation damage using DNA repair enzymes.

    PubMed

    Wu, Yiping; Zhang, Bintian; Guo, Liang-Hong

    2013-07-16

    Exogenous chemicals may produce DNA methylation that is potentially toxic to living systems. Methylated DNA bases are difficult to detect with biosensors because the methyl group is small and chemically inert. In this report, a label-free photoelectrochemical sensor was developed for the selective detection of chemically methylated bases in DNA films. The sensor employed two DNA repair enzymes, human alkyladenine DNA glycosylase and human apurinic/apyrimidinic endonuclease, to convert DNA methylation sites in DNA films on indium tin oxide electrodes into strand breaks. A DNA intercalator, Ru(bpy)2(dppz)(2+) (bpy=2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) was then used as the photoelectrochemical signal indicator to detect the DNA strand breaks. Its photocurrent signal was found to correlate inversely with the amount of 3-methyladenines (metAde) produced with a methylating agent, methylmethane sulfonate (MMS). The sensor detected the methylated bases produced with as low as 1 mM MMS, at which concentration the amount of metAde on the sensor surface was estimated to be 0.5 pg, or 1 metAde in 1.6 × 10(5) normal bases. Other DNA base modification products, such as 5-methylcytosine and DNA adducts with ethyl and styrene groups did not attenuate the photocurrent, demonstrating good selectivity of the sensor. This strategy can be utilized to develop sensors for the detection of other modified DNA bases with specific DNA repair enzymes. PMID:23777269

  9. Chemical method for introducing haptens on to DNA probes

    SciTech Connect

    Keller, G.H.; Cumming, C.U.; Huang, D.P.; Manak, M.M.; Ting, R.

    1988-05-01

    The authors developed a versatile chemical method of attaching hapten moieties onto DNA, for the construction of nonisotopic DNA probes. The DNA is reacted with N-bromosuccinimide at alkaline pH, resulting in bromination of a fraction of the thymine, guanine, and cytosine residues, with adenine modified to a lesser extent. The bromine is subsequently displaced by a primary amino group, attached to a linker arm. The other end of the linker arm has a detectable group preattached to it. They have labeled cloned hepatitis B viral (HBV) DNA with the hapten 2,4-dinitrophenyl (DNP) and used it in combination with a high affinity rabbit anti-DNP antibody, for the detection of hepatitis B DNA by slot blotting. This probe was sensitive enough to specifically detect 1 x 10/sup -17/ mol (1 x 10/sup 6/ copies) of HBV DNA in total DNA from human serum.

  10. Directed Chemical Evolution with an Outsized Genetic Code.

    PubMed

    Krusemark, Casey J; Tilmans, Nicolas P; Brown, Patrick O; Harbury, Pehr B

    2016-01-01

    The first demonstration that macromolecules could be evolved in a test tube was reported twenty-five years ago. That breakthrough meant that billions of years of chance discovery and refinement could be compressed into a few weeks, and provided a powerful tool that now dominates all aspects of protein engineering. A challenge has been to extend this scientific advance into synthetic chemical space: to enable the directed evolution of abiotic molecules. The problem has been tackled in many ways. These include expanding the natural genetic code to include unnatural amino acids, engineering polyketide and polypeptide synthases to produce novel products, and tagging combinatorial chemistry libraries with DNA. Importantly, there is still no small-molecule analog of directed protein evolution, i.e. a substantiated approach for optimizing complex (≥ 10^9 diversity) populations of synthetic small molecules over successive generations. We present a key advance towards this goal: a tool for genetically-programmed synthesis of small-molecule libraries from large chemical alphabets. The approach accommodates alphabets that are one to two orders of magnitude larger than any in Nature, and facilitates evolution within the chemical spaces they create. This is critical for small molecules, which are built up from numerous and highly varied chemical fragments. We report a proof-of-concept chemical evolution experiment utilizing an outsized genetic code, and demonstrate that fitness traits can be passed from an initial small-molecule population through to the great-grandchildren of that population. The results establish the practical feasibility of engineering synthetic small molecules through accelerated evolution. PMID:27508294

  11. Directed Chemical Evolution with an Outsized Genetic Code

    PubMed Central

    Krusemark, Casey J.; Tilmans, Nicolas P.; Brown, Patrick O.; Harbury, Pehr B.

    2016-01-01

    The first demonstration that macromolecules could be evolved in a test tube was reported twenty-five years ago. That breakthrough meant that billions of years of chance discovery and refinement could be compressed into a few weeks, and provided a powerful tool that now dominates all aspects of protein engineering. A challenge has been to extend this scientific advance into synthetic chemical space: to enable the directed evolution of abiotic molecules. The problem has been tackled in many ways. These include expanding the natural genetic code to include unnatural amino acids, engineering polyketide and polypeptide synthases to produce novel products, and tagging combinatorial chemistry libraries with DNA. Importantly, there is still no small-molecule analog of directed protein evolution, i.e. a substantiated approach for optimizing complex (≥ 10^9 diversity) populations of synthetic small molecules over successive generations. We present a key advance towards this goal: a tool for genetically-programmed synthesis of small-molecule libraries from large chemical alphabets. The approach accommodates alphabets that are one to two orders of magnitude larger than any in Nature, and facilitates evolution within the chemical spaces they create. This is critical for small molecules, which are built up from numerous and highly varied chemical fragments. We report a proof-of-concept chemical evolution experiment utilizing an outsized genetic code, and demonstrate that fitness traits can be passed from an initial small-molecule population through to the great-grandchildren of that population. The results establish the practical feasibility of engineering synthetic small molecules through accelerated evolution. PMID:27508294

  12. Site-directed, on-surface assembly of DNA nanostructures.

    PubMed

    Meyer, Rebecca; Saccà, Barbara; Niemeyer, Christof M

    2015-10-01

    Two-dimensional DNA lattices have been assembled from DNA double-crossover (DX) motifs on DNA-encoded surfaces in a site-specific manner. The lattices contained two types of single-stranded protruding arms pointing into opposite directions of the plane. One type of these protruding arms served to anchor the DNA lattice on the solid support through specific hybridization with surface-bound, complementary capture oligomers. The other type of arms allowed for further attachment of DNA-tethered probe molecules on the opposite side of the lattices exposed to the solution. Site-specific lattice assembly and attachment of fluorophore-labeled oligonucleotides and DNA-protein conjugates was demonstrated using DNA microarrays on flat, transparent mica substrates. Owing to their programmable orientation and addressability over a broad dynamic range from the nanometer to the millimeter length scale, such supramolecular architecture might be used for presenting biomolecules on surfaces, for instance, in biosensor applications. PMID:26306556

  13. DNA Charge Transport: from Chemical Principles to the Cell.

    PubMed

    Arnold, Anna R; Grodick, Michael A; Barton, Jacqueline K

    2016-01-21

    The DNA double helix has captured the imagination of many, bringing it to the forefront of biological research. DNA has unique features that extend our interest into areas of chemistry, physics, material science, and engineering. Our laboratory has focused on studies of DNA charge transport (CT), wherein charges can efficiently travel long molecular distances through the DNA helix while maintaining an exquisite sensitivity to base pair π-stacking. Because DNA CT chemistry reports on the integrity of the DNA duplex, this property may be exploited to develop electrochemical devices to detect DNA lesions and DNA-binding proteins. Furthermore, studies now indicate that DNA CT may also be used in the cell by, for example, DNA repair proteins, as a cellular diagnostic, in order to scan the genome to localize efficiently to damage sites. In this review, we describe this evolution of DNA CT chemistry from the discovery of fundamental chemical principles to applications in diagnostic strategies and possible roles in biology. PMID:26933744

  14. Directly labeled fluorescent DNA probes for chromosome mapping

    SciTech Connect

    Marrone, B.L.; Deaven, L.L.; Chen, D.J.; Park, Min S.; MacInnes, M.A.; Salzman, G.C.; Yoshida, T.M.

    1995-12-31

    A new strategy is briefly described for employing nucleic acid probes that are directly labeled with fluorochromes in fluorescence in situ hybridization techniques. These probes will permit the detection, quantitation, and high-precision spatial analysis of multiple DNA sequences along a single chromosome using video-enhanced fluorescence microscopy and digital image processing and analysis. Potential advantages of direct labeled DNA probes for fluorescence in situ hybridization far surpass currently available, indirect DNA probe labeling techniques in ease of use, versatility, and increased signal- to-noise ratio.

  15. RNA-directed repair of DNA double-strand breaks.

    PubMed

    Yang, Yun-Gui; Qi, Yijun

    2015-08-01

    DNA double-strand breaks (DSBs) are among the most deleterious DNA lesions, which if unrepaired or repaired incorrectly can cause cell death or genome instability that may lead to cancer. To counteract these adverse consequences, eukaryotes have evolved a highly orchestrated mechanism to repair DSBs, namely DNA-damage-response (DDR). DDR, as defined specifically in relation to DSBs, consists of multi-layered regulatory modes including DNA damage sensors, transducers and effectors, through which DSBs are sensed and then repaired via DNAprotein interactions. Unexpectedly, recent studies have revealed a direct role of RNA in the repair of DSBs, including DSB-induced small RNA (diRNA)-directed and RNA-templated DNA repair. Here, we summarize the recent discoveries of RNA-mediated regulation of DSB repair and discuss the potential impact of these novel RNA components of the DSB repair pathway on genomic stability and plasticity. PMID:25960340

  16. Direct surface-enhanced Raman scattering analysis of DNA duplexes.

    PubMed

    Guerrini, Luca; Krpetić, Željka; van Lierop, Danny; Alvarez-Puebla, Ramon A; Graham, Duncan

    2015-01-19

    The exploration of the genetic information carried by DNA has become a major scientific challenge. Routine DNA analysis, such as PCR, still suffers from important intrinsic limitations. Surface-enhanced Raman spectroscopy (SERS) has emerged as an outstanding opportunity for the development of DNA analysis, but its application to duplexes (dsDNA) has been largely hampered by reproducibility and/or sensitivity issues. A simple strategy is presented to perform ultrasensitive direct label-free analysis of unmodified dsDNA with the means of SERS by using positively charged silver colloids. Electrostatic adhesion of DNA promotes nanoparticle aggregation into stable clusters yielding intense and reproducible SERS spectra at nanogram level. As potential applications, we report the quantitative recognition of hybridization events as well as the first examples of SERS recognition of single base mismatches and base methylations (5-methylated cytosine and N6-methylated Adenine) in duplexes. PMID:25414148

  17. Novel selection methods for DNA-encoded chemical libraries

    PubMed Central

    Chan, Alix I.; McGregor, Lynn M.; Liu, David R.

    2015-01-01

    Driven by the need for new compounds to serve as biological probes and leads for therapeutic development and the growing accessibility of DNA technologies including high-throughput sequencing, many academic and industrial groups have begun to use DNA-encoded chemical libraries as a source of bioactive small molecules. In this review, we describe the technologies that have enabled the selection of compounds with desired activities from these libraries. These methods exploit the sensitivity of in vitro selection coupled with DNA amplification to overcome some of the limitations and costs associated with conventional screening methods. In addition, we highlight newer techniques with the potential to be applied to the high-throughput evaluation of DNA-encoded chemical libraries. PMID:25723146

  18. DNA-directed mutations. Leading and lagging strand specificity

    NASA Technical Reports Server (NTRS)

    Sinden, R. R.; Hashem, V. I.; Rosche, W. A.

    1999-01-01

    The fidelity of replication has evolved to reproduce B-form DNA accurately, while allowing a low frequency of mutation. The fidelity of replication can be compromised, however, by defined order sequence DNA (dosDNA) that can adopt unusual or non B-DNA conformations. These alternative DNA conformations, including hairpins, cruciforms, triplex DNAs, and slipped-strand structures, may affect enzyme-template interactions that potentially lead to mutations. To analyze the effect of dosDNA elements on spontaneous mutagenesis, various mutational inserts containing inverted repeats or direct repeats were cloned in a plasmid containing a unidirectional origin of replication and a selectable marker for the mutation. This system allows for analysis of mutational events that are specific for the leading or lagging strands during DNA replication in Escherichia coli. Deletions between direct repeats, involving misalignment stabilized by DNA secondary structure, occurred preferentially on the lagging strand. Intermolecular strand switch events, correcting quasipalindromes to perfect inverted repeats, occurred preferentially during replication of the leading strand.

  19. Chemical Biology Probes from Advanced DNA-encoded Libraries.

    PubMed

    Salamon, Hazem; Klika Škopić, Mateja; Jung, Kathrin; Bugain, Olivia; Brunschweiger, Andreas

    2016-02-19

    The identification of bioactive compounds is a crucial step toward development of probes for chemical biology studies. Screening of DNA-encoded small molecule libraries (DELs) has emerged as a validated technology to interrogate vast chemical space. DELs consist of chimeric molecules composed of a low-molecular weight compound that is conjugated to a DNA identifier tag. They are screened as pooled libraries using selection to identify "hits." Screening of DELs has identified numerous bioactive compounds. Some of these molecules were instrumental in gaining a deeper understanding of biological systems. One of the main challenges in the field is the development of synthesis methodology for DELs. PMID:26820267

  20. Mesoporous silica nanoparticles deliver DNA and chemicals into plants

    NASA Astrophysics Data System (ADS)

    Torney, François; Trewyn, Brian G.; Lin, Victor S.-Y.; Wang, Kan

    2007-05-01

    Surface-functionalized silica nanoparticles can deliver DNA and drugs into animal cells and tissues. However, their use in plants is limited by the cell wall present in plant cells. Here we show a honeycomb mesoporous silica nanoparticle (MSN) system with 3-nm pores that can transport DNA and chemicals into isolated plant cells and intact leaves. We loaded the MSN with the gene and its chemical inducer and capped the ends with gold nanoparticles to keep the molecules from leaching out. Uncapping the gold nanoparticles released the chemicals and triggered gene expression in the plants under controlled-release conditions. Further developments such as pore enlargement and multifunctionalization of these MSNs may offer new possibilities in target-specific delivery of proteins, nucleotides and chemicals in plant biotechnology.

  1. Direct Nuclear Delivery of DNA by Photothermal Nanoblade.

    PubMed

    Wu, Ting-Hsiang; Wu, Yi-Chien; Sagullo, Enrico; Teitell, Michael A; Chiou, Pei-Yu

    2015-12-01

    We demonstrate direct nuclear delivery of DNA into live mammalian cells using the photothermal nanoblade. Pulsed laser-triggered cavitation bubbles on a titanium-coated micropipette tip punctured both cellular plasma and nuclear membranes, which was followed by pressure-controlled delivery of DNA into the nucleus. High-level and efficient plasmid expression in different cell types with maintained cell viability was achieved. PMID:25900925

  2. HLA typing by direct DNA sequencing.

    PubMed

    Smith, Linda K

    2012-01-01

    Sequencing-based typing is a high resolution method for the identification of HLA polymorphisms. The majority of HLA Class I alleles can be discriminated by their exon 2 and 3 sequence, and for Class II alleles, exon 2 is generally sufficient. There are polymorphic positions in other exons which may require additional sequencing to exclude certain alleles with differences outside exon 2 and 3, depending on the clinical requirement and relevant accredition guidelines. The process involves selective amplification of target alleles by PCR, agarose gel electrophoresis of the PCR products to assess the quantity and quality, followed by purification of PCR amplicons to remove excess primer and dNTPs. Cycle sequencing reactions using Applied Biosystems™ BigDye(®) Terminator Ready Reaction v1.1 or v3.1 Kit are performed, then purification of sequence reactions before electrophoresing using Applied Biosystems™ 3730 or 3730XL Genetic Analyser (or similar). Data is processed by specialised software packages, which compare the sample sequence to the sequences of all possible theoretical allele combinations to assign an accurate genotype. Examination of all nucleotides, both at conserved and polymorphic positions enables the direct identification of new alleles, which may not be possible with techniques such as SSP and SSO typing. PMID:22665229

  3. Direct-to-PCR tissue preservation for DNA profiling.

    PubMed

    Sorensen, Amy; Berry, Clare; Bruce, David; Gahan, Michelle Elizabeth; Hughes-Stamm, Sheree; McNevin, Dennis

    2016-05-01

    Disaster victim identification (DVI) often occurs in remote locations with extremes of temperatures and humidities. Access to mortuary facilities and refrigeration are not always available. An effective and robust DNA sampling and preservation procedure would increase the probability of successful DNA profiling and allow faster repatriation of bodies and body parts. If the act of tissue preservation also released DNA into solution, ready for polymerase chain reaction (PCR), the DVI process could be further streamlined. In this study, we explored the possibility of obtaining DNA profiles without DNA extraction, by adding aliquots of preservative solutions surrounding fresh human muscle and decomposing human muscle and skin tissue samples directly to PCR. The preservatives consisted of two custom preparations and two proprietary solutions. The custom preparations were a salt-saturated solution of dimethyl sulfoxide (DMSO) with ethylenediaminetetraacetic (EDTA) and TENT buffer (Tris, EDTA, NaCl, Tween 20). The proprietary preservatives were DNAgard (Biomatrica(®)) and Tissue Stabilising Kit (DNA Genotek). We obtained full PowerPlex(®) 21 (Promega) and GlobalFiler(®) (Life Technologies) DNA profiles from fresh and decomposed tissue preserved at 35 °C for up to 28 days for all four preservatives. The preservative aliquots removed from the fresh muscle tissue samples had been stored at -80 °C for 4 years, indicating that long-term archival does not diminish the probability of successful DNA typing. Rather, storage at -80 °C seems to reduce PCR inhibition. PMID:26530406

  4. RNA-directed DNA methylation and demethylation in plants

    PubMed Central

    Viswanathan, CHINNUSAMY; Jian-Kang, ZHU

    2011-01-01

    RNA-directed DNA methylation (RdDM) is a nuclear process in which small interfering RNAs (siRNAs) direct the cytosine methylation of DNA sequences that are complementary to the siRNAs. In plants, double stranded-RNAs (dsRNAs) generated by RNA-dependent RNA polymerase 2 (RDR2) serve as precursors for Dicer-like 3 dependent biogenesis of 24-nt siRNAs. Plant specific RNA polymerase IV (Pol IV) is presumed to generate the initial RNA transcripts that are substrates for RDR2. siRNAs are loaded onto an argonaute4-containing RISC (RNA-induced silencing complex) that targets the de novo DNA methyltransferase DRM2 to RdDM target loci. Nascent RNA transcripts from the target loci are generated by another plant-specific RNA polymerase, Pol V, and these transcripts help recruit complementary siRNAs and the associated RdDM effector complex to the target loci in a transcription-coupled DNA methylation process. Small RNA binding proteins such as ROS3 may direct target-specific DNA demethylation by the ROS1 family of DNA demethylases. Chromatin remodeling enzymes and histone modifying enzymes also participate in DNA methylation and possibly demethylation. One of the well studied functions of RdDM is transposon silencing and genome stability. In addition, RdDM is important for paramutation, imprinting, gene regulation, and plant development. Locus-specific DNA methylation and demethylation, and transposon activation under abiotic stresses suggest that RdDM is also important in stress responses of plants. Further studies will help illuminate the functions of RdDM in the dynamic control of epigenomes during development and environmental stress responses. PMID:19381459

  5. Toxicogenomic evaluation of chemically induced chromosomal imbalance using DNA image analysis.

    PubMed

    Hatzi, Vasiliki I; Terzoudi, Georgia I; Spiliopoulou, Chara A; Stefanidou, Maria E

    2013-06-01

    The study of carcinogenic potential of a variety of chemical agents such as food additives and drugs of abuse via the application of various in vitro methodologies constitutes the first step for the evaluation of their toxicogenomic profile. Considering the chromosomal theories of carcinogenesis, where it is stated that aneuploidy and chromosomal imbalance (instability) are among the main causes of carcinogenesis, chemicals capable to induce such changes in the cells could be considered as potential carcinogens. Chromosomal imbalance and aneuploidy directly affect the overall DNA content of the exposed cell as well as other cellular morpho- and densitometric features. These features can be measured by means of computerized DNA image analysis technologies and include DNA content (DNA Index), Proliferation Index, Ploidy Balance, Degree of Aneuploidy, Skewness and Kurtosis. Considering the enormous number of untested chemicals and drugs of abuse that follow non-genotoxic mechanisms of carcinogenesis, the establishment of a reliable technology for the estimation of chemically induced chromosomal imbalance is of particular importance in toxicogenomic studies. In the present article and based on our previously published work, we highlight the advantages of the applications of DNA image analysis technology in an easy-to-use experimental model for the evaluation of the potential risk of various chemicals. The use of this technology for the detection of chemically induced chromosomal instability will contribute to the development of safer regulatory directives concerning the use of chemicals in food and pharmaceutical industry, as well as in the clarification of mechanisms of action of drugs of abuse. PMID:23215871

  6. Direct nanomaterial-DNA contact effects on DNA and mutation induction.

    PubMed

    Thongkumkoon, P; Sangwijit, K; Chaiwong, C; Thongtem, S; Singjai, P; Yu, L D

    2014-04-01

    The toxicity of nanomaterials has been well known, but mechanisms involved have been little known. This study was aimed at looking at direct interaction between nanomaterials and naked DNA for some fundamental understanding. Two different types of nanomaterials, carbon nanotubes (CNTs) and tungsten trioxide (WO₃) nanoplates, were simply mixed with naked DNA plasmid, respectively, in two different contact modes, dry or wet (in solution), for varied time periods. DNA topological forms were analyzed for changes using gel electrophoresis and fluoro-spectrometry. The nanomaterial-contacted DNA was transferred into bacteria Escherichia coli (E. coli) cells for mutation observation. Certain types and degrees of DNA damage were observed, such as single strand break and double strand break, and bacterial mutation was confirmed. The DNA damage increased with the contacting time in an exponential manner and increased more rapidly in the initial stage for the wet contact. The nanomaterials-contacted DNA transferred bacteria had about less than 10% survival but almost 100% mutation for the surviving cells. The CNTs were more offensive than the metal oxide nanomaterials. The mutation spectrum from the DNA sequencing analysis showed that DNA point mutation was dominated by transversion, which was dominated by guanine changes in the wet contact condition while by cytosine changes in the dry contact condition. The point mutation occurrence in the wet contact was more than in the dry contact, confirming the wet contact more active and thus dangerous than dry contact. This experiment, although as a model study, revealed that direct simple contacts between nanomaterials and DNA could cause DNA changes and thus induce mutations which might potentially lead to cancers, diseases and genetic changes. This could be a mechanism for nanomaterial genotoxicity to the cells and also provided a caution to applications in using nanomaterials for DNA delivery. PMID:24503012

  7. siRNA-directed DNA Methylation in Plants

    PubMed Central

    Xie, Meng; Yu, Bin

    2015-01-01

    DNA cytosine methylationis an important epigenetic process that is correlated with transgene silencing, transposon suppression, and gene imprinting. In plants, small interfering RNAs (siRNAs) can trigger DNA methylation at loci containing their homolog sequences through a process called RNA-directed DNA methylation (RdDM). In canonical RdDM, 24 nucleotide (nt) siRNAs (ra-siRNAs) will be loaded into their effector protein called ARGONAUTE 4 (AGO4) and subsequently targeted to RdDM loci through base-pairing with the non-coding transcripts produced by DNA-directed RNA Polymerase V. Then, the AGO4-ra-siRNA will recruit the DNA methyltransferase to catalyze de novo DNA methylation. Recent studies also identified non-canonical RdDM pathways that involve microRNAs or 21 nt siRNAs. These RdDM pathways are biologically important since they control responses biotic and abiotic stresses, maintain genome stability and regulate development. Here, we summarize recent pro-gresses of mechanisms governing canonical and non-canonical RdDM pathways. PMID:25937811

  8. Chemical Reactions Directed Peptide Self-Assembly

    PubMed Central

    Rasale, Dnyaneshwar B.; Das, Apurba K.

    2015-01-01

    Fabrication of self-assembled nanostructures is one of the important aspects in nanoscience and nanotechnology. The study of self-assembled soft materials remains an area of interest due to their potential applications in biomedicine. The versatile properties of soft materials can be tuned using a bottom up approach of small molecules. Peptide based self-assembly has significant impact in biology because of its unique features such as biocompatibility, straight peptide chain and the presence of different side chain functionality. These unique features explore peptides in various self-assembly process. In this review, we briefly introduce chemical reaction-mediated peptide self-assembly. Herein, we have emphasised enzymes, native chemical ligation and photochemical reactions in the exploration of peptide self-assembly. PMID:25984603

  9. Direct Extraction and Amplification of DNA from Soil.

    ERIC Educational Resources Information Center

    Trevors, Jack T.; Leung, K.

    1998-01-01

    Presents an exercise that describes the direct extraction and purification of DNA from a small soil sample. Also discusses the subsequent amplification of a 343-bp Tn7 transposate A gene fragment (tnsA) from a strain of Pseudomonas aureofaciens 3732RNL11. Contains 21 references. (DDR)

  10. Chromosomal directionality of DNA mismatch repair in Escherichia coli

    PubMed Central

    Hasan, A. M. Mahedi; Leach, David R. F.

    2015-01-01

    Defects in DNA mismatch repair (MMR) result in elevated mutagenesis and in cancer predisposition. This disease burden arises because MMR is required to correct errors made in the copying of DNA. MMR is bidirectional at the level of DNA strand polarity as it operates equally well in the 5′ to 3′ and the 3′ to 5′ directions. However, the directionality of MMR with respect to the chromosome, which comprises parental DNA strands of opposite polarity, has been unknown. Here, we show that MMR in Escherichia coli is unidirectional with respect to the chromosome. Our data demonstrate that, following the recognition of a 3-bp insertion-deletion loop mismatch, the MMR machinery searches for the first hemimethylated GATC site located on its origin-distal side, toward the replication fork, and that resection then proceeds back toward the mismatch and away from the replication fork. This study provides support for a tight coupling between MMR and DNA replication. PMID:26170312

  11. Localization of ASV Integrase-DNA Contacts by Site-Directed Crosslinking and their Structural Analysis

    PubMed Central

    Merkel, George; Alexandratos, Jerry; Zhou, Dongwen; Bojja, Ravi S.; Satoh, Tadashi; Potapov, Mikhail; Kogon, Alex; Potapov, Viktor; Wlodawer, Alexander; Skalka, Anna Marie

    2011-01-01

    Background We applied crosslinking techniques as a first step in preparation of stable avian sarcoma virus (ASV) integrase (IN)-DNA complexes for crystallographic investigations. These results were then compared with the crystal structures of the prototype foamy virus (PFV) intasome and with published data for other retroviral IN proteins. Methodology/Results Photoaffinity crosslinking and site-directed chemical crosslinking were used to localize the sites of contacts with DNA substrates on the surface of ASV IN. Sulfhydryl groups of cysteines engineered into ASV IN and amino-modified nucleotides in DNA substrates were used for attachment of photocrosslinkers. Analysis of photocrosslinking data revealed several specific DNA-protein contacts. To confirm contact sites, thiol-modified nucleotides were introduced into oligo-DNA substrates at suggested points of contact and chemically crosslinked to the cysteines via formation of disulfide bridges. Cysteines incorporated in positions 124 and 146 in the ASV IN core domain were shown to interact directly with host and viral portions of the Y-mer DNA substrate, respectively. Crosslinking of an R244C ASV IN derivative identified contacts at positions 11 and 12 on both strands of viral DNA. The most efficient disulfide crosslinking was observed for complexes of the ASV IN E157C and D64C derivatives with linear viral DNA substrate carrying a thiol-modified scissile phosphate. Conclusion Analysis of our crosslinking results as well as published results of retroviral IN protein from other laboratories shows good agreement with the structure of PFV IN and derived ASV, HIV, and MuLV models for the core domain, but only partial agreement for the N- and C-terminal domains. These differences might be explained by structural variations and evolutionary selection for residues at alternate positions to perform analogous functions, and by methodological differences: i.e., a static picture of a particular assembly from crystallography vs

  12. DNA damage induced by the direct effect of radiation

    NASA Astrophysics Data System (ADS)

    Yokoya, A.; Shikazono, N.; Fujii, K.; Urushibara, A.; Akamatsu, K.; Watanabe, R.

    2008-10-01

    We have studied the nature of DNA damage induced by the direct effect of radiation. The yields of single- (SSB) and double-strand breaks (DSB), base lesions and clustered damage were measured using the agarose gel electrophoresis method after exposing to various kinds of radiations to a simple model DNA molecule, fully hydrated closed-circular plasmid DNA (pUC18). The yield of SSB does not show significant dependence on linear energy transfer (LET) values. On the other hand, the yields of base lesions revealed by enzymatic probes, endonuclease III (Nth) and formamidopyrimidine DNA glycosylase (Fpg), which excise base lesions and leave a nick at the damage site, strongly depend on LET values. Soft X-ray photon (150 kVp) irradiation gives a maximum yield of the base lesions detected by the enzymatic probes as SSB and clustered damage, which is composed of one base lesion and proximate other base lesions or SSBs. The clustered damage is visualized as an enzymatically induced DSB. The yields of the enzymatically additional damages strikingly decrease with increasing levels of LET. These results suggest that in higher LET regions, the repair enzymes used as probes are compromised because of the dense damage clustering. The studies using simple plasmid DNA as a irradiation sample, however, have a technical difficulty to detect multiple SSBs in a plasmid DNA. To detect the additional SSBs induced in opposite strand of the first SSB, we have also developed a novel technique of DNA-denaturation assay. This allows us to detect multiply induced SSBs in both strand of DNA, but not induced DSB.

  13. DNA Conjugation and DNA Directed Self-Assembly of Quantum Dots for Nanophotonic Applications

    NASA Astrophysics Data System (ADS)

    Samanta, Anirban

    Colloidal quantum dots (QDs) or semiconductor nanocrystals are often used to describe 2--20 nm solution processed nanoparticles of various semiconductor materials that display quantum confinement effects. Compared to traditional fluorescent organic dyes, QDs provide many advantages. For biological applications it is necessary to develop reliable methods to functionalize QDs with hydrophilic biomolecules so that they may maintain their stability and functionality in physiological conditions. DNA, a molecule that encodes genetic information, is arguably the smartest molecule that nature has ever produced and one of the most explored bio-macromolecules. QDs that are functionalized with DNA can potentially be organized with nanometer precision by DNA directed self-assembly, and the resulting arrangements may facilitate the display of novel optical properties. The goal of this dissertation was to achieve a robust reliable yet simple strategy to link DNA to QDs so that they can be used for DNA directed self assembly by which we can engineer their optical properties. Presented here is a series of studies to achieve this goal. First we demonstrate the aqueous synthesis of colloidal nanocrystal heterostructures consisting of the CdTe core encapsulated by CdS/ZnS or CdSe/ZnS shells using glutathione (GSH), a tripeptide, as the capping ligand. We next employed this shell synthesis strategy to conjugate PS-PO chimeric DNA to QDs at the time of shell synthesis. We synthesized a library of DNA linked QDs emitting from UV to near IR that are very stable in high salt concentrations. These DNA functionalized QDs were further site-specifically organized on DNA origami in desired patterns directed by DNA self-assembly. We further extended our capability to functionalize DNA to real IR emitting CdxPb 1-xTe alloyed QDs, and demonstrated their stability by self-assembling them on DNA origami. The photo-physical properties of the QDs were further engineered by attaching a QD and a gold

  14. DNA barcoding to fishes: current status and future directions.

    PubMed

    Bhattacharya, Manojit; Sharma, Ashish Ranjan; Patra, Bidhan Chandra; Sharma, Garima; Seo, Eun-Min; Nam, Ju-Suk; Chakraborty, Chiranjib; Lee, Sang-Soo

    2016-07-01

    DNA barcoding appears to be a promising approach for taxonomic identification, characterization, and discovery of newer species, facilitating biodiversity studies. It helps researchers to appreciate genetic and evolutionary associations by collection of molecular, morphological, and distributional data. Fish DNA barcoding, based on the sequencing of a uniform area of Cytochrome C Oxidase type I (COI) gene, has received significant interest as an accurate tool for species identification, authentication, and phylogenetic analysis. The aim of this review article was to investigate recent global status, approaches, and future direction of DNA barcoding in fisheries sectors. We have tried to highlight its possible impacts, complications, and validation issues at species levels for biodiversity analysis. Moreover, an effort has been put forward to understand issues related to various marker genes associated with barcode process as primer sequences and have concluded barcode promotion as an indispensable tool of molecular biology for the development of taxonomic support systems. PMID:26057011

  15. DNA hybridization and ligation for directed colloidal assembly

    NASA Astrophysics Data System (ADS)

    Shyr, Margaret

    Colloidal assembly using DNA hybridization has been pursued as a means assemble non-conventional ordered colloidal structures. However, to date it is undetermined whether DNA hybridization can be used to achieve non-FCC colloidal crystals. Using microcontact printing techniques, we have fabricated covalently bound single stranded DNA (ssDNA) two-dimensional arrays on glass surfaces, which were used to direct the assembly of complementary DNA functionalized polystyrene colloids. Two of the hallmarks of DNA hybridization, sequence specificity and thermal reversibility, were demonstrated. Due to the periodicity of these arrays, laser diffraction was used to directly monitor these structures during assembly. To demonstrate the versatility of the 2D colloidal array assembled via DNA hybridization, a catalytic DNA sequence or DNAzyme was incorporated into the colloidal array system. By tethering the enzymatic strand to the patterned glass surface and the substrate strand to polystyrene colloids, we showed that the DNAzyme could prevent the assembly of the arrays when the required Pb2+ cofactor was provided. Attempts to assemble the colloid arrays and disassemble via the Pb2+-DNAzyme induced cleavage were unsuccessful, likely due to the incomplete cleavage of the multitude of hybridized linkages between each colloid and the surface. Since DNA is not only capable of catalyzing reactions, but also capable of being reacted upon by a variety of biological enzymes, we examined the use of DNA ligase as a means to control the assembly of DNA-functionalized colloids. A three-sequence linker system was used for the hybridization mediated assembly of colloids: one sequence was tethered to the surface of the glass slide or colloids, one was tethered to another colloid surface, and the linker sequence hybridizes simultaneously to both tethered sequences. Once hybridized, the two tethered fragments can be ligated using DNA ligase, resulting in a continuous sequence tethered on one end

  16. BIOASSAY-DIRECTED CHEMICAL ANALYSIS IN ENVIRONMENTAL RESEARCH

    EPA Science Inventory

    The use of short-term bioassay tests in conjunction with analytical measurements, constitute a powerful tool for identifying important environmental contaminants. The authors have coined the terminology 'bioassay directed chemical analysis' to best describe this marriage of analy...

  17. Directional imbibition on a chemically patterned silicon micropillar array.

    PubMed

    Jokinen, Ville

    2016-01-28

    Directional imbibition of oils (hexadecane, tetradecane, and dodecane) and water is demonstrated on a chemically patterned silicon micropillar array. Four different directional imbibition types are shown: unidirectional, two types of bidirectional and tridirectional imbibition. The surfaces consist of a silicon micropillar array with an overlaid surface chemistry pattern. This configuration leads to anisotropic wetting behaviour into various directions of the advancing meniscus. Due to the free energy landscape obtained, the advancing meniscus gets pinned in some directions (determined by the surface chemistry pattern) while it is free to move to the remaining directions. The conditions for directional imbibition and design criteria for the surfaces are derived and discussed. PMID:26576647

  18. Chemically amplified laser direct-writing of aluminum

    NASA Astrophysics Data System (ADS)

    Tsao, J. Y.; Ehrlich, D. J.

    Laser microchemical direct writing has important advantages over other techniques for the deposition of thin-film patterns. Disadvantages, however, are lower throughput and the need to suppress competing processes such as gas phase nucleation of particles or substrate damage. Methods for increasing the overall speed of laser direct writing by microchemistry were investigated. A class of laser deposition techniques has emerged in which laser radiation is used only to enhance or to impede the initial nucleation of a thin film. In general, it is convenient to draw a distinction between nucleation barriers due to physical effects and those due to chemical effects. The first type of barrier is derived from surface tension. The laser deposits a pattern of heterogeneous catalyst to initiate a subsequent transformation that is chemically self-sustaining or autocatalytic. Experiments, in which the laser direct writing of patterned thin films of Al is chemically amplified by subsequent selective pyrolytic chemical vapor deposition are summarized.

  19. Droplet heat transfer and chemical reactions during direct containment heating

    SciTech Connect

    Baker, L. Jr.

    1986-01-01

    A simplified model of heat transfer and chemical reaction has been adapted to evaluate the expected behavior of droplets containing unreacted Zircaloy and stainless steel moving through the containment atmosphere during postulated accidents involving direct containment heating. The model includes internal and external diffusive resistances to reaction. The results indicate that reactions will be incomplete for many conditions characteristic of direct containment heating sequences.

  20. A DNA target of 30 bp is sufficient for RNA-directed DNA methylation.

    PubMed

    Pélissier, T; Wassenegger, M

    2000-01-01

    In higher plants, RNA-DNA interactions can trigger de novo methylation of genomic sequences via a process that is termed RNA-directed DNA methylation (RdDM). In potato spindle tuber viroid (PSTVd)-infected tobacco plants, this process can potentially lead to methylation of all C residues at symmetrical and nonsymmetrical sites within chromosomal inserts that consist of multimers of the 359-bp-long PSTVd cDNA. Using PSTVd cDNA subfragments, we found that genomic targets with as few as 30 nt of sequence complementarity to the viroid RNA are detected and methylated. Genomic sequencing analyses of genome-integrated 30- and 60-bp-long PSTVd subfragments demonstrated that de novo cytosine methylation is not limited to the canonical CpG, CpNpG sites. Sixty-base-pair-long PSTVd cDNA constructs appeared to be densely methylated in nearly all tobacco leaf cells. With the 30-bp-long PSTVd-specific construct, the proportion of cells displaying dense transgene methylation was significantly reduced, suggesting that a minimal target size of about 30 bp is necessary for RdDM. The methylation patterns observed for two different 60-bp constructs further suggested that the sequence identity of the target may influence the methylation mechanism. Finally, a link between viroid pathogenicity and PSTVd RNA-directed methylation of host sequences is proposed. PMID:10668798

  1. A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE INDUCED BY TOXIC INDUSTRIAL CHEMICALS

    EPA Science Inventory

    One of the reported effects for exposure to many of the toxic industrial chemicals is DNA damage. The present study describes a simple, rapid and innovative assay to detect DNA damage resulting from exposure of surrogate DNA to toxic industrial chemicals (acrolein, allylamine, ch...

  2. Label free colorimetric and fluorimetric direct detection of methylated DNA based on silver nanoclusters for cancer early diagnosis.

    PubMed

    Dadmehr, Mehdi; Hosseini, Morteza; Hosseinkhani, Saman; Ganjali, Mohammad Reza; Sheikhnejad, Reza

    2015-11-15

    Epigenetic changes such as DNA methylation of CpG islands located in the promoter region of some tumor suppressor genes are very common in human diseases such as cancer. Detection of aberrant methylation pattern could serve as an excellent diagnostic approach. Recently, the direct detection of methylated DNA sequences without using chemical and enzymatic treatments or antibodies has received great deal of attentions. In this study, we report a colorimetric and fluorimetric technique for direct detection of DNA methylation. Here, the DNA is being used as an effective template for fluorescent silver nanoclusters formation without any chemical modification or DNA labeling. The sensitivity test showed that upon the addition of target methylated DNA, the fluorescence intensity is decreased in a linear range when the concentration of methylated DNA has increased from 2.0×10(-9) to 6.3 ×10(-7) M with the detection limit of 9.4×10(-10) M. The optical and fluorescence spectral behaviors were highly reproducible and clearly discriminated between unmethylated, methylated and even partially methylated DNA in CpG rich sequences. The results were also reproducible when the human plasma was present in our assay system. PMID:26056954

  3. DNA methylation directs functional maturation of pancreatic β cells

    PubMed Central

    Dhawan, Sangeeta; Tschen, Shuen-Ing; Zeng, Chun; Guo, Tingxia; Hebrok, Matthias; Matveyenko, Aleksey; Bhushan, Anil

    2015-01-01

    Pancreatic β cells secrete insulin in response to postprandial increases in glucose levels to prevent hyperglycemia and inhibit insulin secretion under fasting conditions to protect against hypoglycemia. β cells lack this functional capability at birth and acquire glucose-stimulated insulin secretion (GSIS) during neonatal life. Here, we have shown that during postnatal life, the de novo DNA methyltransferase DNMT3A initiates a metabolic program by repressing key genes, thereby enabling the coupling of insulin secretion to glucose levels. In a murine model, β cell–specific deletion of Dnmt3a prevented the metabolic switch, resulting in loss of GSIS. DNMT3A bound to the promoters of the genes encoding hexokinase 1 (HK1) and lactate dehydrogenase A (LDHA) — both of which regulate the metabolic switch — and knockdown of these two key DNMT3A targets restored the GSIS response in islets from animals with β cell–specific Dnmt3a deletion. Furthermore, DNA methylation–mediated repression of glucose-secretion decoupling genes to modulate GSIS was conserved in human β cells. Together, our results reveal a role for DNA methylation to direct the acquisition of pancreatic β cell function. PMID:26098213

  4. Direct chemical evidence for eumelanin pigment from the Jurassic period.

    PubMed

    Glass, Keely; Ito, Shosuke; Wilby, Philip R; Sota, Takayuki; Nakamura, Atsushi; Bowers, C Russell; Vinther, Jakob; Dutta, Suryendu; Summons, Roger; Briggs, Derek E G; Wakamatsu, Kazumasa; Simon, John D

    2012-06-26

    Melanin is a ubiquitous biological pigment found in bacteria, fungi, plants, and animals. It has a diverse range of ecological and biochemical functions, including display, evasion, photoprotection, detoxification, and metal scavenging. To date, evidence of melanin in fossil organisms has relied entirely on indirect morphological and chemical analyses. Here, we apply direct chemical techniques to categorically demonstrate the preservation of eumelanin in two > 160 Ma Jurassic cephalopod ink sacs and to confirm its chemical similarity to the ink of the modern cephalopod, Sepia officinalis. Identification and characterization of degradation-resistant melanin may provide insights into its diverse roles in ancient organisms. PMID:22615359

  5. Direct chemical evidence for eumelanin pigment from the Jurassic period

    PubMed Central

    Glass, Keely; Ito, Shosuke; Wilby, Philip R.; Sota, Takayuki; Nakamura, Atsushi; Bowers, C. Russell; Vinther, Jakob; Dutta, Suryendu; Summons, Roger; Briggs, Derek E. G.; Wakamatsu, Kazumasa; Simon, John D.

    2012-01-01

    Melanin is a ubiquitous biological pigment found in bacteria, fungi, plants, and animals. It has a diverse range of ecological and biochemical functions, including display, evasion, photoprotection, detoxification, and metal scavenging. To date, evidence of melanin in fossil organisms has relied entirely on indirect morphological and chemical analyses. Here, we apply direct chemical techniques to categorically demonstrate the preservation of eumelanin in two > 160 Ma Jurassic cephalopod ink sacs and to confirm its chemical similarity to the ink of the modern cephalopod, Sepia officinalis. Identification and characterization of degradation-resistant melanin may provide insights into its diverse roles in ancient organisms. PMID:22615359

  6. Direct attachment of DNA to semiconducting surfaces for biosensor applications.

    PubMed

    Fahrenkopf, Nicholas M; Shahedipour-Sandvik, Fatemeh; Tokranova, Natalya; Bergkvist, Magnus; Cady, Nathaniel C

    2010-11-01

    In this work we propose a novel method of immobilizing nucleic acids for field effect or high electron mobility transistor-based biosensors. The naturally occurring 5' terminal phosphate group on nucleic acids was used to coordinate with semiconductor and metal oxide surfaces. We demonstrate that DNA can be directly immobilized onto ZrO(2), AlGaN, GaN, and HfO(2) while retaining its ability to hybridize to target sequences with high specificity. By directly immobilizing the probe molecule to the sensor surface, as opposed to conventional crosslinking strategies, the number of steps in device fabrication is reduced. Furthermore, hybridization to target strands occurs closer to the sensor surface, which has the potential to increase device sensitivity by reducing the impact of the Debye screening length. PMID:20869405

  7. Infrared emitting quantum dots: DNA conjugation and DNA origami directed self-assembly

    NASA Astrophysics Data System (ADS)

    Samanta, Anirban; Deng, Zhengtao; Liu, Yan

    2014-04-01

    QDs that emit in the infrared (IR) range are of special interest at the moment because of their potential as tissue imaging reagents. Due to autofluorescence from tissues, QDs that emit in the visible range fail to produce good signal to noise ratios. Here we report the production of CdxPb1-xTe tertiary-alloyed QDs that emit in the 1100-1300 nm wavelength range, capped with the hydrophilic ligands mercaptopropionic acid (MPA) or glutathione (GSH), together with DNA, as specific surface tags. We observed an interesting dependence of the QD emission peaks on the species of capping ligand used. ICP-MS analysis confirmed that changing the identity of the surface ligand in the reaction mixture shifted the elemental composition of the particles and resulted in different Cd/Pb ratios. Further, DNA directed assembly of the particles onto DNA nanostructures ensures that the particle remains stable in high salt conditions, which is crucial to biological applications.QDs that emit in the infrared (IR) range are of special interest at the moment because of their potential as tissue imaging reagents. Due to autofluorescence from tissues, QDs that emit in the visible range fail to produce good signal to noise ratios. Here we report the production of CdxPb1-xTe tertiary-alloyed QDs that emit in the 1100-1300 nm wavelength range, capped with the hydrophilic ligands mercaptopropionic acid (MPA) or glutathione (GSH), together with DNA, as specific surface tags. We observed an interesting dependence of the QD emission peaks on the species of capping ligand used. ICP-MS analysis confirmed that changing the identity of the surface ligand in the reaction mixture shifted the elemental composition of the particles and resulted in different Cd/Pb ratios. Further, DNA directed assembly of the particles onto DNA nanostructures ensures that the particle remains stable in high salt conditions, which is crucial to biological applications. Electronic supplementary information (ESI) available

  8. Direct Measurements of Electrical Transport Through Single DNA Molecules of Complex Sequence

    NASA Astrophysics Data System (ADS)

    Cohen, Hezy; Nogues, Claude

    2005-03-01

    Seemingly contradicting results raised a debate over the ability of DNA to transport charge and the nature of the conduction mechanisms through it. We developed an experimental approach for measuring current through DNA molecules, chemically connected on opposite ends to a metal substrate and to a gold nanoparticle, using a conductive atomic force microscope.^1 Many samples could be made due to the experimental approach adopted here that enabled obtaining reproducible results in various samples, conditions and measurement methods. We present multileveled evidence for charge transport through 26 base-pairs long dsDNA of a complex sequence, characterized by S-shaped I-V curves that show currents higher than 220 nA at 2 V.^2 This significant observation implies that a coherent or band transport mechanism takes over for the high currents (> 1 nA). 1. Claude Nogues, Sidney R. Cohen, Shirley S. Daube, and Ron Naaman, ``Electrical properties of short DNA oligomers characterized by conducting atomic force microscopy,'' PCCP, 2004, 18. 2. Hezy Cohen, Claude Nogues Ron Naaman and Danny Porath. ``Direct Measurement of Electrical Transport Through Single DNA Molecules,'' submitted.

  9. DNA profiles from clothing fibers using direct PCR.

    PubMed

    Blackie, Renée; Taylor, Duncan; Linacre, Adrian

    2016-09-01

    We report on the successful use of direct PCR amplification of single fibers from items of worn clothing. Items of clothing were worn throughout the course of a day, with the individual commencing regular activities. Single fibers were taken from the cuff of the clothing at regular intervals and amplified directly. The same areas were subjected to tape-lifting, and also amplified directly for comparison. The NGM™ kit that amplifies 15 STR loci plus amelogenin was used. A total of 35 single fiber samples were processed and analyzed from five items of clothing, with 81 % of samples returning a profile of 14 alleles or more. All tape-lift samples amplified directly produced DNA profiles of 15 alleles or more. The aim was to develop a simple, operational method that could be used routinely in forensic science casework and that has the potential to generate more complete profiles, which would not be detected using standard extraction methods on this type of sample. For ease of implementation, the process also adheres to standard methods with no increase in the cycle number. PMID:27421265

  10. DNA Oxidation Profiles of Copper Phenanthrene Chemical Nucleases

    NASA Astrophysics Data System (ADS)

    Molphy, Zara; Slator, Creina; Chatgilialoglu, Chryssostomos; Kellett, Andrew

    2015-04-01

    The deleterious effects of metal-catalyzed reactive oxygen species (ROS) in biological systems can be seen in a wide variety of pathological conditions including cancer, cardiovascular disease, ageing, and neurodegenerative disorder. On the other hand however, targeted ROS production in the vicinity of nucleic acids - as demonstrated by metal-activated bleomycin - has paved the way for ROS-active chemotherapeutic drug development. Herein we report mechanistic investigations into the oxidative nuclease activity and redox properties of copper(II) developmental therapeutics [Cu(DPQ)(phen)]2+ (Cu-DPQ-Phen), [Cu(DPPZ)(phen)]2+ (Cu-DPPZ-Phen), and [{Cu(phen)2}2(μ-terph)](terph) (Cu-Terph), with results being compared directly to Sigman’s reagent [Cu(phen)2]2+ throughout (phen = 1,10-phenanthroline; DPQ = dipyridoquinoxaline; DPPZ = dipyridophenazine). Oxidative DNA damage was identified at the minor groove through use of surface bound recognition elements of methyl green, netropsin, and [Co(NH3)6]Cl3 that functioned to control complex accessibility at selected regions. ROS-specific scavengers and stabilisers were employed to identify the cleavage process, the results of which infer hydrogen peroxide produced metal-hydroxo or free hydroxyl radicals (•OH) as the predominant species. The extent of DNA damage owing to these radicals was then quantified through 8-oxo-2'-deoxyguanosine (8-oxo-dG) lesion detection under ELISA protocol with the overall trend following Cu-DPQ-Phen > Cu-Terph > Cu-Phen > Cu-DPPZ. Finally, the effects of oxidative damage on DNA replication processes were investigated using the polymerase chain reaction (PCR) where amplification of 120 base pair DNA sequences of varying base content were inhibited - particularly along A-T rich chains - through oxidative damage of the template strands.

  11. Bioassay-directed chemical analysis in environmental research

    SciTech Connect

    Schuetzle, D.; Lewtas, J.

    1986-01-01

    The use of short-term bioassay tests in conjunction with analytical measurements, constitute a powerful tool for identifying important environmental contaminants. The authors have coined the terminology bioassay directed chemical analysis to best describe this marriage of analytical chemistry and biology. The objective of this methodology is to identify key compounds in various types of air-pollutant samples. Once that task is completed, studies on metabolism, sources, environmental exposure and atmospheric chemistry can be undertaken. The principles and methodologies for bioassay directed chemical analysis are presented and illustrated in this paper. Most of this work has been directed toward the characterization of ambient air and diesel particulates, which are used as examples in this report to illustrate the analytical logic used for identifying the bio-active components of complex mixtures.

  12. Direct transfection of viral and plasmid DNA into the liver or spleen of mice.

    PubMed Central

    Dubensky, T W; Campbell, B A; Villarreal, L P

    1984-01-01

    A method for the direct transfection of polyoma viral DNA and polyoma-plasmid recombinant DNA into the liver or spleen of newborn or adult mice was developed. Calcium phosphate-precipitated DNA was injected directly into mouse organs in combination with hyaluronidase and collagenase. Transfected DNA was shown to replicate at moderate efficiency, relative to direct infection of organs with virus. Transfection with viral DNA rapidly led to an acute infection. A polyoma-bacterial plasmid recombinant DNA also was shown to replicate when transfected into mice. With this plasmid, however, genomic-length polyoma DNA rapidly recombined away from the bacterial component and replicated as viral DNA. This method should allow the direct determination of the biological activity of a cloned DNA within a mouse organ. Images PMID:6095303

  13. Excision of plastid marker genes using directly repeated DNA sequences.

    PubMed

    Mudd, Elisabeth A; Madesis, Panagiotis; Avila, Elena Martin; Day, Anil

    2014-01-01

    Excision of marker genes using DNA direct repeats makes use of the predominant homologous recombination pathways present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and microalgae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required, and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes. Release of selection allows the accumulation of marker-free plastid genomes generated by marker excision, which is spontaneous, random, and a unidirectional process. Positive selection is provided by linking marker excision to restoration of the coding region of an herbicide resistance gene from two overlapping but incomplete coding regions. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat-mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastid genomes during growth, development and flowering of T0 plants allows the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops. PMID:24599849

  14. Ultrasensitive Direct Quantification of Nucleobase Modifications in DNA by Surface-Enhanced Raman Scattering: The Case of Cytosine.

    PubMed

    Morla-Folch, Judit; Xie, Hai-nan; Gisbert-Quilis, Patricia; Gómez-de Pedro, Sara; Pazos-Perez, Nicolas; Alvarez-Puebla, Ramon A; Guerrini, Luca

    2015-11-01

    Recognition of chemical modifications in canonical nucleobases of nucleic acids is of key importance since such modified variants act as different genetic encoders, introducing variability in the biological information contained in DNA. Herein, we demonstrate the feasibility of direct SERS in combination with chemometrics and microfluidics for the identification and relative quantification of 4 different cytosine modifications in both single- and double-stranded DNA. The minute amount of DNA required per measurement, in the sub-nanogram regime, removes the necessity of pre-amplification or enrichment steps (which are also potential sources of artificial DNA damages). These findings show great potentials for the development of fast, low-cost and high-throughput screening analytical devices capable of detecting known and unknown modifications in nucleic acids (DNA and RNA) opening new windows of activity in several fields such as biology, medicine and forensic sciences. PMID:26447808

  15. Direct visualization of a DNA glycosylase searching for damage.

    PubMed

    Chen, Liwei; Haushalter, Karl A; Lieber, Charles M; Verdine, Gregory L

    2002-03-01

    DNA glycosylases preserve the integrity of genetic information by recognizing damaged bases in the genome and catalyzing their excision. It is unknown how DNA glycosylases locate covalently modified bases hidden in the DNA helix amongst vast numbers of normal bases. Here we employ atomic-force microscopy (AFM) with carbon nanotube probes to image search intermediates of human 8-oxoguanine DNA glycosylase (hOGG1) scanning DNA. We show that hOGG1 interrogates DNA at undamaged sites by inducing drastic kinks. The sharp DNA bending angle of these non-lesion-specific search intermediates closely matches that observed in the specific complex of 8-oxoguanine-containing DNA bound to hOGG1. These findings indicate that hOGG1 actively distorts DNA while searching for damaged bases. PMID:11927259

  16. Large-area spatially ordered arrays of gold nanoparticles directed by lithographically confined DNA origami.

    PubMed

    Hung, Albert M; Micheel, Christine M; Bozano, Luisa D; Osterbur, Lucas W; Wallraff, Greg M; Cha, Jennifer N

    2010-02-01

    The development of nanoscale electronic and photonic devices will require a combination of the high throughput of lithographic patterning and the high resolution and chemical precision afforded by self-assembly. However, the incorporation of nanomaterials with dimensions of less than 10 nm into functional devices has been hindered by the disparity between their size and the 100 nm feature sizes that can be routinely generated by lithography. Biomolecules offer a bridge between the two size regimes, with sub-10 nm dimensions, synthetic flexibility and a capability for self-recognition. Here, we report the directed assembly of 5-nm gold particles into large-area, spatially ordered, two-dimensional arrays through the site-selective deposition of mesoscopic DNA origami onto lithographically patterned substrates and the precise binding of gold nanocrystals to each DNA structure. We show organization with registry both within an individual DNA template and between components on neighbouring DNA origami, expanding the generality of this method towards many types of patterns and sizes. PMID:20023644

  17. Direct Radiation Damage to Crystalline DNA: What is the Source of Unaltered Base Release?

    PubMed Central

    Razskazovskiy, Yuriy; Debije, Michael G.; Bernhard, William A.

    2008-01-01

    The radiation chemical yields of unaltered base release have been measured in three crystalline double-stranded DNA oligomers after X irradiation at 4 K. The yields of released bases are between 10 and 20% of the total free radical yields measured at 4 K. Using these numbers, we estimate that the yield of DNA strand breaks due to the direct effect is about 0.1 μmol J−1. The damage responsible for base release is independent of the base type (C, G, A or T) and is not scavenged by anthracycline drugs intercalated in the DNA. For these reasons, reactions initiated by the hydroxyl radical have been ruled out as the source of base release. Since the intercalated anthracycline scavenges electrons and holes completely but does not inhibit base release, the possibility for damage transfer from the bases to the sugars can also be ruled out. The results are consistent with a model in which primary radical cations formed directly on the sugar-phosphate backbone react by two competing pathways: deprotonation, which localizes the damage on the sugar, and hole tunneling, which transfers the damage to the base stack. Quantitative estimates indicate that these two processes are approximately equally efficient. PMID:10761004

  18. The direct peptide reactivity assay: selectivity of chemical respiratory allergens.

    PubMed

    Lalko, Jon F; Kimber, Ian; Gerberick, G Frank; Foertsch, Leslie M; Api, Anne Marie; Dearman, Rebecca J

    2012-10-01

    It is well known that some chemicals are capable of causing allergic diseases of the skin and respiratory tract. Commonly, though not exclusively, chemical allergens are associated with the selective development of skin or respiratory sensitization. The reason for this divergence is unclear, although it is hypothesized that the nature of interactions between the chemical hapten and proteins is influential. The direct peptide reactivity assay (DPRA) has been developed as a screen for the identification of skin-sensitizing chemicals, and here we describe the use of this method to explore whether differences exist between skin and respiratory allergens with respect to their peptide-binding properties. Known skin and respiratory sensitizers were reacted with synthetic peptides containing either lysine (Lys) or cysteine (Cys) for 24 h. The samples were analyzed by HPLC/UV, and the loss of peptide from the reaction mixture was expressed as the percent depletion compared with the control. The potential for preferential reactivity was evaluated by comparing the ratio of Lys to Cys depletion (Lys:Cys ratio). The results demonstrate that the majority of respiratory allergens are reactive in the DPRA, and that in contrast to most skin-sensitizing chemicals, preferentially react with the Lys peptide. These data suggest that skin and respiratory chemical allergens can result in different protein conjugates, which may in turn influence the quality of induced immune responses. Overall, these investigations reveal that the DPRA has considerable potential to be incorporated into tiered testing approaches for the identification and characterization of chemical respiratory allergens. PMID:22713598

  19. A microsuspension adaptation of the Bacillus subtilis ''rec'' assay. [Detection of chemically induced DNA damage

    SciTech Connect

    McCarroll, N.E.; Keech, B.H.; Piper, C.E.

    1981-01-01

    We have demonstrated the utility of an Escherichia coli microsuspension assay to detect and characterize chemical mediation of DNA damage by a wide variety of mutagens and carcinogens. The assay have been improved by the development of a microsuspension modification to the Bacillus subtilis ''rec'' assay. The addition of these gram-positive organisms has allowed detection of DNA damage induced by benzo(a)pyrene (B(a)P), 3-aminopyrene (3-AP), 7, 12-dimethylbenz(a)anthrancene (DMBA), 3-methylcholanthrene (3-MC), and 4-nitrobiphenyl (4-NBP). Data presented in this paper from tests of 61 additional compounds, including a representative number of direct and promutagenic agents, indicate that the B subtilis H17 and M45 strains provide an effective microbial system for identification of DNA damage susceptible to postreplicational repair. The results of this study further suggests that the inclusion of these strains in the microsuspension assay for DNA damage will markedly enhance the detection of agents which cannot readily penetrate the intact cell wall of E coli.

  20. Chemical Bonding Technology: Direct Investigation of Interfacial Bonds

    NASA Technical Reports Server (NTRS)

    Koenig, J. L.; Boerio, F. J.; Plueddemann, E. P.; Miller, J.; Willis, P. B.; Cuddihy, E. F.

    1986-01-01

    This is the third Flat-Plate Solar Array (FSA) Project document reporting on chemical bonding technology for terrestrial photovoltaic (PV) modules. The impetus for this work originated in the late 1970s when PV modules employing silicone encapsulation materials were undergoing delamination during outdoor exposure. At that time, manufacturers were not employing adhesion promoters and, hence, module interfaces in common with the silicone materials were only in physical contact and therefore easily prone to separation if, for example, water were to penetrate to the interfaces. Delamination with silicone materials virtually vanished when adhesion promoters, recommended by silicone manufacturers, were used. The activities related to the direct investigation of chemically bonded interfaces are described.

  1. Direct Evidence for the Formation of Precatenanes during DNA Replication*

    PubMed Central

    Cebrián, Jorge; Castán, Alicia; Martínez, Víctor; Kadomatsu-Hermosa, Maridian J.; Parra, Cristina; Fernández-Nestosa, María José; Schaerer, Christian; Hernández, Pablo; Krimer, Dora B.; Schvartzman, Jorge B.

    2015-01-01

    The dynamics of DNA topology during replication are still poorly understood. Bacterial plasmids are negatively supercoiled. This underwinding facilitates strand separation of the DNA duplex during replication. Leading the replisome, a DNA helicase separates the parental strands that are to be used as templates. This strand separation causes overwinding of the duplex ahead. If this overwinding persists, it would eventually impede fork progression. In bacteria, DNA gyrase and topoisomerase IV act ahead of the fork to keep DNA underwound. However, the processivity of the DNA helicase might overcome DNA gyrase and topoisomerase IV. It was proposed that the overwinding that builds up ahead of the fork could force it to swivel and diffuse this positive supercoiling behind the fork where topoisomerase IV would also act to maintain replicating the DNA underwound. Putative intertwining of sister duplexes in the replicated region are called precatenanes. Fork swiveling and the formation of precatenanes, however, are still questioned. Here, we used classical genetics and high resolution two-dimensional agarose gel electrophoresis to examine the torsional tension of replication intermediates of three bacterial plasmids with the fork stalled at different sites before termination. The results obtained indicated that precatenanes do form as replication progresses before termination. PMID:25829493

  2. Brownian dynamics simulation of directional sliding of histone octamers caused by DNA bending

    NASA Astrophysics Data System (ADS)

    Li, Wei; Dou, Shuo-Xing; Xie, Ping; Wang, Peng-Ye

    2006-05-01

    Chromatin-remodeling complexes such as SWI/SNF and RSC of yeast can perturb the structure of nucleosomes in an ATP-dependent manner. Experimental results prove that this chromatin remodeling process involves DNA bending. We simulate the effect of DNA bending, caused by chromatin-remodeling complexes, on directional sliding of histone octamers by Brownian dynamics simulation. The simulation results show that, after a DNA loop being generated at the side of a nucleosome, the histone octamer slides towards this DNA loop until the loop disappears. The DNA loop size is an important factor affecting the process of directional sliding of the histone octamer.

  3. Directed organization of DNA filaments in a soft matter template.

    PubMed

    De Sio, Luciano; D'Aquila, Patrizia; Brunelli, Elvira; Strangi, Giuseppe; Bellizzi, Dina; Passarino, Giuseppe; Umeton, Cesare; Bartolino, Roberto

    2013-03-12

    We have developed a noninvasive, all-optical, holographic technique for permanently aligning liquid crystalline DNA filaments in a microperiodic template realized in soft-composite (polymeric) materials. By combining optical intensity holography with a selective microfluidic etching process, a channelled microstructure has been realized which enables self-assembly of DNA. The striking chemicophysical properties of the structure immobilize the DNA filaments within the microchannels without the need of any kind of surface chemistry or functionalization. Polarized optical, confocal, and electronic microscopies have been used for characterizing the DNA geometry inside the microchannels in terms of birefringence, fluorescence, and nanoscale organization properties. In particular, observation of a far-field diffraction pattern confirms a periodic organization of the DNA filaments inside the polymeric template. PMID:23425153

  4. Comparison of six commercially-available DNA polymerases for direct PCR.

    PubMed

    Miura, Masashi; Tanigawa, Chihiro; Fujii, Yoshito; Kaneko, Satoshi

    2013-01-01

    The use of a "direct PCR" DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens. PMID:24213192

  5. {beta}-carboline derivatives: Novel photosensitizers that intercalate into DNA to cause direct DNA damage in photodynamic therapy

    SciTech Connect

    Guan Huaji; Liu Xiaodong; Peng Wenlie; Cao Rihui; Ma Yan; Chen Hongsheng; Xu Anlong . E-mail: ls36@zsu.edu.cn

    2006-04-14

    Novel 1,3,9-trisubstituted {beta}-carboline derivatives were found to exhibit DNA photocleavage properties under visible light irradiation in a cell-free system, which could be reduced by antioxidant vitamin E. Their photo-cytotoxicity to human tumor cell line HeLa was confirmed, in which apoptosis only contributed a small part to the cell death, and necrosis was the dominating outcome of HeLa cells in photodynamic therapy (PDT) using {beta}-carboline derivatives. Different from other clinical PDT drugs, {beta}-carboline derivatives were demonstrated to be able to distribute in the nucleus and intercalate into DNA, and consequently cause direct DNA damage by photochemical reaction products in PDT, which was proved by the distinct DNA tails in the comet assay and the considerable amount of DNA damaged cells quantified by flow cytometry. This mechanism could be the explanation for the delay of cell proliferation at DNA synthesis and mitosis.

  6. Direct simulations of chemically reacting turbulent mixing layers, part 2

    NASA Astrophysics Data System (ADS)

    Metcalfe, Ralph W.; McMurtry, Patrick A.; Jou, Wen-Huei; Riley, James J.; Givi, Peyman

    1988-06-01

    The results of direct numerical simulations of chemically reacting turbulent mixing layers are presented. This is an extension of earlier work to a more detailed study of previous three dimensional simulations of cold reacting flows plus the development, validation, and use of codes to simulate chemically reacting shear layers with heat release. Additional analysis of earlier simulations showed good agreement with self similarity theory and laboratory data. Simulations with a two dimensional code including the effects of heat release showed that the rate of chemical product formation, the thickness of the mixing layer, and the amount of mass entrained into the layer all decrease with increasing rates of heat release. Subsequent three dimensional simulations showed similar behavior, in agreement with laboratory observations. Baroclinic torques and thermal expansion in the mixing layer were found to produce changes in the flame vortex structure that act to diffuse the pairing vortices, resulting in a net reduction in vorticity. Previously unexplained anomalies observed in the mean velocity profiles of reacting jets and mixing layers were shown to result from vorticity generation by baroclinic torques.

  7. Direct chemical oxidation of mixed or toxic wastes

    SciTech Connect

    Balazs, G B; Cooper, J F; Farmer, J C; Lewis, P

    1999-05-01

    Direct Chemical Oxidation (DCO) is an ambient-pressure, low-temperature (<100 C), and aqueous-based process for general-purpose destruction of the organic fraction of hazardous or mixed waste. It uses the peroxydisulfate anion (S{sub 2}O{sub 8}{sup 2{minus}}) in acid or base solutions. The byproduct of the oxidation reaction, typically sodium or ammonium hydrogen sulfate, may be recycled electrolytically to produce the oxidant. The oxidation kinetic reaction is first order with respect to the peroxydisulfate concentration, expressed in equivalents. The rate constant is constant for nearly all dissolved organic compounds: k{sub a} = 0.01 {+-} 0.005 min{sup {minus}1}. This reflects a common rate-determining step, which is the decomposition of the peroxydisulfate anion into the chemically active derivative, the sulfate radical anion, SO{sub 4}{sup {minus}}. This decomposition is promoted in DCO by raising the operating temperature into the range of 80-100 C. Rates are given for approximately 30 substances with diverse functional groups at low concentrations, and for a number of solid and liquid wastes typical of nuclear and chemical industries. The process has been scale up for treatment studies on chlorinated hydrocarbons, in which the hydrolysis of solvent mixtures was followed by oxidation of products in a series of stirred tank reactors. Cost estimates, safety considerations, and a comprehensive bibliography are given.

  8. Direct simulations of chemically reacting turbulent mixing layers, part 2

    NASA Technical Reports Server (NTRS)

    Metcalfe, Ralph W.; Mcmurtry, Patrick A.; Jou, Wen-Huei; Riley, James J.; Givi, Peyman

    1988-01-01

    The results of direct numerical simulations of chemically reacting turbulent mixing layers are presented. This is an extension of earlier work to a more detailed study of previous three dimensional simulations of cold reacting flows plus the development, validation, and use of codes to simulate chemically reacting shear layers with heat release. Additional analysis of earlier simulations showed good agreement with self similarity theory and laboratory data. Simulations with a two dimensional code including the effects of heat release showed that the rate of chemical product formation, the thickness of the mixing layer, and the amount of mass entrained into the layer all decrease with increasing rates of heat release. Subsequent three dimensional simulations showed similar behavior, in agreement with laboratory observations. Baroclinic torques and thermal expansion in the mixing layer were found to produce changes in the flame vortex structure that act to diffuse the pairing vortices, resulting in a net reduction in vorticity. Previously unexplained anomalies observed in the mean velocity profiles of reacting jets and mixing layers were shown to result from vorticity generation by baroclinic torques.

  9. Retroviral DNA Integration Directed by HIV Integration Protein in Vitro

    NASA Astrophysics Data System (ADS)

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

    1990-09-01

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

  10. Direct observation of DNA overwinding by reverse gyrase

    PubMed Central

    Ogawa, Taisaku; Yogo, Katsunori; Furuike, Shou; Sutoh, Kazuo; Kikuchi, Akihiko; Kinosita, Kazuhiko

    2015-01-01

    Reverse gyrase, found in hyperthermophiles, is the only enzyme known to overwind (introduce positive supercoils into) DNA. The ATP-dependent activity, detected at >70 °C, has so far been studied solely by gel electrophoresis; thus, the reaction dynamics remain obscure. Here, we image the overwinding reaction at 71 °C under a microscope, using DNA containing consecutive 30 mismatched base pairs that serve as a well-defined substrate site. A single reverse gyrase molecule processively winds the DNA for >100 turns. Bound enzyme shows moderate temperature dependence, retaining significant activity down to 50 °C. The unloaded reaction rate at 71 °C exceeds five turns per second, which is >102-fold higher than hitherto indicated but lower than the measured ATPase rate of 20 s−1, indicating loose coupling. The overwinding reaction sharply slows down as the torsional stress accumulates in DNA and ceases at stress of mere ∼5 pN⋅nm, where one more turn would cost only sixfold the thermal energy. The enzyme would thus keep DNA in a slightly overwound state to protect, but not overprotect, the genome of hyperthermophiles against thermal melting. Overwinding activity is also highly sensitive to DNA tension, with an effective interaction length exceeding the size of reverse gyrase, implying requirement for slack DNA. All results point to the mechanism where strand passage relying on thermal motions, as in topoisomerase IA, is actively but loosely biased toward overwinding. PMID:26023188

  11. Mechano-chemical selections of two competitive unfolding pathways of a single DNA i-motif

    NASA Astrophysics Data System (ADS)

    Xu, Yue; Chen, Hu; Qu, Yu-Jie; Artem, K. Efremov; Li, Ming; Ouyang, Zhong-Can; Liu, Dong-Sheng; Yan, Jie

    2014-06-01

    The DNA i-motif is a quadruplex structure formed in tandem cytosine-rich sequences in slightly acidic conditions. Besides being considered as a building block of DNA nano-devices, it may also play potential roles in regulating chromosome stability and gene transcriptions. The stability of i-motif is crucial for these functions. In this work, we investigated the mechanical stability of a single i-motif formed in the human telomeric sequence 5'-(CCCTAA)3CCC, which revealed a novel pH and loading rate-dependent bimodal unfolding force distribution. Although the cause of the bimodal unfolding force species is not clear, we proposed a phenomenological model involving a direct unfolding favored at lower loading rate or higher pH value, which is subject to competition with another unfolding pathway through a mechanically stable intermediate state whose nature is yet to be determined. Overall, the unique mechano—chemical responses of i-motif-provide a new perspective to its stability, which may be useful to guide designing new i-motif-based DNA mechanical nano-devices.

  12. Evolution of DNA and RNA as catalysts for chemical reactions.

    PubMed

    Jäschke, A; Seelig, B

    2000-06-01

    In vitro selection from combinatorial nucleic acid libraries has provided new RNA and DNA molecules that have catalytic properties. Catalyzed reactions now go far beyond self-modifying reactions of nucleic acid molecules. The future application of in vitro selected RNA and DNA catalysts in bioorganic synthesis appears promising. PMID:10826969

  13. Fabrication of Ultrasensitive Field-Effect Transistor DNA Biosensors by a Directional Transfer Technique Based on CVD-Grown Graphene.

    PubMed

    Zheng, Chao; Huang, Le; Zhang, Hong; Sun, Zhongyue; Zhang, Zhiyong; Zhang, Guo-Jun

    2015-08-12

    Most graphene field-effect transistor (G-FET) biosensors are fabricated through a routine process, in which graphene is transferred onto a Si/SiO2 substrate and then devices are subsequently produced by micromanufacture processes. However, such a fabrication approach can introduce contamination onto the graphene surface during the lithographic process, resulting in interference for the subsequent biosensing. In this work, we have developed a novel directional transfer technique to fabricate G-FET biosensors based on chemical-vapor-deposition- (CVD-) grown single-layer graphene (SLG) and applied this biosensor for the sensitive detection of DNA. A FET device with six individual array sensors was first fabricated, and SLG obtained by the CVD-growth method was transferred onto the sensor surface in a directional manner. Afterward, peptide nucleic acid (PNA) was covalently immobilized on the graphene surface, and DNA detection was realized by applying specific target DNA to the PNA-functionalized G-FET biosensor. The developed G-FET biosensor was able to detect target DNA at concentrations as low as 10 fM, which is 1 order of magnitude lower than those reported in a previous work. In addition, the biosensor was capable of distinguishing the complementary DNA from one-base-mismatched DNA and noncomplementary DNA. The directional transfer technique for the fabrication of G-FET biosensors is simple, and the as-constructed G-FET DNA biosensor shows ultrasensitivity and high specificity, indicating its potential application in disease diagnostics as a point-of-care tool. PMID:26203889

  14. Construction of a DNA Nano-Object Directly Demonstrates Computation

    PubMed Central

    Wu, Gang; Jonoska, Natasha; Seeman, Nadrian C.

    2009-01-01

    We demonstrate a computing method in which a DNA nano-object representing the solution of a problem emerges as a result of self-assembly. We report an experiment in which three-vertex colorability for a 6-vertex graph with 9 edges is solved by constructing a DNA molecule representing the colored graph itself. Our findings show that computation based on “shape processing” is a viable alternative to symbol processing when computing by molecular self-assembly. PMID:19607875

  15. Direct measurement of the dielectric polarization properties of DNA.

    PubMed

    Cuervo, Ana; Dans, Pablo D; Carrascosa, José L; Orozco, Modesto; Gomila, Gabriel; Fumagalli, Laura

    2014-09-01

    The electric polarizability of DNA, represented by the dielectric constant, is a key intrinsic property that modulates DNA interaction with effector proteins. Surprisingly, it has so far remained unknown owing to the lack of experimental tools able to access it. Here, we experimentally resolved it by detecting the ultraweak polarization forces of DNA inside single T7 bacteriophages particles using electrostatic force microscopy. In contrast to the common assumption of low-polarizable behavior like proteins (εr ∼ 2-4), we found that the DNA dielectric constant is ∼ 8, considerably higher than the value of ∼ 3 found for capsid proteins. State-of-the-art molecular dynamic simulations confirm the experimental findings, which result in sensibly decreased DNA interaction free energy than normally predicted by Poisson-Boltzmann methods. Our findings reveal a property at the basis of DNA structure and functions that is needed for realistic theoretical descriptions, and illustrate the synergetic power of scanning probe microscopy and theoretical computation techniques. PMID:25136104

  16. Direct measurement of the dielectric polarization properties of DNA

    PubMed Central

    Cuervo, Ana; Dans, Pablo D.; Carrascosa, José L.; Orozco, Modesto; Gomila, Gabriel; Fumagalli, Laura

    2014-01-01

    The electric polarizability of DNA, represented by the dielectric constant, is a key intrinsic property that modulates DNA interaction with effector proteins. Surprisingly, it has so far remained unknown owing to the lack of experimental tools able to access it. Here, we experimentally resolved it by detecting the ultraweak polarization forces of DNA inside single T7 bacteriophages particles using electrostatic force microscopy. In contrast to the common assumption of low-polarizable behavior like proteins (εr ∼ 2–4), we found that the DNA dielectric constant is ∼8, considerably higher than the value of ∼3 found for capsid proteins. State-of-the-art molecular dynamic simulations confirm the experimental findings, which result in sensibly decreased DNA interaction free energy than normally predicted by Poisson–Boltzmann methods. Our findings reveal a property at the basis of DNA structure and functions that is needed for realistic theoretical descriptions, and illustrate the synergetic power of scanning probe microscopy and theoretical computation techniques. PMID:25136104

  17. DNA-directed capture of primary cells from a complex mixture and controlled orthogonal release monitored by SPR imaging.

    PubMed

    Bombera, Radoslaw; Leroy, Loïc; Livache, Thierry; Roupioz, Yoann

    2012-03-15

    Many biological samples are composed of several cell types. Qualitative and quantitative analysis of these complex mixtures is of major interest for both diagnostic and biomedical applications. Because large amounts of biological material are often challenging to collect, tremendous efforts have been made for a decade to design miniaturized platforms-such as lab-on-a-chip or microarrays-to run sensitive and reliable analysis from tiny quantities of starting material. Although barely explored so far, the release of resolved cellular samples constitutes an exciting strategy for further cell analysis. Herein, we propose a DNA-based biochip suitable for cell-type analysis in a label-free manner. The DNA-array is firstly converted into antibody-array using antibody-DNA conjugates. These protein-DNA hybrid molecules are chemically synthesized by covalent coupling of short oligonucleotides to antibodies directed against cell-type specific markers. We show not only specific capture of primary spleen cells on protein-DNA microarray spots but also their fast and specific orthogonal release according to the antibody-DNA combinations by incorporating restriction sites in DNA. Both molecular and cellular interactions occurring on the biochip are monitored by surface plasmon resonance (SPR) imaging. This optical technique turns out to be a powerful way to monitor, in real-time, biological interactions occurring on the microarrayed features. PMID:22236780

  18. Inter-laboratory evaluation of the ISO standard 11063 "Soil quality - Method to directly extract DNA from soil samples".

    PubMed

    Petric, I; Philippot, L; Abbate, C; Bispo, A; Chesnot, T; Hallin, S; Laval, K; Lebeau, T; Lemanceau, P; Leyval, C; Lindström, K; Pandard, P; Romero, E; Sarr, A; Schloter, M; Simonet, P; Smalla, K; Wilke, B-M; Martin-Laurent, F

    2011-03-01

    Extracting DNA directly from micro-organisms living in soil is a crucial step for the molecular analysis of soil microbial communities. However, the use of a plethora of different soil DNA extraction protocols, each with its own bias, makes accurate data comparison difficult. To overcome this problem, a method for soil DNA extraction was proposed to the International Organization for Standardization (ISO) in 2006. This method was evaluated by 13 independent European laboratories actively participating in national and international ring tests. The reproducibility of the standardized method for molecular analyses was evaluated by comparing the amount of DNA extracted, as well as the abundance and genetic structure of the total bacterial community in the DNA extracted from 12 different soils by the 13 laboratories. High quality DNA was successfully extracted from all 12 soils, despite different physical and chemical characteristics and a range of origins from arable soils, through forests to industrial sites. Quantification of the 16S rRNA gene abundances by real time PCR and analysis of the total bacterial community structure by automated ribosomal intergenic spacer analysis (A-RISA) showed acceptable to good levels of reproducibility. Based on the results of both ring-tests, the method was unanimously approved by the ISO as an international standard method and the normative protocol will now be disseminated within the scientific community. Standardization of a soil DNA extraction method will improve data comparison, facilitating our understanding of soil microbial diversity and soil quality monitoring. PMID:21256879

  19. The shape of the DNA minor groove directs binding by the DNA-bending protein Fis

    SciTech Connect

    Stella, Stefano; Cascio, Duilio; Johnson, Reid C.

    2010-06-21

    The bacterial nucleoid-associated protein Fis regulates diverse reactions by bending DNA and through DNA-dependent interactions with other control proteins and enzymes. In addition to dynamic nonspecific binding to DNA, Fis forms stable complexes with DNA segments that share little sequence conservation. Here we report the first crystal structures of Fis bound to high- and low-affinity 27-base-pair DNA sites. These 11 structures reveal that Fis selects targets primarily through indirect recognition mechanisms involving the shape of the minor groove and sequence-dependent induced fits over adjacent major groove interfaces. The DNA shows an overall curvature of {approx}65{sup o}, and the unprecedented close spacing between helix-turn-helix motifs present in the apodimer is accommodated by severe compression of the central minor groove. In silico DNA structure models show that only the roll, twist, and slide parameters are sufficient to reproduce the changes in minor groove widths and recreate the curved Fis-bound DNA structure. Models based on naked DNA structures suggest that Fis initially selects DNA targets with intrinsically narrow minor grooves using the separation between helix-turn-helix motifs in the Fis dimer as a ruler. Then Fis further compresses the minor groove and bends the DNA to generate the bound structure.

  20. Asymmetric Unwrapping of Nucleosomes under Tension Directed by DNA Local Flexibility

    PubMed Central

    Ngo, Thuy T. M.; Zhang, Qiucen; Zhou, Ruobo

    2015-01-01

    Summary Dynamics of the nucleosome and exposure of nucleosomal DNA play key roles in many nuclear processes but local dynamics of the nucleosome and its modulation by DNA sequence are poorly understood. Using single-molecule assays we observed that the nucleosome can unwrap asymmetrically and directionally under force. The relative DNA flexibility of the inner quarters of nucleosomal DNA controls the unwrapping direction such that the nucleosome unwraps from the stiffer side. If the DNA flexibility is similar on two sides, it stochastically unwraps from either side. The two ends of the nucleosome are orchestrated such that the opening of one end helps to stabilize the other end, providing a mechanism to amplify even small differences in flexibility to a large asymmetry in nucleosome stability. Our discovery of DNA flexibility as a critical factor for nucleosome dynamics and mechanical stability suggests a novel mechanism of gene regulation by DNA sequence and modifications. PMID:25768909

  1. Asymmetric unwrapping of nucleosomes under tension directed by DNA local flexibility.

    PubMed

    Ngo, Thuy T M; Zhang, Qiucen; Zhou, Ruobo; Yodh, Jaya G; Ha, Taekjip

    2015-03-12

    Dynamics of the nucleosome and exposure of nucleosomal DNA play key roles in many nuclear processes, but local dynamics of the nucleosome and its modulation by DNA sequence are poorly understood. Using single-molecule assays, we observed that the nucleosome can unwrap asymmetrically and directionally under force. The relative DNA flexibility of the inner quarters of nucleosomal DNA controls the unwrapping direction such that the nucleosome unwraps from the stiffer side. If the DNA flexibility is similar on two sides, it stochastically unwraps from either side. The two ends of the nucleosome are orchestrated such that the opening of one end helps to stabilize the other end, providing a mechanism to amplify even small differences in flexibility to a large asymmetry in nucleosome stability. Our discovery of DNA flexibility as a critical factor for nucleosome dynamics and mechanical stability suggests a novel mechanism of gene regulation by DNA sequence and modifications. PMID:25768909

  2. Chemical carcinogenesis — the price for DNA-repair?

    NASA Astrophysics Data System (ADS)

    Wintersberger, Ulrike

    1982-03-01

    This essay examines the possibility of merging the mutation theory of cancer with the hypothesis that cancer is a change in the state of the differentiation of cells. It is suggested that during normal development DNA rearrangements occur, concerning genes which code for differentiation specific cell communication proteins. These proteins are responsible for the proper functioning of growth control in a multicellular organism. DNA-damaging agents — mutagens — induce DNA repair enzymes, some of which may catalyse illegitimate genome rearrangements, thus leading to a change of the balance between growth and differentiation. A cell with a selective advantage may arise and become the origin of a tumor.

  3. New approach for direct chemical synthesis of hexagonal Co nanoparticles

    NASA Astrophysics Data System (ADS)

    Abel, Frank M.; Tzitzios, Vasilis; Hadjipanayis, George C.

    2016-02-01

    In this paper, we explore the possibility of producing hexagonal Cobalt nanoparticles, with high saturation magnetization by direct chemical synthesis. The nanoparticles were synthesized by reduction of anhydrous cobalt (II) chloride by NaBH4 in tetraglyme at temperatures in the range of 200-270 °C under a nitrogen-hydrogen atmosphere. The reactions were done at high temperatures to allow for the formation of as-made hexagonal cobalt. The size of the particles was controlled by the addition of different surfactants. The best magnetic properties so far were obtained on spherical hexagonal Co nanoparticles with an average size of 45 nm, a saturation magnetization of 143 emu/g and coercivity of 500 Oe. the saturation magnetization and coercivity were further improved by annealing the Co nanoparticles leading to saturation magnetization of 160 emu/g and coercivity of 540 Oe.

  4. Characterization of glycosphingolipids by direct inlet chemical ionization mass spectrometry.

    PubMed

    Ariga, T; Murata, T; Oshima, M; Maezawa, M; Miyatake, T

    1980-09-01

    Permethylated derivatives of cerebrosides and ceramide di-, tri-, tetra-, and penta-hexosides were analyzed by the direct inlet ammonia chemical ionization (CI) mass spectrometry. In the CI mass spectra, the fragment ions produced by the loss of methanol from the protonated molecular ion were observed in all of the glycosphingolipids. Other fragment ions due to the cleavage of glycosidic moiety were major ones under the CI conditions. These ions provide information on the molecular species of glycosphingolipids and the sugar sequence of their oligosaccharides. Glycosphingolipids with hydroxy fatty acids could also be differentiated from those with nonhydroxy fatty acids by comparing the intensities of characteristic fragment ions. The CI method should be particularly useful in structural studies of glycosphingolipids from natural sources. PMID:7441059

  5. Direct simulations of chemically reacting turbulent mixing layers

    NASA Technical Reports Server (NTRS)

    Riley, J. J.; Metcalfe, R. W.

    1984-01-01

    The report presents the results of direct numerical simulations of chemically reacting turbulent mixing layers. The work consists of two parts: (1) the development and testing of a spectral numerical computer code that treats the diffusion reaction equations; and (2) the simulation of a series of cases of chemical reactions occurring on mixing layers. The reaction considered is a binary, irreversible reaction with no heat release. The reacting species are nonpremixed. The results of the numerical tests indicate that the high accuracy of the spectral methods observed for rigid body rotation are also obtained when diffusion, reaction, and more complex flows are considered. In the simulations, the effects of vortex rollup and smaller scale turbulence on the overall reaction rates are investigated. The simulation results are found to be in approximate agreement with similarity theory. Comparisons of simulation results with certain modeling hypotheses indicate limitations in these hypotheses. The nondimensional product thickness computed from the simulations is compared with laboratory values and is found to be in reasonable agreement, especially since there are no adjustable constants in the method.

  6. Delay chemical master equation: direct and closed-form solutions

    PubMed Central

    Leier, Andre; Marquez-Lago, Tatiana T.

    2015-01-01

    The stochastic simulation algorithm (SSA) describes the time evolution of a discrete nonlinear Markov process. This stochastic process has a probability density function that is the solution of a differential equation, commonly known as the chemical master equation (CME) or forward-Kolmogorov equation. In the same way that the CME gives rise to the SSA, and trajectories of the latter are exact with respect to the former, trajectories obtained from a delay SSA are exact representations of the underlying delay CME (DCME). However, in contrast to the CME, no closed-form solutions have so far been derived for any kind of DCME. In this paper, we describe for the first time direct and closed solutions of the DCME for simple reaction schemes, such as a single-delayed unimolecular reaction as well as chemical reactions for transcription and translation with delayed mRNA maturation. We also discuss the conditions that have to be met such that such solutions can be derived. PMID:26345616

  7. Proton-induced direct and indirect damage of plasmid DNA.

    PubMed

    Vyšín, Luděk; Pachnerová Brabcová, Kateřina; Štěpán, Václav; Moretto-Capelle, Patrick; Bugler, Beatrix; Legube, Gaelle; Cafarelli, Pierre; Casta, Romain; Champeaux, Jean Philippe; Sence, Martine; Vlk, Martin; Wagner, Richard; Štursa, Jan; Zach, Václav; Incerti, Sebastien; Juha, Libor; Davídková, Marie

    2015-08-01

    Clustered DNA damage induced by 10, 20 and 30 MeV protons in pBR322 plasmid DNA was investigated. Besides determination of strand breaks, additional lesions were detected using base excision repair enzymes. The plasmid was irradiated in dry form, where indirect radiation effects were almost fully suppressed, and in water solution containing only minimal residual radical scavenger. Simultaneous irradiation of the plasmid DNA in the dry form and in the solution demonstrated the contribution of the indirect effect as prevalent. The damage composition slightly differed when comparing the results for liquid and dry samples. The obtained data were also subjected to analysis concerning different methodological approaches, particularly the influence of irradiation geometry, models used for calculation of strand break yields and interpretation of the strand breaks detected with the enzymes. It was shown that these parameters strongly affect the results. PMID:26007308

  8. Directed assembly of discrete gold nanoparticle groupings usingbranched DNA scaffolds

    SciTech Connect

    Claridge, Shelley A.; Goh, Sarah L.; Frechet, Jean M.J.; Williams, Shara C.; Micheel, Christine M.; Alivisatos, A. Paul

    2004-09-14

    The concept of self-assembled dendrimers is explored for the creation of discrete nanoparticle assemblies. Hybridization of branched DNA trimers and nanoparticle-DNA conjugates results in the synthesis of nanoparticle trimer and tetramer complexes. Multiple tetramer architectures are investigated, utilizing Au-DNA conjugates with varying secondary structural motifs. Hybridization products are analyzed by gel electrophoresis, and discrete bands are observed corresponding to structures with increasing numbers of hybridization events. Samples extracted from each band are analyzed by transmission electron microscopy, and statistics compiled from micrographs are used to compare assembly characteristics for each architecture. Asymmetric structures are also produced in which both 5 and 10 nm Au particles are assembled on branched scaffolds.

  9. Increment of DNA topoisomerases in chemically and virally transformed cells

    SciTech Connect

    Crespi, M.D.; Mladovan, A.G.; Baldi, A. )

    1988-03-01

    The activities of topoisomerases I and II were assayed in subcellular extracts obtained from nontumorigenic BALB/c 3T3 A31 and normal rat kidney (NRK) cell lines and from the same cells transformed by benzo(a)pyrene (BP-A31), Moloney (M-MSV-A31) and Kirsten (K-A31) sarcoma viruses, and simian virus 40 (SV-NRK). The enzymatic activity of topoisomerase I was monitored by the relaxation of negatively supercoiled pBR322 DNA and by the formation of covalent complexes between {sup 32}P-labeled DNA and topoisomerase I. Topoisomerase II activity was determined by decatenation of kinetoplast DNA (k-DNA). It was found that nuclear and cytoplasmic type I topoisomerase specific activities were higher in every transformed cell line than in the normal counterparts. These differences cannot be attributed to an inhibitory factor present in A31 cells. When chromatin was treated at increasing ionic strengths, the 0.4 M NaCl extract showed the highest topoisomerase I specific activity. Spontaneously transformed A31 cells showed topoisomerase I activity similar to that of extracts of cells transformed by benzo(a)pyrene. Topoisomerase II specific activity was also increased in SV-NRK cells, as judged by the assay for decatenation of k-DNA to yield minicircle DNA.

  10. Theoretical studies of chemical reactivity of metabolically activated forms of aromatic amines toward DNA.

    PubMed

    Shamovsky, Igor; Ripa, Lena; Blomberg, Niklas; Eriksson, Leif A; Hansen, Peter; Mee, Christine; Tyrchan, Christian; O'Donovan, Mike; Sjö, Peter

    2012-10-15

    The metabolism of aromatic and heteroaromatic amines (ArNH₂) results in nitrenium ions (ArNH⁺) that modify nucleobases of DNA, primarily deoxyguanosine (dG), by forming dG-C8 adducts. The activated amine nitrogen in ArNH⁺ reacts with the C8 of dG, which gives rise to mutations in DNA. For the most mutagenic ArNH₂, including the majority of known genotoxic carcinogens, the stability of ArNH⁺ is of intermediate magnitude. To understand the origin of this observation as well as the specificity of reactions of ArNH⁺ with guanines in DNA, we investigated the chemical reactivity of the metabolically activated forms of ArNH₂, that is, ArNHOH and ArNHOAc, toward 9-methylguanine by DFT calculations. The chemical reactivity of these forms is determined by the rate constants of two consecutive reactions leading to cationic guanine intermediates. The formation of ArNH⁺ accelerates with resonance stabilization of ArNH⁺, whereas the formed ArNH⁺ reacts with guanine derivatives with the constant diffusion-limited rate until the reaction slows down when ArNH⁺ is about 20 kcal/mol more stable than PhNH⁺. At this point, ArNHOH and ArNHOAc show maximum reactivity. The lowest activation energy of the reaction of ArNH⁺ with 9-methylguanine corresponds to the charge-transfer π-stacked transition state (π-TS) that leads to the direct formation of the C8 intermediate. The predicted activation barriers of this reaction match the observed absolute rate constants for a number of ArNH⁺. We demonstrate that the mutagenic potency of ArNH₂ correlates with the rate of formation and the chemical reactivity of the metabolically activated forms toward the C8 atom of dG. On the basis of geometric consideration of the π-TS complex made of genotoxic compounds with long aromatic systems, we propose that precovalent intercalation in DNA is not an essential step in the genotoxicity pathway of ArNH₂. The mechanism-based reasoning suggests rational design strategies to

  11. A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE: INDUCED BY RADIATION, CHEMICALS AND ENZYMES

    EPA Science Inventory

    A simple and rapid assay to detect DNA damage is reported. This assay is based on the ability of certain dyes to fluoresce upon intercalation with dsDNA. Damage caused by ultraviolet (UV) radiation, chemicals or restriction enzymes is detected using this assay. UV radiation at...

  12. DEVELOPMENT OF RAPID TECHNIQUES FOR DETECTION OF CHEMICALLY-INDUCED DNA DAMAGE

    EPA Science Inventory

    Rapid and cost-effective indicator assays are being developed which may be used as a rapid screen to assess the potential for exposure to hazardous compounds that can be related to a biological target (e.g., DNA). Chemically-induced DNA damage will be measured using surrogate DN...

  13. The direct application of the polymerase chain reaction to DNA extracted from foods.

    PubMed

    Dickinson, J H; Kroll, R G; Grant, K A

    1995-04-01

    Two methods for the successful extraction of DNA from foods are described. The rapid lysis method uses a proteinase K buffer system to lyse cells and solubilize food samples. DNA is then precipitated using isopropanol. The second method achieves cell lysis using toluene and mutanolysin, and solubilization using guanidium thiocyanate. Following protein removal with organic solvents DNA is precipitated with isopropanol. Both methods enabled the polymerase chain reaction to be applied directly to DNA extracted from samples of cheese, coleslaw and raw chicken and allowed the direct rapid, sensitive and specific detection of Yersinia enterocolitica, Aerococcus viridans and Listeria monocytogenes in these foods. PMID:7766115

  14. DNA binding fluorescent proteins for the direct visualization of large DNA molecules.

    PubMed

    Lee, Seonghyun; Oh, Yeeun; Lee, Jungyoon; Choe, Sojeong; Lim, Sangyong; Lee, Hyun Soo; Jo, Kyubong; Schwartz, David C

    2016-01-01

    Fluorescent proteins that also bind DNA molecules are useful reagents for a broad range of biological applications because they can be optically localized and tracked within cells, or provide versatile labels for in vitro experiments. We report a novel design for a fluorescent, DNA-binding protein (FP-DBP) that completely 'paints' entire DNA molecules, whereby sequence-independent DNA binding is accomplished by linking a fluorescent protein to two small peptides (KWKWKKA) using lysine for binding to the DNA phosphates, and tryptophan for intercalating between DNA bases. Importantly, this ubiquitous binding motif enables fluorescent proteins (Kd = 14.7 μM) to confluently stain DNA molecules and such binding is reversible via pH shifts. These proteins offer useful robust advantages for single DNA molecule studies: lack of fluorophore mediated photocleavage and staining that does not perturb polymer contour lengths. Accordingly, we demonstrate confluent staining of naked DNA molecules presented within microfluidic devices, or localized within live bacterial cells. PMID:26264666

  15. DNA binding fluorescent proteins for the direct visualization of large DNA molecules

    PubMed Central

    Lee, Seonghyun; Oh, Yeeun; Lee, Jungyoon; Choe, Sojeong; Lim, Sangyong; Lee, Hyun Soo; Jo, Kyubong; Schwartz, David C.

    2016-01-01

    Fluorescent proteins that also bind DNA molecules are useful reagents for a broad range of biological applications because they can be optically localized and tracked within cells, or provide versatile labels for in vitro experiments. We report a novel design for a fluorescent, DNA-binding protein (FP-DBP) that completely ‘paints’ entire DNA molecules, whereby sequence-independent DNA binding is accomplished by linking a fluorescent protein to two small peptides (KWKWKKA) using lysine for binding to the DNA phosphates, and tryptophan for intercalating between DNA bases. Importantly, this ubiquitous binding motif enables fluorescent proteins (Kd = 14.7 μM) to confluently stain DNA molecules and such binding is reversible via pH shifts. These proteins offer useful robust advantages for single DNA molecule studies: lack of fluorophore mediated photocleavage and staining that does not perturb polymer contour lengths. Accordingly, we demonstrate confluent staining of naked DNA molecules presented within microfluidic devices, or localized within live bacterial cells. PMID:26264666

  16. Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase

    PubMed Central

    Morin, José A.; Cao, Francisco J.; Lázaro, José M.; Arias-Gonzalez, J. Ricardo; Valpuesta, José M.; Carrascosa, José L.; Salas, Margarita; Ibarra, Borja

    2015-01-01

    During DNA replication replicative polymerases move in discrete mechanical steps along the DNA template. To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication. We determine the main kinetic parameters of the nucleotide incorporation cycle and their dependence on external load and nucleotide (dNTP) concentration. The data is inconsistent with power stroke models for translocation, instead supports a loose-coupling mechanism between chemical catalysis and mechanical translocation during DNA replication. According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state. We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (∼50 pN). PMID:25800740

  17. Chemically directed assembly of nanoparticles for material and biological applications

    NASA Astrophysics Data System (ADS)

    Park, Myoung-Hwan

    The unique electronic, magnetic, and optical properties of nanoparticles (NPs) make them useful building blocks for nanodevices and biofabrication. Site-selective immobilization/deposition of NPs on surfaces at desired positions is an important fabrication step in realizing the potential of nanomaterials in these applications. In this thesis, my research has focused on developing new strategies for mono- and multilayered-NP deposition on surfaces, increasing the stability of NP-assembles upon various surfaces for practical use of NP-based devices. Chemically directed dithiocarbamate binding of amine groups to NPs in the presence of CS2 was used for enhancing the robustness of NP assembles. Such patterning methodologies have allowed me to use site-directed NP immobilization in applications as diverse as microcontact printing, nanomolding in capillaries, nanoimprint lithography, and photolithography. Also, I have developed a simple and reliable one-step technique to form robust dendrimer-NP nanocomposites using dithiocarbamate-based chemistry. These composites are able to encapsulate and release various therapeutics, providing controllable sustained release and to separate small molecules and biomacromolecules.

  18. Direct observation of DNA threading in flap endonuclease complexes.

    PubMed

    AlMalki, Faizah A; Flemming, Claudia S; Zhang, Jing; Feng, Min; Sedelnikova, Svetlana E; Ceska, Tom; Rafferty, John B; Sayers, Jon R; Artymiuk, Peter J

    2016-07-01

    Maintenance of genome integrity requires that branched nucleic acid molecules be accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki-fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates and products, at resolutions of 1.9-2.2 Å. They reveal single-stranded DNA threading through a hole in the enzyme, which is enclosed by an inverted V-shaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate, thereby juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate's single-stranded branch approaches, threads through and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual 'fly-casting, thread, bend and barb' mechanism. PMID:27273516

  19. Direct interaction between cohesin complex and DNA replication machinery

    SciTech Connect

    Ryu, Min-Jung; Kim, Beom-Jun; Lee, Jeong-Won; Lee, Min-Woo; Choi, Hyun-Kyung; Kim, Seong-Tae . E-mail: stkim@med.skku.ac.kr

    2006-03-17

    Structural maintenance of chromosome 1 (Smc1) is a multifunctional protein, which has been implicated in sister chromatid cohesion, DNA recombination and repair, and the activation of cell cycle checkpoints by ionizing radiation, ultraviolet light, and other genotoxic agents. In order to identify the proteins that interact with Smc1, we conducted the Tandem affinity purification (TAP) technique and analyzed the Smc1-interacting proteins via MALDI-TOF mass spectrometry. We identified minichromosome maintenance 7 (Mcm7), an essential component of the pre-replication complex, as a novel Smc1-interacting protein. Co-immunoprecipitation revealed an interaction occurring between Smc1 and Mcm7, both in vitro and in vivo. Using a GST pull-down assay, we determined that Smc1 interacts physically with Mcm7 via its N-terminal and hinge regions, and Mcm7 interacts with Smc1 via its middle region. Interestingly, we also discovered that Smc1 interacts with other DNA replication proteins, including Mcm6, RFC1, and DNA polymerase {alpha}. These results suggest that a functional link exists between the cohesin complex and DNA replication proteins.

  20. The Influence of DNA Configuration on the Direct Strand Break Yield

    PubMed Central

    Bernal, M. A.; deAlmeida, C. E.; Incerti, S.; Champion, C.; Ivanchenko, V.; Francis, Z.

    2015-01-01

    Purpose. To study the influence of DNA configuration on the direct damage yield. No indirect effect has been accounted for. Methods. The GEANT4-DNA code was used to simulate the interactions of protons and alpha particles with geometrical models of the A-, B-, and Z-DNA configurations. The direct total, single, and double strand break yields and site-hit probabilities were determined. Certain features of the energy deposition process were also studied. Results. A slight increase of the site-hit probability as a function of the incident particle linear energy transfer was found for each DNA configuration. Each DNA form presents a well-defined site-hit probability, independently of the particle linear energy transfer. Approximately 70% of the inelastic collisions and ~60% of the absorbed dose are due to secondary electrons. These fractions are slightly higher for protons than for alpha particles at the same incident energy. Conclusions. The total direct strand break yield for a given DNA form depends weakly on DNA conformation topology. This yield is practically determined by the target volume of the DNA configuration. However, the double strand break yield increases with the packing ratio of the DNA double helix; thus, it depends on the DNA conformation. PMID:26124855

  1. Direct measurement of DNA-mediated adhesion between lipid bilayers.

    PubMed

    Shimobayashi, S F; Mognetti, B M; Parolini, L; Orsi, D; Cicuta, P; Di Michele, L

    2015-06-28

    Multivalent interactions between deformable mesoscopic units are ubiquitous in biology, where membrane macromolecules mediate the interactions between neighbouring living cells and between cells and solid substrates. Lately, analogous artificial materials have been synthesised by functionalising the outer surface of compliant Brownian units, for example emulsion droplets and lipid vesicles, with selective linkers, in particular short DNA sequences. This development extended the range of applicability of DNA as a selective glue, originally applied to solid nano and colloidal particles. On very deformable lipid vesicles, the coupling between statistical effects of multivalent interactions and mechanical deformation of the membranes gives rise to complex emergent behaviours, as we recently contributed to demonstrate [Parolini et al., Nat. Commun., 2015, 6, 5948]. Several aspects of the complex phenomenology observed in these systems still lack a quantitative experimental characterisation and a fundamental understanding. Here we focus on the DNA-mediated multivalent interactions of a single liposome adhering to a flat supported bilayer. This simplified geometry enables the estimate of the membrane tension induced by the DNA-mediated adhesive forces acting on the liposome. Our experimental investigation is completed by morphological measurements and the characterisation of the DNA-melting transition, probed by in situ Förster Resonant Energy Transfer spectroscopy. Experimental results are compared with the predictions of an analytical theory that couples the deformation of the vesicle to a full description of the statistical mechanics of mobile linkers. With at most one fitting parameter, our theory is capable of semi-quantitatively matching experimental data, confirming the quality of the underlying assumptions. PMID:25989828

  2. The interplay of primer-template DNA phosphorylation status and single-stranded DNA binding proteins in directing clamp loaders to the appropriate polarity of DNA

    PubMed Central

    Hayner, Jaclyn N.; Douma, Lauren G.; Bloom, Linda B.

    2014-01-01

    Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3′OH (3′DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3′DNA, fluorescent β and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5′phosphate (5′P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5′DNA). The 5′P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3′DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5′DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3′DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role. PMID:25159615

  3. DNA glycosylase enzymes induced during chemical adaptation of M. luteus.

    PubMed Central

    Riazuddin, S; Athar, A; Ahmed, Z; Lali, S M; Sohail, A

    1987-01-01

    Five peaks of DNA glycosylase activity showing a preference for MNNG alkylated DNA have been identified from extracts of adapted M. luteus. They are numerically designated as GI to GV in order of their decreasing molecular weights. The first two of these peaks have been highly purified. GI, is a constitutive heat labile protein, 35% stimulated by the presence of 50 mM NaCl, acts exclusively on 3 MeA residues in alkylated DNA, 60-70% inhibited by the presence of 2 mM free 3MeA and has been designated as 3MeA DNA glycosylase enzyme. GII, which is an inducible protein, is heat stable, 28% inhibited by the presence of 50 mM NaCl, removes 3MeA, 3MeG, 7MeA & 7MeG with different efficiency, and has been designated as 3,7 methylpurine DNA glycosylase enzyme. The rate of release of 3 methylpurines is 30 times that of 7MeG. There is no activity of either enzyme on O2-MeC, O2-MeT, O4-MeT or O6-MeG. The apparent molecular weights of GI and GII proteins are 28 Kd and 22 Kd respectively. PMID:3628000

  4. Rates of Chemical Cleavage of DNA and RNA Oligomers Containing Guanine Oxidation Products

    PubMed Central

    2016-01-01

    The nucleobase guanine in DNA (dG) and RNA (rG) has the lowest standard reduction potential of the bases, rendering it a major site of oxidative damage in these polymers. Mapping the sites at which oxidation occurs in an oligomer via chemical reagents utilizes hot piperidine for cleaving oxidized DNA and aniline (pH 4.5) for cleaving oxidized RNA. In the present studies, a series of time-dependent cleavages of DNA and RNA strands containing various guanine lesions were examined to determine the strand scission rate constants. The guanine base lesions 8-oxo-7,8-dihydroguanine (OG), spiroiminodihydantoin (Sp), 5-guanidinohydantoin (Gh), 2,2,4-triamino-2H-oxazol-5-one (Z), and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) were evaluated in piperidine-treated DNA and aniline-treated RNA. These data identified wide variability in the chemical lability of the lesions studied in both DNA and RNA. Further, the rate constants for cleaving lesions in RNA were generally found to be significantly smaller than for lesions in DNA. The OG nucleotides were poorly cleaved in DNA and RNA; Sp nucleotides were slowly cleaved in DNA and did not cleave significantly in RNA; Gh and Z nucleotides cleaved in both DNA and RNA at intermediate rates; and 2Ih oligonucleotides cleaved relatively quickly in both DNA and RNA. The data are compared and contrasted with respect to future experimental design. PMID:25853314

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

    PubMed Central

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

    2015-01-01

    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. PMID:26649734

  6. Physisorption of DNA molecules on chemically modified single-walled carbon nanotubes with and without sonication.

    PubMed

    Umemura, Kazuo; Ishibashi, Yu; Oura, Shusuke

    2016-09-01

    We investigated the physisorption phenomenon of single-stranded DNA (ssDNA) molecules onto two types of commercially available chemically functionalized single-walled carbon nanotubes (SWNTs) by atomic force microscopy (AFM) and agarose gel electrophoresis. We found that DNA molecules can adsorb on the water-soluble SWNT surfaces without sonication, although sonication treatment has been used for hybridization of DNA and SWNTs in many previous studies. Using our method, damage of DNA molecules by sonication can be avoided. On the other hand, the amount of DNA molecules adsorbed on SWNT surfaces increased when the samples were sonicated. This fact suggests that the sonication is effective not only at debundling of SWNTs, but also at assisting DNA adsorption. Furthermore, DNA adsorption was affected by the types of functionalized SWNTs. In the case of SWNTs functionalized with polyethylene glycol (PEG-SWNT), physisorption of ssDNA molecules was confirmed only by agarose-gel electrophoresis. In contrast, amino-terminated SWNTs (NH2-SWNTs) showed a change in the height distribution profile based on AFM observations. These results suggest that DNA molecules tended to adsorb to NH2-SWNT surfaces, although DNA molecules can also adsorb on PEG-SWNT surfaces. Our results revealed fundamental information for developing nanobiodevices using hybrids of DNA and SWNTs. PMID:26846296

  7. GADD45α inhibition of DNMT1 dependent DNA methylation during homology directed DNA repair

    PubMed Central

    Lee, Bongyong; Morano, Annalisa; Porcellini, Antonio; Muller, Mark T.

    2012-01-01

    In this work, we examine regulation of DNA methyltransferase 1 (DNMT1) by the DNA damage inducible protein, GADD45α. We used a system to induce homologous recombination (HR) at a unique double-strand DNA break in a GFP reporter in mammalian cells. After HR, the repaired DNA is hypermethylated in recombinant clones showing low GFP expression (HR-L expressor class), while in high expressor recombinants (HR-H clones) previous methylation patterns are erased. GADD45α, which is transiently induced by double-strand breaks, binds to chromatin undergoing HR repair. Ectopic overexpression of GADD45α during repair increases the HR-H fraction of cells (hypomethylated repaired DNA), without altering the recombination frequency. Conversely, silencing of GADD45α increases methylation of the recombined segment and amplifies the HR-L expressor (hypermethylated) population. GADD45α specifically interacts with the catalytic site of DNMT1 and inhibits methylation activity in vitro. We propose that double-strand DNA damage and the resulting HR process involves precise, strand selected DNA methylation by DNMT1 that is regulated by GADD45α. Since GADD45α binds with high avidity to hemimethylated DNA intermediates, it may also provide a barrier to spreading of methylation during or after HR repair. PMID:22135303

  8. DNA directed self-assembly of shape-controlled hydrogels

    PubMed Central

    Qi, Hao; Ghodousi, Majid; Du, Yanan; Grun, Casey; Bae, Hojae; Yin, Peng; Khademhosseini, Ali

    2013-01-01

    Using DNA as programmable, sequence specific ‘glues’, shape-controlled hydrogel units are self-assembled into prescribed structures. Here we report that aggregates are produced using hydrogel cubes with edge length ranging from 30 micrometers to 1 millimeter, demonstrating assembly across scales. In a simple one-pot agitation reaction, 25 dimers are constructed in parallel from 50 distinct hydrogel cube species, demonstrating highly multiplexed assembly. Using hydrogel cuboids displaying face-specific DNA glues, diverse structures are achieved in aqueous and in interfacial agitation systems. These include dimers, extended chains, and open network structures in an aqueous system; and dimers, chains of fixed length, T-junctions, and square shapes in the interfacial system, demonstrating the versatility of the assembly system. PMID:24013352

  9. Directed enzymatic activation of 1-D DNA tiles.

    PubMed

    Garg, Sudhanshu; Chandran, Harish; Gopalkrishnan, Nikhil; LaBean, Thomas H; Reif, John

    2015-02-24

    The tile assembly model is a Turing universal model of self-assembly where a set of square shaped tiles with programmable sticky sides undergo coordinated self-assembly to form arbitrary shapes, thereby computing arbitrary functions. Activatable tiles are a theoretical extension to the Tile assembly model that enhances its robustness by protecting the sticky sides of tiles until a tile is partially incorporated into a growing assembly. In this article, we experimentally demonstrate a simplified version of the Activatable tile assembly model. In particular, we demonstrate the simultaneous assembly of protected DNA tiles where a set of inert tiles are activated via a DNA polymerase to undergo linear assembly. We then demonstrate stepwise activated assembly where a set of inert tiles are activated sequentially one after another as a result of attachment to a growing 1-D assembly. We hope that these results will pave the way for more sophisticated demonstrations of activated assemblies. PMID:25625898

  10. Direct imaging of single UvrD helicase dynamics on long single-stranded DNA.

    PubMed

    Lee, Kyung Suk; Balci, Hamza; Jia, Haifeng; Lohman, Timothy M; Ha, Taekjip

    2013-01-01

    Fluorescence imaging of single-protein dynamics on DNA has been largely limited to double-stranded DNA or short single-stranded DNA. We have developed a hybrid approach for observing single proteins moving on laterally stretched kilobase-sized ssDNA. Here we probed the single-stranded DNA translocase activity of Escherichia coli UvrD by single fluorophore tracking, while monitoring DNA unwinding activity with optical tweezers to capture the entire sequence of protein binding, single-stranded DNA translocation and multiple pathways of unwinding initiation. The results directly demonstrate that the UvrD monomer is a highly processive single-stranded DNA translocase that is stopped by a double-stranded DNA, whereas two monomers are required to unwind DNA to a detectable degree. The single-stranded DNA translocation rate does not depend on the force applied and displays a remarkable homogeneity, whereas the unwinding rate shows significant heterogeneity. These findings demonstrate that UvrD assembly state regulates its DNA helicase activity with functional implications for its stepping mechanism, and also reveal a previously unappreciated complexity in the active species during unwinding. PMID:23695672

  11. Direct detection of DNA conformation in hybridization processes.

    PubMed

    Papadakis, George; Tsortos, Achilleas; Bender, Florian; Ferapontova, Elena E; Gizeli, Electra

    2012-02-21

    DNA hybridization studies at surfaces normally rely on the detection of mass changes as a result of the addition of the complementary strand. In this work we propose a mass-independent sensing principle based on the quantitative monitoring of the conformation of the immobilized single-strand probe and of the final hybridized product. This is demonstrated by using a label-free acoustic technique, the quartz crystal microbalance (QCM-D), and oligonucleotides of specific sequences which, upon hybridization, result in DNAs of various shapes and sizes. Measurements of the acoustic ratio ΔD/ΔF in combination with a "discrete molecule binding" approach are used to confirm the formation of straight hybridized DNA molecules of specific lengths (21, 75, and 110 base pairs); acoustic results are also used to distinguish between single- and double-stranded molecules as well as between same-mass hybridized products with different shapes, i.e., straight or "Y-shaped". Issues such as the effect of mono- and divalent cations to hybridization and the mechanism of the process (nucleation, kinetics) when it happens on a surface are carefully considered. Finally, this new sensing principle is applied to single-nucleotide polymorphism detection: a DNA hairpin probe hybridized to the p53 target gene gave products of distinct geometrical features depending on the presence or absence of the SNP, both readily distinguishable. Our results suggest that DNA conformation probing with acoustic wave sensors is a much more improved detection method over the popular mass-related, on/off techniques offering higher flexibility in the design of solid-phase hybridization assays. PMID:22248021

  12. Mapping site-specific endonuclease binding to DNA by direct imaging with AFM

    SciTech Connect

    Allison, D.P.; Thundat, T.; Doktycz, M.J.; Kerper, P.S.; Warmack, R.J.; Modrich, P.; Isfort, R.J.

    1995-12-31

    Physical mapping of DNA can be accomplished by direct AFM imaging of site specific proteins bound to DNA molecules. Using Gln-111, a mutant of EcoRI endonuclease with a specific affinity for EcoRI sites 1,000 times greater than wild type enzyme but with cleavage rate constants reduced by a factor of 10{sup 4}, the authors demonstrate site-specific mapping by direct AFM imaging. Images are presented showing specific-site binding of Gln-111 to plasmids having either one (pBS{sup +}) or two (pMP{sup 32}) EcoRI sites. Identification of the Gln-111/DNA complex is greatly enhanced by biotinylation of the complex followed by reaction with streptavidin gold prior to imaging. Image enhancement coupled with improvements in the preparation techniques for imaging large DNA molecules, such as lambda DNA (47 kb), has the potential to contribute to direct AFM restriction mapping of cosmid-sized genomic DNAs.

  13. Liposome-based chemical barcodes for single molecule DNA detection using imaging mass spectrometry.

    PubMed

    Gunnarsson, Anders; Sjövall, Peter; Höök, Fredrik

    2010-02-10

    We report on a mass-spectrometry (time-of-flight secondary ion mass spectrometry, TOF-SIMS) based method for multiplexed DNA detection utilizing a random array, where the lipid composition of small unilamellar liposomes act as chemical barcodes to identify unique DNA target sequences down to the single molecule level. In a sandwich format, suspended target-DNA to be detected mediates the binding of capture-DNA modified liposomes to surface-immobilized probe-DNA. With the lipid composition of each liposome encoding a unique target-DNA sequence, TOF-SIMS analysis was used to determine the chemical fingerprint of the bound liposomes. Using high-resolution TOF-SIMS imaging, providing sub-200 nm spatial resolution, single DNA targets could be detected and identified via the chemical fingerprint of individual liposomes. The results also demonstrate the capability of TOF-SIMS to provide multiplexed detection of DNA targets on substrate areas in the micrometer range. Together with a high multiplexing capacity, this makes the concept an interesting alternative to existing barcode concepts based on fluorescence, Raman, or graphical codes for small-scale bioanalysis. PMID:20085369

  14. Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

    PubMed Central

    Kulczyk, Arkadiusz W.; Tanner, Nathan A.; Loparo, Joseph J.; Richardson, Charles C.; van Oijen, Antoine M.

    2010-01-01

    We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized λ DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2). PMID:20332766

  15. RecG Directs DNA Synthesis during Double-Strand Break Repair

    PubMed Central

    Azeroglu, Benura; Mawer, Julia S. P.; Cockram, Charlotte A.; White, Martin A.; Hasan, A. M. Mahedi; Filatenkova, Milana; Leach, David R. F.

    2016-01-01

    Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR) that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300), arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability. PMID:26872352

  16. Physical controls on directed virus assembly at nanoscale chemical templates

    SciTech Connect

    Cheung, C L; Chung, S; Chatterji, A; Lin, T; Johnson, J E; Hok, S; Perkins, J; De Yoreo, J

    2006-05-10

    Viruses are attractive building blocks for nanoscale heterostructures, but little is understood about the physical principles governing their directed assembly. In-situ force microscopy was used to investigate organization of Cowpea Mosaic Virus engineered to bind specifically and reversibly at nanoscale chemical templates with sub-30nm features. Morphological evolution and assembly kinetics were measured as virus flux and inter-viral potential were varied. The resulting morphologies were similar to those of atomic-scale epitaxial systems, but the underlying thermodynamics was analogous to that of colloidal systems in confined geometries. The 1D templates biased the location of initial cluster formation, introduced asymmetric sticking probabilities, and drove 1D and 2D condensation at subcritical volume fractions. The growth kinetics followed a t{sup 1/2} law controlled by the slow diffusion of viruses. The lateral expansion of virus clusters that initially form on the 1D templates following introduction of polyethylene glycol (PEG) into the solution suggests a significant role for weak interaction.

  17. Direct Atom Imaging by Chemical-Sensitive Holography.

    PubMed

    Lühr, Tobias; Winkelmann, Aimo; Nolze, Gert; Krull, Dominique; Westphal, Carsten

    2016-05-11

    In order to understand the physical and chemical properties of advanced materials, functional molecular adsorbates, and protein structures, a detailed knowledge of the atomic arrangement is essential. Up to now, if subsurface structures are under investigation, only indirect methods revealed reliable results of the atoms' spatial arrangement. An alternative and direct method is three-dimensional imaging by means of holography. Holography was in fact proposed for electron waves, because of the electrons' short wavelength at easily accessible energies. Further, electron waves are ideal structure probes on an atomic length scale, because electrons have a high scattering probability even for light elements. However, holographic reconstructions of electron diffraction patterns have in the past contained severe image artifacts and were limited to at most a few tens of atoms. Here, we present a general reconstruction algorithm that leads to high-quality atomic images showing thousands of atoms. Additionally, we show that different elements can be identified by electron holography for the example of FeS2. PMID:27070050

  18. DNA unwinding step-size of E. coli RecBCD helicase determined from single turnover chemical quenched-flow kinetic studies.

    PubMed

    Lucius, Aaron L; Vindigni, Alessandro; Gregorian, Razmic; Ali, Janid A; Taylor, Andrew F; Smith, Gerald R; Lohman, Timothy M

    2002-11-29

    The mechanism by which Escherichia coli RecBCD DNA helicase unwinds duplex DNA was examined in vitro using pre-steady-state chemical quenched-flow kinetic methods. Single turnover DNA unwinding experiments were performed by addition of ATP to RecBCD that was pre-bound to a series of DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. In each case, the time-course for formation of completely unwound DNA displayed a distinct lag phase that increased with duplex length, reflecting the transient formation of partially unwound DNA intermediates during unwinding catalyzed by RecBCD. Quantitative analysis of five independent sets of DNA unwinding time courses indicates that RecBCD unwinds duplex DNA in discrete steps, with an average unwinding "step-size", m=3.9(+/-1.3)bp step(-1), with an average unwinding rate of k(U)=196(+/-77)steps s(-1) (mk(U)=790(+/-23)bps(-1)) at 25.0 degrees C (10mM MgCl(2), 30 mM NaCl (pH 7.0), 5% (v/v) glycerol). However, additional steps, not linked directly to DNA unwinding are also detected. This kinetic DNA unwinding step-size is similar to that determined for the E.coli UvrD helicase, suggesting that these two SF1 superfamily helicases may share similar mechanisms of DNA unwinding. PMID:12445778

  19. Unravelling Microbial Communities with DNA-Microarrays: Challengesand Future Directions.

    SciTech Connect

    Wagner, Michael; Smidt, Hauke; Loy, Alexander; Zhou, Jizhong

    2007-03-08

    High-throughput technologies are urgently needed formonitoring the formidable biodiversity and functional capabilities ofmicroorganisms in the environment. Ten years ago, DNA microarrays,miniaturized platforms for highly parallel hybridization reactions, foundtheir way into environmental microbiology and raised great expectationsamong researchers in the field. In this article, we briefly summarize thestate-of-the-art of microarray approaches in microbial ecology researchand discuss in more detail crucial problems and promising solutions.Finally, we outline scenarios for an innovative combination ofmicroarrays with other molecular tools for structure-function analysis ofcomplex microbial communities.

  20. Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation

    NASA Astrophysics Data System (ADS)

    Yoo, Jejoong; Kim, Hajin; Aksimentiev, Aleksei; Ha, Taekjip

    2016-03-01

    Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA-DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA-DNA interactions that we report here may play a role in the chromosome organization and gene regulation.

  1. Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation.

    PubMed

    Yoo, Jejoong; Kim, Hajin; Aksimentiev, Aleksei; Ha, Taekjip

    2016-01-01

    Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA-DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA-DNA interactions that we report here may play a role in the chromosome organization and gene regulation. PMID:27001929

  2. Chemical Morphing of DNA Containing Four Noncanonical Bases.

    PubMed

    Eremeeva, Elena; Abramov, Michail; Margamuljana, Lia; Rozenski, Jef; Pezo, Valerie; Marlière, Philippe; Herdewijn, Piet

    2016-06-20

    The ability of alternative nucleic acids, in which all four nucleobases are substituted, to replicate in vitro and to serve as genetic templates in vivo was evaluated. A nucleotide triphosphate set of 5-chloro-2'-deoxyuridine, 7-deaza-2'-deoxyadenosine, 5-fluoro-2'-deoxycytidine, and 7-deaza-2'deoxyguanosine successfully underwent polymerase chain reaction (PCR) amplification using templates of different lengths (57 or 525mer) and Taq or Vent (exo-) DNA polymerases as catalysts. Furthermore, a fully morphed gene encoding a dihydrofolate reductase was generated by PCR using these fully substituted nucleotides and was shown to transform and confer trimethoprim resistance to E. coli. These results demonstrated that fully modified templates were accurately read by the bacterial replication machinery and provide the first example of a long fully modified DNA molecule being functional in vivo. PMID:27159019

  3. Chemical and Biological Consequences of Oxidatively Damaged Guanine in DNA

    PubMed Central

    Delaney, Sarah; Jarem, Daniel A.; Volle, Catherine B.; Yennie, Craig J.

    2013-01-01

    Of the four native nucleosides, 2′-deoxyguanosine (dGuo) is most easily oxidized. Two lesions derived from dGuo are 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy)·dGuo. Furthermore, while steady-state levels of 8-oxodGuo can be detected in genomic DNA, it is also known that 8-oxodGuo is more easily oxidized than dGuo. Thus, 8-oxodGuo is susceptible to further oxidation to form several hyperoxidized dGuo products. This review addresses the structural impact, the mutagenic and genotoxic potential, and biological implications of oxidatively damaged DNA, in particular 8-oxodGuo, Fapy·dGuo, and the hyperoxidized dGuo products. PMID:22239655

  4. Direct detection of linker DNA bending in defined-length oligomers of chromatin.

    PubMed Central

    Yao, J; Lowary, P T; Widom, J

    1990-01-01

    Linker DNA, which connects between nucleosomes in chromatin, is short and, therefore, may be essentially straight and inflexible. We have carried out hydrodynamic and electron microscopic studies of dinucleosomes--fragments of chromatin containing just two nucleosomes--to test the ability of linker DNA to bend. We find that ionic conditions that stabilize the folding of long chromatin cause linker DNA in dinucleosomes to bend, bringing the two nucleosomes into contact. The results uphold a key prediction of the solenoid model of chromosome folding and suggest a mechanism by which proteins that are separated along the DNA can interact by direct contact. Images PMID:2217191

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

    PubMed

    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. PMID:27057775

  6. A unified matrix hypothesis of DNA-directed morphogenesis, protodynamism and growth control.

    PubMed

    Scherrer, K

    1989-04-01

    A theoretical concept is proposed, in order to explain some enigmatic aspects of cellular and molecular biology of eukaryotic organisms. Among these are the C-value paradox of DNA redundancy, the correlation of DNA content and cell size, the disruption of genes at DNA level, the "Chromosome field" data of Lima de Faria (Hereditas 93:1, 1980), the "quantal mitosis" proposition of Holtzer et al. (Curr. Top. Dev. Biol. 7:229 1972), the inheritance of morphological patterns, the relations of DNA and chromosome organisation to cellular structure and function, the molecular basis of speciation, etc. The basic proposition of the "Unified Matrix Hypothesis" is that the nuclear DNA has a direct morphogenic function, in addition to its coding function in protein synthesis. This additional genetic information is thought to be largely contained in the non-protein coding transcribed DNA, and in the untranscribed part of the genome. PMID:2765661

  7. Electrochemical direct immobilization of DNA sequences for label-free herpes virus detection

    NASA Astrophysics Data System (ADS)

    Tam, Phuong Dinh; Trung, Tran; Tuan, Mai Anh; Chien, Nguyen Duc

    2009-09-01

    DNA sequences/bio-macromolecules of herpes virus (5'-AT CAC CGA CCC GGA GAG GGA C-3') were directly immobilized into polypyrrole matrix by using the cyclic voltammetry method, and grafted onto arrays of interdigitated platinum microelectrodes. The morphology surface of the obtained PPy/DNA of herpes virus composite films was investigated by a FESEM Hitachi-S 4800. Fourier transform infrared spectroscopy (FTIR) was used to characterize the PPy/DNA film and to study the specific interactions that may exist between DNA biomacromolecules and PPy chains. Attempts are made to use these PPy/DNA composite films for label-free herpes virus detection revealed a response time of 60 s in solutions containing as low as 2 nM DNA concentration, and self life of six months when immerged in double distilled water and kept refrigerated.

  8. 33 CFR 95.035 - Reasonable cause for directing a chemical test.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... chemical test. 95.035 Section 95.035 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND... DANGEROUS DRUG § 95.035 Reasonable cause for directing a chemical test. (a) Only a law enforcement officer or a marine employer may direct an individual operating a vessel to undergo a chemical test...

  9. Directed self-organization of single DNA molecules in a nanoslit via embedded nanopit arrays

    PubMed Central

    Reisner, Walter; Larsen, Niels B.; Flyvbjerg, Henrik; Tegenfeldt, Jonas O.; Kristensen, Anders

    2009-01-01

    We show that arrays of nanopit structures etched in a nanoslit can control the positioning and conformation of single DNA molecules in nanofluidic devices. By adjusting the spacing, organization and placement of the nanopits it is possible to immobilize DNA at predetermined regions of a device without additional chemical modification and achieve a high degree of control over local DNA conformation. DNA can be extended between two nanopits and in closely spaced arrays will self-assemble into “connect-the-dots” conformations consisting of locally pinned segments joined by fluctuating linkers. These results have broad implications for nanotechnology fields that require methods for the nanoscale positioning and manipulation of DNA. PMID:19122138

  10. Incorporation of viral DNA packaging motor channel in lipid bilayers for real-time, single-molecule sensing of chemicals and double-stranded DNA

    PubMed Central

    Haque, Farzin; Geng, Jia; Montemagno, Carlo; Guo, Peixuan

    2013-01-01

    Over the past decade, nanopores have rapidly emerged as stochastic biosensors. This protocol describes the cloning, expression, and purification of the channel of bacteriophage phi29 DNA packaging nanomotor and its subsequent incorporation into lipid membranes for single-pore sensing of dsDNA and chemicals. The membrane-embedded phi29 nanochannels remain functional and structurally intact under a range of conditions. When ions and macromolecules translocate through these nanochannels, reliable fingerprint changes in conductance are observed. Compared with other well studied biological pores, the phi29 nanochannel has a larger cross-sectional area, which enables the translocation of dsDNA. Furthermore, specific amino acids can be introduced by site-directed mutagenesis within the large cavity of the channel to conjugate receptors that are able to bind specific ligands or analytes for desired applications. The lipid membrane embedded nanochannel system has immense potential nanotechnological and biomedical applications in bioreactors, environmental sensing, drug monitoring, controlled drug delivery, early disease diagnosis, and high-throughput DNA sequencing. The total time required for completing one round of this protocol is around one month. PMID:23348364

  11. Development of a Daphnia magna DNA microarray for evaluating the toxicity of environmental chemicals.

    PubMed

    Watanabe, Hajime; Takahashi, Eri; Nakamura, Yuko; Oda, Shigeto; Tatarazako, Norihisa; Iguchi, Taisen

    2007-04-01

    Toxic chemical contaminants have a variety of detrimental effects on various species, and the impact of pollutants on ecosystems has become an urgent issue. However, the majority of studies regarding the effects of chemical contaminants have focused on vertebrates. Among aquatic organisms, Daphnia magna has been used extensively to evaluate organism- and population-level responses of invertebrates to pollutants in acute toxicity or reproductive toxicity tests. Although these types of tests can provide information concerning hazardous concentrations of chemicals, they provide no information about their mode of action. Recent advances in molecular genetic techniques have provided tools to better understand the responses of aquatic organisms to pollutants. In the present study, we adapted some of the techniques of molecular genetics to develop new tools, which form the basis for an ecotoxicogenomic assessment of D. magna. Based on a Daphnia expressed sequence tag database, we developed an oligonucleotide-based DNA microarray with high reproducibility. The DNA microarray was used to evaluate gene expression profiles of neonatal daphnids exposed to several different chemicals: Copper sulfate, hydrogen peroxide, pentachlorophenol, or beta-naphthoflavone. Exposure to these chemicals resulted in characteristic patterns of gene expression that were chemical-specific, indicating that the Daphnia DNA microarray can be used for classification of toxic chemicals and for development of a mechanistic understanding of chemical toxicity on a common freshwater organism. PMID:17447551

  12. DNA-decorated carbon-nanotube-based chemical sensors on complementary metal oxide semiconductor circuitry

    NASA Astrophysics Data System (ADS)

    Chen, Chia-Ling; Yang, Chih-Feng; Agarwal, Vinay; Kim, Taehoon; Sonkusale, Sameer; Busnaina, Ahmed; Chen, Michelle; Dokmeci, Mehmet R.

    2010-03-01

    We present integration of single-stranded DNA (ss-DNA)-decorated single-walled carbon nanotubes (SWNTs) onto complementary metal oxide semiconductor (CMOS) circuitry as nanoscale chemical sensors. SWNTs were assembled onto CMOS circuitry via a low voltage dielectrophoretic (DEP) process. Besides, bare SWNTs are reported to be sensitive to various chemicals, and functionalization of SWNTs with biomolecular complexes further enhances the sensing specificity and sensitivity. After decorating ss-DNA on SWNTs, we have found that the sensing response of the gas sensor was enhanced (up to ~ 300% and ~ 250% for methanol vapor and isopropanol alcohol vapor, respectively) compared with bare SWNTs. The SWNTs coupled with ss-DNA and their integration on CMOS circuitry demonstrates a step towards realizing ultra-sensitive electronic nose applications.

  13. DNA-decorated carbon-nanotube-based chemical sensors on complementary metal oxide semiconductor circuitry.

    PubMed

    Chen, Chia-Ling; Yang, Chih-Feng; Agarwal, Vinay; Kim, Taehoon; Sonkusale, Sameer; Busnaina, Ahmed; Chen, Michelle; Dokmeci, Mehmet R

    2010-03-01

    We present integration of single-stranded DNA (ss-DNA)-decorated single-walled carbon nanotubes (SWNTs) onto complementary metal oxide semiconductor (CMOS) circuitry as nanoscale chemical sensors. SWNTs were assembled onto CMOS circuitry via a low voltage dielectrophoretic (DEP) process. Besides, bare SWNTs are reported to be sensitive to various chemicals, and functionalization of SWNTs with biomolecular complexes further enhances the sensing specificity and sensitivity. After decorating ss-DNA on SWNTs, we have found that the sensing response of the gas sensor was enhanced (up to approximately 300% and approximately 250% for methanol vapor and isopropanol alcohol vapor, respectively) compared with bare SWNTs. The SWNTs coupled with ss-DNA and their integration on CMOS circuitry demonstrates a step towards realizing ultra-sensitive electronic nose applications. PMID:20139486

  14. New directions: Atmospheric chemical mechanisms for the future

    EPA Science Inventory

    The chemical reaction scheme or mechanism used to represent atmospheric chemical reactions is at the heart of each air quality model used in research and policy applications to predict and analyse the complex air pollutants: ozone, air toxics and PM2.5. This is necessarily only a...

  15. A radioimmunoassay method for detection of DNA based on chemical immobilization of anti-DNA antibody.

    PubMed

    Yoo, S K; Yoon, M; Park, U J; Han, H S; Kim, J H; Hwang, H J

    1999-09-30

    High selectivity provided by biomolecules such as antibodies and enzymes has been exploited during the last two decades for development of biosensors. Of particular importance are efficient immobilization methods for biomolecules in order to preserve their biological activities. In this study, we have evaluated immobilization strategies for an anti-DNA antibody on a self-assembled monolayer of omega-functionalized thiols. The antibody was immobilized via peptide bond formation between the primary amines in the antibody and the carboxyl groups on the self-assembled monolayer. The peptide bond coupling was achieved by activating COOH groups on the surface through N-Hydroxysuccimide (NHS)-ester formation, followed by acylation of NH2 group in the antibody. DNA binding activity of the immobilized antibody was examined by counting beta emission from 35S-labeled DNA. PMID:10551259

  16. Direct Sequencing from the Minimal Number of DNA Molecules Needed to Fill a 454 Picotiterplate

    PubMed Central

    Martínez-Priego, Llúcia; D’Auria, Giussepe; Calafell, Francesc; Moya, Andrés

    2014-01-01

    The large amount of DNA needed to prepare a library in next generation sequencing protocols hinders direct sequencing of small DNA samples. This limitation is usually overcome by the enrichment of such samples with whole genome amplification (WGA), mostly by multiple displacement amplification (MDA) based on φ29 polymerase. However, this technique can be biased by the GC content of the sample and is prone to the development of chimeras as well as contamination during enrichment, which contributes to undesired noise during sequence data analysis, and also hampers the proper functional and/or taxonomic assignments. An alternative to MDA is direct DNA sequencing (DS), which represents the theoretical gold standard in genome sequencing. In this work, we explore the possibility of sequencing the genome of Escherichia coli from the minimum number of DNA molecules required for pyrosequencing, according to the notion of one-bead-one-molecule. Using an optimized protocol for DS, we constructed a shotgun library containing the minimum number of DNA molecules needed to fill a selected region of a picotiterplate. We gathered most of the reference genome extension with uniform coverage. We compared the DS method with MDA applied to the same amount of starting DNA. As expected, MDA yielded a sparse and biased read distribution, with a very high amount of unassigned and unspecific DNA amplifications. The optimized DS protocol allows unbiased sequencing to be performed from samples with a very small amount of DNA. PMID:24887077

  17. New Modeling Approaches to Study DNA Damage by the Direct and Indirect Effects of Ionizing Radiation

    NASA Technical Reports Server (NTRS)

    Plante, Ianik; Cucinotta, Francis A.

    2012-01-01

    DNA is damaged both by the direct and indirect effects of radiation. In the direct effect, the DNA itself is ionized, whereas the indirect effect involves the radiolysis of the water molecules surrounding the DNA and the subsequent reaction of the DNA with radical products. While this problem has been studied for many years, many unknowns still exist. To study this problem, we have developed the computer code RITRACKS [1], which simulates the radiation track structure for heavy ions and electrons, calculating all energy deposition events and the coordinates of all species produced by the water radiolysis. In this work, we plan to simulate DNA damage by using the crystal structure of a nucleosome and calculations performed by RITRACKS. The energy deposition events are used to calculate the dose deposited in nanovolumes [2] and therefore can be used to simulate the direct effect of the radiation. Using the positions of the radiolytic species with a radiation chemistry code [3] it will be possible to simulate DNA damage by indirect effect. The simulation results can be compared with results from previous calculations such as the frequencies of simple and complex strand breaks [4] and with newer experimental data using surrogate markers of DNA double ]strand breaks such as . ]H2AX foci [5].

  18. Endonucleolytic activity directed towards 8-(2-hydroxy-2-propyl) purines in double-stranded DNA.

    PubMed Central

    Livneh, Z; Elad, D; Sperling, J

    1979-01-01

    Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of lambda greater than 305 nm led to the specific 8-substitution of purine moieties in the DNA, yielding 8-(2-hydroxy-2-propyl)adenine and 8-(2-hydroxy-2-propyl)guanine as the only detectable damage in the DNA. Using this specifically photoalkylated DNA as a substrate, we discovered in extracts of Micrococcus luteus an endonucleolytic activity that is directed towards 8-(2-hydroxy-2-propyl) purines in DNA. The activity is not a combination of a DNA-glycosylase and an apurinic site endonuclease. It is not inhibited by single-stranded DNA, by UV- or gamma-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA. however, gamma- or UV-(254 nm) irradiated double-stranded DNAs to inhibit the activity, hinting at the possibility of a common type of lesion in these damaged DNAs. Divalent cations are not required for the incising activity, and it is fully active in 1 mM EDTA, whereas caffeine and ATP cause inhibition. Extracts of mutant M. luteus lacking pyrimidine-dimer-directed endonucleases were found to contain the endonucleolytic activity in levels comparable to those present in the wild type. After the incision, we could demonstrate the specific excision of the 8-alkylated purines from the damaged DNA. The special conformational consequences of the 8-alkylation of purines, at the nucleotide level, namely their nonregular syn conformation, suggest that it is the distortion in the DNA that is recognized by the endonuclease. PMID:293658

  19. Endonucleolytic activity directed towards 8-(2-hydroxy-2-propyl) purines in double-stranded DNA.

    PubMed

    Livneh, Z; Elad, D; Sperling, J

    1979-11-01

    Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of lambda greater than 305 nm led to the specific 8-substitution of purine moieties in the DNA, yielding 8-(2-hydroxy-2-propyl)adenine and 8-(2-hydroxy-2-propyl)guanine as the only detectable damage in the DNA. Using this specifically photoalkylated DNA as a substrate, we discovered in extracts of Micrococcus luteus an endonucleolytic activity that is directed towards 8-(2-hydroxy-2-propyl) purines in DNA. The activity is not a combination of a DNA-glycosylase and an apurinic site endonuclease. It is not inhibited by single-stranded DNA, by UV- or gamma-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA. however, gamma- or UV-(254 nm) irradiated double-stranded DNAs to inhibit the activity, hinting at the possibility of a common type of lesion in these damaged DNAs. Divalent cations are not required for the incising activity, and it is fully active in 1 mM EDTA, whereas caffeine and ATP cause inhibition. Extracts of mutant M. luteus lacking pyrimidine-dimer-directed endonucleases were found to contain the endonucleolytic activity in levels comparable to those present in the wild type. After the incision, we could demonstrate the specific excision of the 8-alkylated purines from the damaged DNA. The special conformational consequences of the 8-alkylation of purines, at the nucleotide level, namely their nonregular syn conformation, suggest that it is the distortion in the DNA that is recognized by the endonuclease. PMID:293658

  20. Fluorescence, XPS, and TOF-SIMS surface chemical state image analysis of DNA microarrays.

    PubMed

    Lee, Chi-Ying; Harbers, Gregory M; Grainger, David W; Gamble, Lara J; Castner, David G

    2007-08-01

    Performance improvements in DNA-modified surfaces required for microarray and biosensor applications rely on improved capabilities to accurately characterize the chemistry and structure of immobilized DNA molecules on micropatterned surfaces. Recent innovations in imaging X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) now permit more detailed studies of micropatterned surfaces. We have exploited the complementary information provided by imaging XPS and imaging TOF-SIMS to detail the chemical composition, spatial distribution, and hybridization efficiency of amine-terminated single-stranded DNA (ssDNA) bound to commercial polyacrylamide-based, amine-reactive microarray slides, immobilized in both macrospot and microarray diagnostic formats. Combinations of XPS imaging and small spot analysis were used to identify micropatterned DNA spots within printed DNA arrays on slide surfaces and quantify DNA elements within individual microarray spots for determination of probe immobilization and hybridization efficiencies. This represents the first report of imaging XPS of DNA immobilization and hybridization efficiencies for arrays fabricated on commercial microarray slides. Imaging TOF-SIMS provided distinct analytical data on the lateral distribution of DNA within single array microspots before and after target hybridization. Principal component analysis (PCA) applied to TOF-SIMS imaging datasets demonstrated that the combination of these two techniques provides information not readily observable in TOF-SIMS images alone, particularly in identifying species associated with array spot nonuniformities (e.g., "halo" or "donut" effects often observed in fluorescence images). Chemically specific spot images were compared to conventional fluorescence scanned images in microarrays to provide new information on spot-to-spot DNA variations that affect current diagnostic reliability, assay variance, and sensitivity. PMID:17625851

  1. Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation

    PubMed Central

    Yoo, Jejoong; Kim, Hajin; Aksimentiev, Aleksei; Ha, Taekjip

    2016-01-01

    Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA–DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA–DNA interactions that we report here may play a role in the chromosome organization and gene regulation. PMID:27001929

  2. A Pol V–Mediated Silencing, Independent of RNA–Directed DNA Methylation, Applies to 5S rDNA

    PubMed Central

    Douet, Julien; Tutois, Sylvie; Tourmente, Sylvette

    2009-01-01

    The plant-specific RNA polymerases Pol IV and Pol V are essential to RNA–directed DNA methylation (RdDM), which also requires activities from RDR2 (RNA–Dependent RNA Polymerase 2), DCL3 (Dicer-Like 3), AGO4 (Argonaute), and DRM2 (Domains Rearranged Methyltransferase 2). RdDM is dedicated to the methylation of target sequences which include transposable elements, regulatory regions of several protein-coding genes, and 5S rRNA–encoding DNA (rDNA) arrays. In this paper, we have studied the expression of the 5S-210 transcript, a marker of silencing release at 5S RNA genes, to show a differential impact of RNA polymerases IV and V on 5S rDNA arrays during early development of the plant. Using a combination of molecular and cytological assays, we show that Pol IV, RDR2, DRM2, and Pol V, actors of the RdDM, are required to maintain a transcriptional silencing of 5S RNA genes at chromosomes 4 and 5. Moreover, we have shown a derepression associated to chromatin decondensation specific to the 5S array from chromosome 4 and restricted to the Pol V–loss of function. In conclusion, our results highlight a new role for Pol V on 5S rDNA, which is RdDM–independent and comes specifically at chromosome 4, in addition to the RdDM pathway. PMID:19834541

  3. Invited Review: DNA-mediated artificial nanobiostructures: state of the art and future directions

    NASA Astrophysics Data System (ADS)

    Bashir, R.

    2001-01-01

    DNA (deoxyribonucleic acid)-mediated assembly of nanometer and micrometer scale structures can have a profound impact in the fields of nanoelectronics and nanotechnology. Such structures can also find applications in microelectromechanical systems, hybrid bio-sensors, and the potential to continue the scaling of Moore's law beyond the 50 nm node. While engineers and scientists have been long aspiring to controllably and specifically manipulate structures at the micrometer and nanometer scale, nature has been performing these tasks and assembling structures with great accuracy and high efficiency using highly specific biological molecules such as DNA and proteins. This paper describes the motivations and fundamentals behind these assembly concepts, with a focus on DNA hybridization-mediated assembly, and presents the state of the art in this field. In addition, new ideas and directions for future research on DNA-mediated assembly of active devices and DNA-based molecular devices are also presented.

  4. Quantitative Field Testing Rotylenchulus reniformis DNA from Metagenomic Samples Isolated Directly from Soil

    PubMed Central

    Showmaker, Kurt; Lawrence, Gary W.; Lu, Shien; Balbalian, Clarissa; Klink, Vincent P.

    2011-01-01

    A quantitative PCR procedure targeting the β-tubulin gene determined the number of Rotylenchulus reniformis Linford & Oliveira 1940 in metagenomic DNA samples isolated from soil. Of note, this outcome was in the presence of other soil-dwelling plant parasitic nematodes including its sister genus Helicotylenchus Steiner, 1945. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated directly from soil. PMID:22194958

  5. A comparison of RNA with DNA in template-directed synthesis

    NASA Technical Reports Server (NTRS)

    Zielinski, M.; Kozlov, I. A.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    2000-01-01

    Nonenzymatic template-directed copying of RNA sequences rich in cytidylic acid using nucleoside 5'-(2-methylimidazol-1-yl phosphates) as substrates is substantially more efficient than the copying of corresponding DNA sequences. However, many sequences cannot be copied, and the prospect of replication in this system is remote, even for RNA. Surprisingly, wobble-pairing leads to much more efficient incorporation of G opposite U on RNA templates than of G opposite T on DNA templates.

  6. Bacterial origin recognition complexes direct assembly of higher-order DnaA oligomeric structures.

    PubMed

    Miller, Diana T; Grimwade, Julia E; Betteridge, Thu; Rozgaja, Tania; Torgue, Julien J-C; Leonard, Alan C

    2009-11-01

    Eukaryotic initiator proteins form origin recognition complexes (ORCs) that bind to replication origins during most of the cell cycle and direct assembly of prereplication complexes (pre-RCs) before the onset of S phase. In the eubacterium Escherichia coli, there is a temporally similar nucleoprotein complex comprising the initiator protein DnaA bound to three high-affinity recognition sites in the unique origin of replication, oriC. At the time of initiation, this high-affinity DnaA-oriC complex (the bacterial ORC) accumulates additional DnaA that interacts with lower-affinity sites in oriC, forming a pre-RC. In this paper, we investigate the functional role of the bacterial ORC and examine whether it mediates low-affinity DnaA-oriC interactions during pre-RC assembly. We report that E. coli ORC is essential for DnaA occupation of low-affinity sites. The assistance given by ORC is directed primarily to proximal weak sites and requires oligomerization-proficient DnaA. We propose that in bacteria, DnaA oligomers of limited length and stability emerge from single high-affinity sites and extend toward weak sites to facilitate their loading as a key stage of prokaryotic pre-RC assembly. PMID:19833870

  7. Efficiency, error and yield in light-directed maskless synthesis of DNA microarrays

    PubMed Central

    2011-01-01

    Background Light-directed in situ synthesis of DNA microarrays using computer-controlled projection from a digital micromirror device--maskless array synthesis (MAS)--has proved to be successful at both commercial and laboratory scales. The chemical synthetic cycle in MAS is quite similar to that of conventional solid-phase synthesis of oligonucleotides, but the complexity of microarrays and unique synthesis kinetics on the glass substrate require a careful tuning of parameters and unique modifications to the synthesis cycle to obtain optimal deprotection and phosphoramidite coupling. In addition, unintended deprotection due to scattering and diffraction introduce insertion errors that contribute significantly to the overall error rate. Results Stepwise phosphoramidite coupling yields have been greatly improved and are now comparable to those obtained in solid phase synthesis of oligonucleotides. Extended chemical exposure in the synthesis of complex, long oligonucleotide arrays result in lower--but still high--final average yields which approach 99%. The new synthesis chemistry includes elimination of the standard oxidation until the final step, and improved coupling and light deprotection. Coupling Insertions due to stray light are the limiting factor in sequence quality for oligonucleotide synthesis for gene assembly. Diffraction and local flare are by far the largest contributors to loss of optical contrast. Conclusions Maskless array synthesis is an efficient and versatile method for synthesizing high density arrays of long oligonucleotides for hybridization- and other molecular binding-based experiments. For applications requiring high sequence purity, such as gene assembly, diffraction and flare remain significant obstacles, but can be significantly reduced with straightforward experimental strategies. PMID:22152062

  8. DNA methylation: a mechanism linking environmental chemical exposures to risk of autism spectrum disorders?

    PubMed Central

    Keil, Kimberly P.; Lein, Pamela J.

    2016-01-01

    There is now compelling evidence that gene by environment interactions are important in the etiology of autism spectrum disorders (ASDs). However, the mechanisms by which environmental factors interact with genetic susceptibilities to confer individual risk for ASD remain a significant knowledge gap in the field. The epigenome, and in particular DNA methylation, is a critical gene expression regulatory mechanism in normal and pathogenic brain development. DNA methylation can be influenced by environmental factors such as diet, hormones, stress, drugs, or exposure to environmental chemicals, suggesting that environmental factors may contribute to adverse neurodevelopmental outcomes of relevance to ASD via effects on DNA methylation in the developing brain. In this review, we describe epidemiological and experimental evidence implicating altered DNA methylation as a potential mechanism by which environmental chemicals confer risk for ASD, using polychlorinated biphenyls (PCBs), lead, and bisphenol A (BPA) as examples. Understanding how environmental chemical exposures influence DNA methylation and how these epigenetic changes modulate the risk and/or severity of ASD will not only provide mechanistic insight regarding gene-environment interactions of relevance to ASD but may also suggest potential intervention strategies for these and potentially other neurodevelopmental disorders. PMID:27158529

  9. Direct electrochemical stripping detection of cystic-fibrosis-related DNA linked through cadmium sulfide quantum dots

    NASA Astrophysics Data System (ADS)

    Marin, Sergio; Merkoçi, Arben

    2009-02-01

    Electrochemical detection of a cadmium sulfide quantum dots (CdS QDs)-DNA complex connected to paramagnetic microbeads (MB) was performed without the need for chemical dissolving. The method is based on dropping 20 µl of CdS QD-DNA-MB suspension on the surface of a screen-printed electrode. It is followed by magnetic collection on the surface of the working electrode and electrochemical detection using square-wave voltammetry (SWV), giving a well-shaped and sensitive analytical signal. A cystic-fibrosis-related DNA sequence was sandwiched between the two DNA probes. One DNA probe is linked via biotin-streptavidin bonding with MB and the other one via thiol groups with the CdS QD used as tags. Nonspecific signals of DNA were minimized using a blocking agent and the results obtained were successfully employed in a model DNA sensor with an interest in future applications in the clinical field. The developed nanoparticle biosensing system may offer numerous opportunities in other fields where fast, low cost and efficient detection of small volume samples is required.

  10. Having a direct look: Analysis of DNA damage and repair mechanisms by next generation sequencing

    PubMed Central

    Meier, Bettina; Gartner, Anton

    2014-01-01

    Genetic information is under constant attack from endogenous and exogenous sources, and the use of model organisms has provided important frameworks to understand how genome stability is maintained and how various DNA lesions are repaired. The advance of high throughput next generation sequencing (NGS) provides new inroads for investigating mechanisms needed for genome maintenance. These emerging studies, which aim to link genetic toxicology and mechanistic analyses of DNA repair processes in vivo, rely on defining mutational signatures caused by faulty replication, endogenous DNA damaging metabolites, or exogenously applied genotoxins; the analysis of their nature, their frequency and distribution. In contrast to classical studies, where DNA repair deficiency is assessed by reduced cellular survival, the localization of DNA repair factors and their interdependence as well as limited analysis of single locus reporter assays, NGS based approaches reveal the direct, quantal imprint of mutagenesis genome-wide, at the DNA sequence level. As we will show, such investigations require the analysis of DNA derived from single genotoxin treated cells, or DNA from cell populations regularly passaged through single cell bottlenecks when naturally occurring mutation accumulation is investigated. We will argue that the life cycle of the nematode Caenorhabditis elegans, its genetic malleability combined with whole genome sequencing provides an exciting model system to conduct such analysis. PMID:25131498

  11. Mismatch repair inhibits homeologous recombination via coordinated directional unwinding of trapped DNA structures.

    PubMed

    Tham, Khek-Chian; Hermans, Nicolaas; Winterwerp, Herrie H K; Cox, Michael M; Wyman, Claire; Kanaar, Roland; Lebbink, Joyce H G

    2013-08-01

    Homeologous recombination between divergent DNA sequences is inhibited by DNA mismatch repair. In Escherichia coli, MutS and MutL respond to DNA mismatches within recombination intermediates and prevent strand exchange via an unknown mechanism. Here, using purified proteins and DNA substrates, we find that in addition to mismatches within the heteroduplex region, secondary structures within the displaced single-stranded DNA formed during branch migration within the recombination intermediate are involved in the inhibition. We present a model that explains how higher-order complex formation of MutS, MutL, and DNA blocks branch migration by preventing rotation of the DNA strands within the recombination intermediate. Furthermore, we find that the helicase UvrD is recruited to directionally resolve these trapped intermediates toward DNA substrates. Thus, our results explain on a mechanistic level how the coordinated action between MutS, MutL, and UvrD prevents homeologous recombination and maintains genome stability. PMID:23932715

  12. Direct Correlation of DNA Binding and Single Protein Domain Motion via Dual Illumination Fluorescence Microscopy

    PubMed Central

    2015-01-01

    We report a dual illumination, single-molecule imaging strategy to dissect directly and in real-time the correlation between nanometer-scale domain motion of a DNA repair protein and its interaction with individual DNA substrates. The strategy was applied to XPD, an FeS cluster-containing DNA repair helicase. Conformational dynamics was assessed via FeS-mediated quenching of a fluorophore site-specifically incorporated into XPD. Simultaneously, binding of DNA molecules labeled with a spectrally distinct fluorophore was detected by colocalization of the DNA- and protein-derived signals. We show that XPD undergoes thermally driven conformational transitions that manifest in spatial separation of its two auxiliary domains. DNA binding does not strictly enforce a specific conformation. Interaction with a cognate DNA damage, however, stabilizes the compact conformation of XPD by increasing the weighted average lifetime of this state by 140% relative to an undamaged DNA. Our imaging strategy will be a valuable tool to study other FeS-containing nucleic acid processing enzymes. PMID:25204359

  13. Direct non transcriptional role of NF-Y in DNA replication.

    PubMed

    Benatti, Paolo; Belluti, Silvia; Miotto, Benoit; Neusiedler, Julia; Dolfini, Diletta; Drac, Marjorie; Basile, Valentina; Schwob, Etienne; Mantovani, Roberto; Blow, J Julian; Imbriano, Carol

    2016-04-01

    NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication. PMID:26732297

  14. Direct transformation of rodent fibroblasts by jaagsiekte sheep retrovirus DNA

    PubMed Central

    Maeda, Naoyoshi; Palmarini, Massimo; Murgia, Claudio; Fan, Hung

    2001-01-01

    Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary carcinoma, a unique animal model for human bronchioalveolar carcinoma. We previously isolated a JSRV proviral clone and showed that it was both infectious and oncogenic. Thus JSRV is necessary and sufficient for the development of ovine pulmonary carcinoma, but no data are available on the mechanisms of transformation. Inspection of the JSRV genome reveals standard retroviral genes, but no evidence for a viral oncogene. However, an alternate ORF in pol (orf-x) might be a candidate for a transforming gene. We tested whether the JSRV genome might encode a transforming gene by transfecting an expression plasmid for JSRV [pCMVJS21, driven by the cytomegalovirus (CMV) immediate early promoter] into mouse NIH 3T3 cells. Foci of transformed cells appeared in the transfected cultures 2–3 weeks posttransfection; cloned transformants showed anchorage independence for growth, and they expressed JSRV RNA. These results indicate that the JRSV genome contains information with direct transforming potential for NIH 3T3 cells. Transfection of a mutated version of pCMVJS21 in which the orf-x protein was terminated by two stop codons also gave transformed foci. Thus, orf-x was eliminated as the candidate transforming gene. In addition, another derivative of pCMVJS21 (pCMVJS21ΔGP) in which the gag, pol (and orf-x) coding sequences were deleted also gave transformed foci. These results indicate that the envelope gene carries the transforming potential. This is an unusual example of a native retroviral structural protein with transformation potential. PMID:11296288

  15. Rapid DNA Sequencing by Direct Nanoscale Reading of Nucleotide Bases on Individual DNA Chains

    SciTech Connect

    Lee, James Weifu; Meller, Amit

    2007-01-01

    Since the independent invention of DNA sequencing by Sanger and by Gilbert 30 years ago, it has grown from a small scale technique capable of reading several kilobase-pair of sequence per day into today's multibillion dollar industry. This growth has spurred the development of new sequencing technologies that do not involve either electrophoresis or Sanger sequencing chemistries. Sequencing by Synthesis (SBS) involves multiple parallel micro-sequencing addition events occurring on a surface, where data from each round is detected by imaging. New High Throughput Technologies for DNA Sequencing and Genomics is the second volume in the Perspectives in Bioanalysis series, which looks at the electroanalytical chemistry of nucleic acids and proteins, development of electrochemical sensors and their application in biomedicine and in the new fields of genomics and proteomics. The authors have expertly formatted the information for a wide variety of readers, including new developments that will inspire students and young scientists to create new tools for science and medicine in the 21st century. Reviews of complementary developments in Sanger and SBS sequencing chemistries, capillary electrophoresis and microdevice integration, MS sequencing and applications set the framework for the book.

  16. Chemical Reaction Engineering: Current Status and Future Directions.

    ERIC Educational Resources Information Center

    Dudukovic, M. P.

    1987-01-01

    Describes Chemical Reaction Engineering (CRE) as the discipline that quantifies the interplay of transport phenomena and kinetics in relating reactor performance to operating conditions and input variables. Addresses the current status of CRE in both academic and industrial settings and outlines future trends. (TW)

  17. New directions: Atmospheric chemical mechanisms for the future

    NASA Astrophysics Data System (ADS)

    Kaduwela, Ajith; Luecken, Deborah; Carter, William; Derwent, Richard

    2015-12-01

    The atmospheric chemical reaction scheme, commonly referred to as the chemical mechanism, is the portion of an air quality model that represents the atmospheric chemistry of the pollutants. It is at the heart of every air quality model used in research and policy applications to predict and analyse the complex air pollutants: ozone, air toxics and PM2.5. The chemical mechanism should incorporate available information on chemical kinetics and reaction pathways and be the conduit through which the fundamental science of atmospheric chemistry is applied to solve real-world problems. The efficiency and effectiveness of policies developed to reduce exposure to harmful pollutants depend on how well the mechanisms reflect the actual chemistry. If the mechanism has reaction pathways that are incorrectly characterised or completely missing, the resulting predictions may underestimate emission reduction requirements needed to meet public health and ecosystem protection targets, or may overstate the emission reductions needed and cause unnecessary implementation costs. It is therefore essential that mechanisms utilise the best, most up-to-date atmospheric chemistry information available so that policy development is based on air quality model predictions that are robust, transparent and free from scientific challenge. We are concerned that this may not continue to be the case.

  18. DNA-damaging agents in cancer chemotherapy: serendipity and chemical biology.

    PubMed

    Cheung-Ong, Kahlin; Giaever, Guri; Nislow, Corey

    2013-05-23

    DNA-damaging agents have a long history of use in cancer chemotherapy. The full extent of their cellular mechanisms, which is essential to balance efficacy and toxicity, is often unclear. In addition, the use of many anticancer drugs is limited by dose-limiting toxicities as well as the development of drug resistance. Novel anticancer compounds are continually being developed in the hopes of addressing these limitations; however, it is essential to be able to evaluate these compounds for their mechanisms of action. This review covers the current DNA-damaging agents used in the clinic, discusses their limitations, and describes the use of chemical genomics to uncover new information about the DNA damage response network and to evaluate novel DNA-damaging compounds. PMID:23706631

  19. Automated screening for small organic ligands using DNA-encoded chemical libraries.

    PubMed

    Decurtins, Willy; Wichert, Moreno; Franzini, Raphael M; Buller, Fabian; Stravs, Michael A; Zhang, Yixin; Neri, Dario; Scheuermann, Jörg

    2016-04-01

    DNA-encoded chemical libraries (DECLs) are collections of organic compounds that are individually linked to different oligonucleotides, serving as amplifiable identification barcodes. As all compounds in the library can be identified by their DNA tags, they can be mixed and used in affinity-capture experiments on target proteins of interest. In this protocol, we describe the screening process that allows the identification of the few binding molecules within the multiplicity of library members. First, the automated affinity selection process physically isolates binding library members. Second, the DNA codes of the isolated binders are PCR-amplified and subjected to high-throughput DNA sequencing. Third, the obtained sequencing data are evaluated using a C++ program and the results are displayed using MATLAB software. The resulting selection fingerprints facilitate the discrimination of binding from nonbinding library members. The described procedures allow the identification of small organic ligands to biological targets from a DECL within 10 d. PMID:26985574

  20. Suppression of the chemically transformed phenotype of BHK cells by a human cDNA

    SciTech Connect

    Eiden, M.V.; MacArthur, L.; Okayama, Hiroto )

    1991-10-01

    Transformation of baby hamster kidney cell line BHK SN-10 by chemical carcinogens such as nitrosylmethylurea (NMU) is mediated by the loss of a gene product critical for the suppression of malignant transformation. Somatic cell hybrids between chemically transformed BHK SN-10 cells and either normal hamster kidney or human fibroblast cells are nontransformed; therefore, a recessive mechanism underlies the malignant transformation of BHK SN-10 cells after chemical carcinogenesis. A human fibroblast cDNA library was constructed and introduced into NMU-transformed BHK SN-10 cells (NMU 34m) in order to identify a human cDNA capable of suppressing cellular transformation. NMU-transformed BHK cells were analyzed for reversion to an anchorage-dependent stable reversion of NMU 34m cells encodes the intermediate filament protein vimentin, which is apparently required for maintenance of the normal phenotype in BHK SN-10 cells.

  1. DNA-decorated carbon nanotube-based FETs as ultrasensitive chemical sensors: Discrimination of homologues, structural isomers, and optical isomers

    NASA Astrophysics Data System (ADS)

    Khamis, S. M.; Jones, R. A.; Johnson, A. T. C.; Preti, G.; Kwak, J.; Gelperin, A.

    2012-06-01

    We have explored the abilities of all-electronic DNA-carbon nanotube (DNA-NT) vapor sensors to discriminate very similar classes of molecules. We screened hundreds of DNA-NT devices against a panel of compounds chosen because of their similarities. We demonstrated that DNA-NT vapor sensors readily discriminate between series of chemical homologues that differ by single methyl groups. DNA-NT devices also discriminate among structural isomers and optical isomers, a trait common in biological olfactory systems, but only recently demonstrated for electronic FET based chemical sensors.

  2. Understanding of DNA directed nanoparticle superlattices in bulk and thin film

    NASA Astrophysics Data System (ADS)

    Lee, Byeongdu

    Over the years, there have been significant advances in assembling nanoparticles with DNA into superlattices. Since the first reports on DNA directed FCC and BCC superlattices consisting of single type of spherical nanoparticles, building blocks for the DNA-nanoparticle superlattices have been extended from a spherical gold nanoparticle to various types of other particles including quantum dots, magnetic, hollow, or polyhedral particles. Not only single component, but superlattices of binary and ternary components have also been synthesized. Although still many details are unclear, now there is a general consensus about thermodynamics of this type of assembly, which led us to fabricate thin films of DNA directed nanoparticle superlattices on substrate for applications such as optical materials. Since the structures are formed in aqueous condition, small angle x-ray scattering (SAXS) that does not disturb the system has been a critical tool to determine structural and thermodynamic characteristics of the assemblies. Thus, we have also been improving SAXS instrumentations and computational methods to calculate scattering profiles for the nanoparticle superlattices. In this talk, we will summarize our works with a focus on some structural details of these superlattices and DNA and understanding about the role of DNA in the crystallization processes in bulk and thin film.

  3. A Binary-Encounter-Bethe Approach to Simulate DNA Damage by the Direct Effect

    NASA Technical Reports Server (NTRS)

    Plante, Ianik; Cucinotta, Francis A.

    2013-01-01

    The DNA damage is of crucial importance in the understanding of the effects of ionizing radiation. The main mechanisms of DNA damage are by the direct effect of radiation (e.g. direct ionization) and by indirect effect (e.g. damage by.OH radicals created by the radiolysis of water). Despite years of research in this area, many questions on the formation of DNA damage remains. To refine existing DNA damage models, an approach based on the Binary-Encounter-Bethe (BEB) model was developed[1]. This model calculates differential cross sections for ionization of the molecular orbitals of the DNA bases, sugars and phosphates using the electron binding energy, the mean kinetic energy and the occupancy number of the orbital. This cross section has an analytic form which is quite convenient to use and allows the sampling of the energy loss occurring during an ionization event. To simulate the radiation track structure, the code RITRACKS developed at the NASA Johnson Space Center is used[2]. This code calculates all the energy deposition events and the formation of the radiolytic species by the ion and the secondary electrons as well. We have also developed a technique to use the integrated BEB cross section for the bases, sugar and phosphates in the radiation transport code RITRACKS. These techniques should allow the simulation of DNA damage by ionizing radiation, and understanding of the formation of double-strand breaks caused by clustered damage in different conditions.

  4. Fluorescent marker for direct detection of specific dsDNA sequences

    PubMed Central

    Dylla-Spears, Rebecca; Townsend, Jacqueline E.; Sohn, Lydia L.; Jen-Jacobson, Linda; Muller, Susan J.

    2009-01-01

    We have created a fluorescent marker using a mutant EcoRI restriction endonuclease (K249C) that enables prolonged, direct visualization of specific sequences on genomic lengths of double-stranded (ds) DNA. The marker consists of a biotinylated enzyme, attached through the biotin-avidin interaction to a fluorescent nanosphere. Control over biotin position with respect to the enzyme’s binding pocket is achieved by biotinylating the mutant EcoRI at the mutation site. Biotinylated enzyme is incubated with dsDNA and NeutrAvidin-coated, fluorescent nanospheres under conditions that allow enzyme binding but prevent cleavage. Marker-laden DNA is then fluorescently stained and stretched on polylysine-coated glass slides so that the positions of the bound markers along individual DNA molecules can be measured. We demonstrate the marker’s ability to bind specifically to its target sequence using both bulk gel-shift assays and single-molecule methods. PMID:19908852

  5. Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase

    NASA Astrophysics Data System (ADS)

    Harada, Yoshie; Ohara, Osamu; Takatsuki, Akira; Itoh, Hiroyasu; Shimamoto, Nobuo; Kinosita, Kazuhiko

    2001-01-01

    Helical filaments driven by linear molecular motors are anticipated to rotate around their axis, but rotation consistent with the helical pitch has not been observed. 14S dynein and non-claret disjunctional protein (ncd) rotated a microtubule more efficiently than expected for its helical pitch, and myosin rotated an actin filament only poorly. For DNA-based motors such as RNA polymerase, transcription-induced supercoiling of DNA supports the general picture of tracking along the DNA helix. Here we report direct and real-time optical microscopy measurements of rotation rate that are consistent with high-fidelity tracking. Single RNA polymerase molecules attached to a glass surface rotated DNA for >100 revolutions around the right-handed screw axis of the double helix with a rotary torque of >5pNnm. This real-time observation of rotation opens the possibility of resolving individual transcription steps.

  6. Fluorescent marker for direct detection of specific dsDNA sequences.

    PubMed

    Dylla-Spears, Rebecca; Townsend, Jacqueline E; Sohn, Lydia L; Jen-Jacobson, Linda; Muller, Susan J

    2009-12-15

    We have created a fluorescent marker using a mutant EcoRI restriction endonuclease (K249C) that enables prolonged, direct visualization of specific sequences on genomic lengths of double-stranded (ds) DNA. The marker consists of a biotinylated enzyme, attached through the biotin-avidin interaction to a fluorescent nanosphere. Control over biotin position with respect to the enzyme's binding pocket is achieved by biotinylating the mutant EcoRI at the mutation site. Biotinylated enzyme is incubated with dsDNA and NeutrAvidin-coated, fluorescent nanospheres under conditions that allow enzyme binding but prevent cleavage. Marker-laden DNA is then fluorescently stained and stretched on polylysine-coated glass slides so that the positions of the bound markers along individual DNA molecules can be measured. We demonstrate the marker's ability to bind specifically to its target sequence using both bulk gel-shift assays and single-molecule methods. PMID:19908852

  7. Direct chemical synthesis of L10 FePt nanoparticles

    NASA Astrophysics Data System (ADS)

    Tzitzios, Vasilis; Basina, Georgia; Colak, Levent; Niarchos, Dimitrios; Hadjipanayis, George

    2011-04-01

    Ordered faced-centered tetragonal (fct) FePt nanoparticles were successfully synthesized by a chemical method using presynthesized Au nanoparticles as the "catalyst." The reaction temperature was also studied and it seems that there is an optimum value of 360 °C where the fct structure is formed. The particles have a mean diameter of 3.5-15 nm and the x-ray diffraction patterns exhibited (001) and (110), which signified the tetragonal phase formation. Room temperature magnetic hysteresis loops show that the FePt particles have coercive fields between 0.68 and 2.8 kOe.

  8. Direct action of endocrine disrupting chemicals on human sperm.

    PubMed

    Schiffer, Christian; Müller, Astrid; Egeberg, Dorte L; Alvarez, Luis; Brenker, Christoph; Rehfeld, Anders; Frederiksen, Hanne; Wäschle, Benjamin; Kaupp, U Benjamin; Balbach, Melanie; Wachten, Dagmar; Skakkebaek, Niels E; Almstrup, Kristian; Strünker, Timo

    2014-07-01

    Synthetic endocrine disrupting chemicals (EDCs), omnipresent in food, household, and personal care products, have been implicated in adverse trends in human reproduction, including infertility and increasing demand for assisted reproduction. Here, we study the action of 96 ubiquitous EDCs on human sperm. We show that structurally diverse EDCs activate the sperm-specific CatSper channel and, thereby, evoke an intracellular Ca(2+) increase, a motility response, and acrosomal exocytosis. Moreover, EDCs desensitize sperm for physiological CatSper ligands and cooperate in low-dose mixtures to elevate Ca(2+) levels in sperm. We conclude that EDCs interfere with various sperm functions and, thereby, might impair human fertilization. PMID:24820036

  9. Direct action of endocrine disrupting chemicals on human sperm

    PubMed Central

    Schiffer, Christian; Müller, Astrid; Egeberg, Dorte L; Alvarez, Luis; Brenker, Christoph; Rehfeld, Anders; Frederiksen, Hanne; Wäschle, Benjamin; Kaupp, U Benjamin; Balbach, Melanie; Wachten, Dagmar; Skakkebaek, Niels E; Almstrup, Kristian; Strünker, Timo

    2014-01-01

    Synthetic endocrine disrupting chemicals (EDCs), omnipresent in food, household, and personal care products, have been implicated in adverse trends in human reproduction, including infertility and increasing demand for assisted reproduction. Here, we study the action of 96 ubiquitous EDCs on human sperm. We show that structurally diverse EDCs activate the sperm-specific CatSper channel and, thereby, evoke an intracellular Ca2+ increase, a motility response, and acrosomal exocytosis. Moreover, EDCs desensitize sperm for physiological CatSper ligands and cooperate in low-dose mixtures to elevate Ca2+ levels in sperm. We conclude that EDCs interfere with various sperm functions and, thereby, might impair human fertilization. PMID:24820036

  10. Direct-Dispense Polymeric Waveguides Platform for Optical Chemical Sensors

    PubMed Central

    Hajj-Hassan, Mohamad; Gonzalez, Timothy; Ghafar-Zadeh, Ebrahim; Djeghelian, Hagop; Chodavarapu, Vamsy; Andrews, Mark; Therriault, Daniel

    2008-01-01

    We describe an automated robotic technique called direct-dispense to fabricate a polymeric platform that supports optical sensor arrays. Direct-dispense, which is a type of the emerging direct-write microfabrication techniques, uses fugitive organic inks in combination with cross-linkable polymers to create microfluidic channels and other microstructures. Specifically, we describe an application of direct-dispensing to develop optical biochemical sensors by fabricating planar ridge waveguides that support sol-gel-derived xerogel-based thin films. The xerogel-based sensor materials act as host media to house luminophore biochemical recognition elements. As a prototype implementation, we demonstrate gaseous oxygen (O2) responsive optical sensors that operate on the basis of monitoring luminescence intensity signals. The optical sensor employs a Light Emitting Diode (LED) excitation source and a standard silicon photodiode as the detector. The sensor operates over the full scale (0%-100%) of O2 concentrations with a response time of less than 1 second. This work has implications for the development of miniaturized multi-sensor platforms that can be cost-effectively and reliably mass-produced.

  11. Silver-mediated base pairings: towards dynamic DNA nanostructures with enhanced chemical and thermal stability

    NASA Astrophysics Data System (ADS)

    Swasey, Steven M.; Gwinn, Elisabeth G.

    2016-04-01

    The thermal and chemical fragility of DNA nanomaterials assembled by Watson–Crick (WC) pairing constrain the settings in which these materials can be used and how they can be functionalized. Here we investigate use of the silver cation, Ag+, as an agent for more robust, metal-mediated self-assembly, focusing on the simplest duplex building blocks that would be required for more elaborate Ag+–DNA nanostructures. Our studies of Ag+-induced assembly of non-complementary DNA oligomers employ strands of 2–24 bases, with varied base compositions, and use electrospray ionization mass spectrometry to determine product compositions. High yields of duplex products containing narrowly distributed numbers of Ag+ can be achieved by optimizing solution conditions. These Ag+-mediated duplexes are stable to at least 60 mM Mg2+, higher than is necessary for WC nanotechnology schemes such as tile assemblies and DNA origami, indicating that sequential stages of Ag+-mediated and WC-mediated assembly may be feasible. Circular dichroism spectroscopy suggests simple helical structures for Ag+-mediated duplexes with lengths to at least 20 base pairs, and further indicates that the structure of cytosine-rich duplexes is preserved at high urea concentrations. We therefore propose an approach towards dynamic DNA nanomaterials with enhanced thermal and chemical stability through designs that combine sturdy silver-mediated ‘frames’ with WC paired ‘pictures’.

  12. DNA-Directed Antibody Immobilization for Enhanced Detection of Single Viral Pathogens.

    PubMed

    Seymour, Elif; Daaboul, George G; Zhang, Xirui; Scherr, Steven M; Ünlü, Nese Lortlar; Connor, John H; Ünlü, M Selim

    2015-10-20

    Here, we describe the use of DNA-conjugated antibodies for rapid and sensitive detection of whole viruses using a single-particle interferometric reflectance imaging sensor (SP-IRIS), a simple, label-free biosensor capable of imaging individual nanoparticles. First, we characterize the elevation of the antibodies conjugated to a DNA sequence on a three-dimensional (3-D) polymeric surface using a fluorescence axial localization technique, spectral self-interference fluorescence microscopy (SSFM). Our results indicate that using DNA linkers results in significant elevation of the antibodies on the 3-D polymeric surface. We subsequently show the specific detection of pseudotyped vesicular stomatitis virus (VSV) as a model virus on SP-IRIS platform. We demonstrate that DNA-conjugated antibodies improve the capture efficiency by achieving the maximal virus capture for an antibody density as low as 0.72 ng/mm(2), whereas for unmodified antibody, the optimal virus capture requires six times greater antibody density on the sensor surface. We also show that using DNA conjugated anti-EBOV GP (Ebola virus glycoprotein) improves the sensitivity of EBOV-GP carrying VSV detection compared to directly immobilized antibodies. Furthermore, utilizing a DNA surface for conversion to an antibody array offers an easier manufacturing process by replacing the antibody printing step with DNA printing. The DNA-directed immobilization technique also has the added advantages of programmable sensor surface generation based on the need and resistance to high temperatures required for microfluidic device fabrication. These capabilities improve the existing SP-IRIS technology, resulting in a more robust and versatile platform, ideal for point-of-care diagnostics applications. PMID:26378807

  13. Software-supported USER cloning strategies for site-directed mutagenesis and DNA assembly.

    PubMed

    Genee, Hans Jasper; Bonde, Mads Tvillinggaard; Bagger, Frederik Otzen; Jespersen, Jakob Berg; Sommer, Morten O A; Wernersson, Rasmus; Olsen, Lars Rønn

    2015-03-20

    USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes. To demonstrate the utility, we designed primers for a simultaneous two-position site-directed mutagenesis of green fluorescent protein (GFP) to yellow fluorescent protein (YFP), which in a single step reaction resulted in a 94% cloning efficiency. AMUSER also supports degenerate nucleotide primers, single insert combinatorial assembly, and flexible parameters for PCR amplification. AMUSER is freely available online at http://www.cbs.dtu.dk/services/AMUSER/. PMID:24847672

  14. Dependence of 5-fluorouracil-mediated radiosensitization on DNA-directed effects

    SciTech Connect

    Lawrence, T.S.; Davis, M.A.; Maybaum, J. )

    1994-06-15

    Although 5-fluorouracil (FUra) has been demonstrated to be a radiation sensitizer both in the laboratory and the clinic, it is not known whether radiosensitization results primary from FUra's DNA or RNA-directed effects. The authors studied the radiosensitizing effects of FUra [+-] thymidine (dThd) on HT29 human colon cancer cells, which are relatively sensitive to the DNA-directed action of FUra, in comparison to SW620 and HuTu80 human colon cancer cells, which are relatively resistant to FUra's DNA-directed effects. They hypothesized that if FUra were acting chiefly through DNA dependent mechanisms, HT29 cells would (a) show greater radiosensitization than SW620 and HuTu80 cells under the same conditions of exposure; and (b) demonstrate selective reversal of radiation sensitivity (compared to cytotoxicity) in the presence of FUra + dThd, compared to FUra alone. They found that the enhancement ratio produced by a 24 h exposure to 10 [mu]M FUra was significantly greater in HT29 cells compared to SW620 and HuTu80 cells (enhancement ratios of 2.1 [+-] 0.1; 1.1 [+-] 0.1, and 1.3 [+-] 0.1, respectively). Furthermore, in HT29 cells, dThd blocked FUra-mediated radiosensitization to a greater extent than FUra-mediated cytotoxicity. Thus, the hypotheses were confirmed. These findings support the concept that the manipulation of FUra's DNA-dependent actions, for example, through modulators of thymidylate synthase (TS) activity, may increase radiosensitization in clinical trials in the treatment of gastrointestinal cancers. However, since resistance to the DNA-directed effects of fluoropyrimidines can result from mechanisms unrelated to TS inhibition, additional strategies will be required to potentiate fluoropyrimidine-mediated radiosensitization. 15 refs., 2 figs., 1 tab.

  15. Macroscopic response and directional disorder dynamics in chemically substituted ferroelectrics

    NASA Astrophysics Data System (ADS)

    Parravicini, Jacopo; DelRe, Eugenio; Agranat, Aharon J.; Parravicini, Gianbattista

    2016-03-01

    Using temperature-resolved dielectric spectroscopy in the range 25-320 K we investigate the macroscopic response, phase symmetry, and order/disorder states in bulk ferroelectric K1-yLiyTa1-xNbx (KLTN). Four long-range symmetry phases are identified with their relative transitions. Directional analysis of the order/disorder states using Fröhlich entropy indicates global symmetry breaking along the growth axis and an anisotropic dipolar effective thermodynamic behavior, which ranges from disordered to ordered at the same temperature for different directions in the sample. Results indicate that the macroscopic polarization, driven by nanosized polar regions, follows a microscopic perovskite eight-sites lattice model.

  16. DNA-protein cross-links produced by various chemicals in cultured human lymphoma cells.

    PubMed

    Costa, M; Zhitkovich, A; Harris, M; Paustenbach, D; Gargas, M

    1997-04-11

    Chemicals such as cis-platinum, formaldehyde, chromate, copper, and certain arsenic compounds have been shown to produce DNA-protein cross-links in human in vitro cell systems at high doses, such as those in the cytotoxic range. Thus far there have only been a limited number of other chemicals evaluated for their ability to produce cross-links. The purpose of the work described here was to evaluate whether select industrial chemicals can form DNA-protein cross-links in human cells in vitro. We evaluated acetaldehyde, acrolein, diepoxybutane, paraformaldehyde, 2-furaldehyde, propionaldehyde, chloroacetaldehyde, sodium arsenite, and a deodorant tablet [Mega Blue; hazardous component listed as tris(hydroxymethyl)nitromethane]. Short- and long-term cytotoxicity was evaluated and used to select appropriate doses for in vitro testing. DNA-protein cross-linking was evaluated at no fewer than three doses and two cell lysate washing temperatures (45 and 65 degrees C) in Epstein-Barr virus (EBV) human Burkitt's lymphoma cells. The two washing temperatures were used to assess the heat stability of the DNA-protein cross-link, 2-Furaldehyde, acetaldehyde, and propionaldehyde produced statistically significant increases in DNA-protein cross-links at washing temperatures of 45 degrees C, but not 65 degrees C, and at or above concentrations of 5, 17.5, and 75 mM, respectively. Acrolein, diepoxybutane, paraformaldehyde, and Mega Blue produced statistically significant increases in DNA-protein cross-links washed at 45 and 65 degrees C at or above concentrations of 0.15 mM, 12.5 mM, 0.003%, and 0.1%, respectively. Sodium arsenite and chloroacetaldehyde did not produce significantly increased DNA-protein cross-links at either temperature nor at any dose tested. Excluding paraformaldehyde and 2-furaldehyde treatments, significant increases in DNA-protein cross-links were observed only at doses that resulted in complete cell death within 4 d following dosing. This work demonstrates that

  17. Direct evaluation of airborne contamination in chemically amplified resist films

    NASA Astrophysics Data System (ADS)

    Yamashita, Yoshio; Taguchi, Takao; Watanabe, Takeo

    1995-06-01

    Airborne contamination in chemically amplified resist films was evaluated by monitoring deprotection reaction using an IR spectrometer. T-BOC protected (20, 50 and 100 mol%) m- and p-cresol novolak resins and triphenyltriflate were used as a matrix polymer and a photoacid generator (PAG), respectively. Three levels of clean environments whose base contaminant (NH4+) concentrations were 50 - 80, 5 - 10 and less than 1 ppb, were prepared for the experiments. In order to determine the delay effects precisely, other processes including baking, exposure, and storage during process intervals were conducted in a base-free environment. The PEB delay effect as well as radiation sensitivity without delay depended on the t-BOC content, and the best results were obtained at 50% and 25 - 50% t- BOC contents in m-cresol novolak and p-cresol novolak systems, respectively.

  18. Environmental chemicals and DNA methylation in adults: a systematic review of the epidemiologic evidence.

    PubMed

    Ruiz-Hernandez, Adrian; Kuo, Chin-Chi; Rentero-Garrido, Pilar; Tang, Wan-Yee; Redon, Josep; Ordovas, Jose M; Navas-Acien, Ana; Tellez-Plaza, Maria

    2015-01-01

    Current evidence supports the notion that environmental exposures are associated with DNA-methylation and expression changes that can impact human health. Our objective was to conduct a systematic review of epidemiologic studies evaluating the association between environmental chemicals with DNA methylation levels in adults. After excluding arsenic, recently evaluated in a systematic review, we identified a total of 17 articles (6 on cadmium, 4 on lead, 2 on mercury, 1 on nickel, 1 on antimony, 1 on tungsten, 5 on persistent organic pollutants and perfluorinated compounds, 1 on bisphenol A, and 3 on polycyclic aromatic hydrocarbons). The selected articles reported quantitative methods to determine DNA methylation including immunocolorimetric assays for total content of genomic DNA methylation, and microarray technologies, methylation-specific quantitative PCR, Luminometric Methylation Assay (LUMA), and bisulfite pyrosequencing for DNA methylation content of genomic sites such as gene promoters, LINE-1, Alu elements, and others. Considering consistency, temporality, strength, dose-response relationship, and biological plausibility, we concluded that the current evidence is not sufficient to provide inference because differences across studies and limited samples sizes make it difficult to compare across studies and to evaluate sources of heterogeneity. Important questions for future research include the need for larger and longitudinal studies, the validation of findings, and the systematic evaluation of the dose-response relationships. Future studies should also consider the evaluation of epigenetic marks recently in the research spotlight such as DNA hydroxymethylation and the role of underlying genetic variants. PMID:25984247

  19. Direct numerical simulation of turbulent, chemically reacting flows

    NASA Astrophysics Data System (ADS)

    Doom, Jeffrey Joseph

    This dissertation: (i) develops a novel numerical method for DNS/LES of compressible, turbulent reacting flows, (ii) performs several validation simulations, (iii) studies auto-ignition of a hydrogen vortex ring in air and (iv) studies a hydrogen/air turbulent diffusion flame. The numerical method is spatially non-dissipative, implicit and applicable over a range of Mach numbers. The compressible Navier-Stokes equations are rescaled so that the zero Mach number equations are discretely recovered in the limit of zero Mach number. The dependent variables are co--located in space, and thermodynamic variables are staggered from velocity in time. The algorithm discretely conserves kinetic energy in the incompressible, inviscid, non--reacting limit. The chemical source terms are implicit in time to allow for stiff chemical mechanisms. The algorithm is readily applicable to complex chemical mechanisms. Good results are obtained for validation simulations. The algorithm is used to study auto-ignition in laminar vortex rings. A nine species, nineteen reaction mechanism for H2/air combustion proposed by Mueller et al. [37] is used. Diluted H 2 at ambient temperature (300 K) is injected into hot air. The simulations study the effect of fuel/air ratio, oxidizer temperature, Lewis number and stroke ratio (ratio of piston stroke length to diameter). Results show that auto--ignition occurs in fuel lean, high temperature regions with low scalar dissipation at a 'most reactive' mixture fraction, zeta MR (Mastorakos et al. [32]). Subsequent evolution of the flame is not predicted by zetaMR; a most reactive temperature TMR is defined and shown to predict both the initial auto-ignition as well as subsequent evolution. For stroke ratios less than the formation number, ignition in general occurs behind the vortex ring and propagates into the core. At higher oxidizer temperatures, ignition is almost instantaneous and occurs along the entire interface between fuel and oxidizer. For stroke

  20. 33 CFR 95.035 - Reasonable cause for directing a chemical test.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Reasonable cause for directing a... DANGEROUS DRUG § 95.035 Reasonable cause for directing a chemical test. (a) Only a law enforcement officer... reasonable cause exists. Reasonable cause exists when: (1) The individual was directly involved in...

  1. Line scanning system for direct digital chemiluminescence imaging of DNA sequencing blots

    SciTech Connect

    Karger, A.E.; Weiss, R.; Gesteland, R.F. Eccles Inst. of Human Genetics, Salt Lake City, UT )

    1993-07-01

    A cryogenically cooled charge-coupled device (CCD) camera equipped with an area CCD array is used in a line scanning system for low-light-level imaging of chemiluminescent DNA sequencing blots. Operating the CCD camera in time-delayed integration (TDI) mode results in continuous data acquisition independent of the length of the CCD array. Scanning is possible with a resolution of 1.4 line pairs/mm at the 50% level of the modulation transfer function. High-sensitivity, low-light-level scanning of chemiluminescent direct-transfer electrophoresis (DTE) DNA sequencing blots is shown. The detection of DNA fragments on the blot involves DNA-DNA hybridization with oligonucleotide-alkaline phosphatase conjugate and 1,2-dioxetane-based chemiluminescence. The width of the scan allows the recording of up to four sequencing reactions (16 lanes) on one scan. The scan speed of 52 cm/h used for the sequencing blots corresponds to a data acquisition rate of 384 pixels/s. The chemiluminescence detection limit on the scanned images is 3.9 [times] 10[sup [minus]18] mol of plasmid DNA. A conditional median filter is described to remove spikes caused by cosmic ray events from the CCD images. 39 refs., 9 refs.

  2. INVOLVED IN DE NOVO 2-containing complex involved in RNA-directed DNA methylation in Arabidopsis

    SciTech Connect

    Ausin, Israel; Greenberg, Maxim V.C.; Simanshu, Dhirendra K.; Hale, Christopher J.; Vashisht, Ajay A.; Simon, Stacey A.; Lee, Tzuu-fen; Feng, Suhua; Española, Sophia D.; Meyers, Blake C.; Wohlschlegel, James A.; Patel, Dinshaw J.; Jacobsen, Steven E.

    2012-10-23

    At least three pathways control maintenance of DNA cytosine methylation in Arabidopsis thaliana. However, the RNA-directed DNA methylation (RdDM) pathway is solely responsible for establishment of this silencing mark. We previously described INVOLVED IN DE NOVO 2 (IDN2) as being an RNA-binding RdDM component that is required for DNA methylation establishment. In this study, we describe the discovery of two partially redundant proteins that are paralogous to IDN2 and that form a stable complex with IDN2 in vivo. Null mutations in both genes, termed IDN2-LIKE 1 and IDN2-LIKE 2 (IDNL1 and IDNL2), result in a phenotype that mirrors, but does not further enhance, the idn2 mutant phenotype. Genetic analysis suggests that this complex acts in a step in the downstream portion of the RdDM pathway. We also have performed structural analysis showing that the IDN2 XS domain adopts an RNA recognition motif (RRM) fold. Finally, genome-wide DNA methylation and expression analysis confirms the placement of the IDN proteins in an RdDM pathway that affects DNA methylation and transcriptional control at many sites in the genome. Results from this study identify and describe two unique components of the RdDM machinery, adding to our understanding of DNA methylation control in the Arabidopsis genome.

  3. Directly observing the motion of DNA molecules near solid-state nanopores.

    PubMed

    Ando, Genki; Hyun, Changbae; Li, Jiali; Mitsui, Toshiyuki

    2012-11-27

    We investigate the diffusion and the drift motion of λ DNA molecules near solid-state nanopores prior to their translocation through the nanopores using fluorescence microscopy. The radial dependence of the electric field near a nanopore generated by an applied voltage in ionic solution can be estimated quantitatively in 3D by analyzing the motion of negatively charged DNA molecules. We find that the electric field is approximately spherically symmetric around the nanopore under the conditions investigated. In addition, DNA clogging at the nanopore was directly observed. Surprisingly, the probability of the clogging event increases with increasing external bias voltage. We also find that DNA molecules clogging the nanopore reduce the electric field amplitude at the nanopore membrane surface. To better understand these experimental results, analytical method with Ohm's law and computer simulation with Poisson and Nernst-Planck (PNP) equations are used to calculate the electric field near the nanopore. These results are of great interest in both experimental and theoretical considerations of the motion of DNA molecules near voltage-biased nanopores. These findings will also contribute to the development of solid-state nanopore-based DNA sensing devices. PMID:23046052

  4. Electroporation mediated DNA vaccination directly to a mucosal surface results in improved immune responses

    PubMed Central

    Kichaev, Gleb; Mendoza, Janess M; Amante, Dinah; Smith, Trevor RF; McCoy, Jay R; Sardesai, Niranjan Y; Broderick, Kate E

    2013-01-01

    In vivo electroporation (EP) has been shown to be a highly efficient non-viral method for enhancing DNA vaccine delivery and immunogenicity, when the site of immunization is the skin or muscle of animals and humans. However, the route of entry for many microbial pathogens is via the mucosal surfaces of the human body. We have previously reported on minimally invasive, surface and contactless EP devices for enhanced DNA delivery to dermal tissue. Robust antibody responses were induced following vaccine delivery in several tested animal models using these devices. Here, we investigated extending the modality of the surface device to efficiently deliver DNA vaccines to mucosal tissue. Initially, we demonstrated reporter gene expression in the epithelial layer of buccal mucosa in a guinea pig model. There was minimal tissue damage in guinea pig mucosal tissue resulting from EP. Delivery of a DNA vaccine encoding influenza virus nucleoprotein (NP) of influenza H1N1 elicited robust and sustained systemic IgG antibody responses following EP-enhanced delivery in the mucosa. Upon further analysis, IgA antibody responses were detected in vaginal washes and sustained cellular immune responses were detected in animals immunized at the oral mucosa with the surface EP device. This data confirms that DNA delivery and EP targeting mucosal tissue directly results in both robust and sustainable humoral as well as cellular immune responses without tissue damage. These responses are seen both in the mucosa and systemically in the blood. Direct DNA vaccine delivery enhanced by EP in mucosa may have important clinical applications for delivery of prophylactic and therapeutic DNA vaccines against diseases such as HIV, HPV and pneumonia that enter at mucosal sites and require both cellular and humoral immune responses for protection. PMID:23954979

  5. Electroporation mediated DNA vaccination directly to a mucosal surface results in improved immune responses.

    PubMed

    Kichaev, Gleb; Mendoza, Janess M; Amante, Dinah; Smith, Trevor R F; McCoy, Jay R; Sardesai, Niranjan Y; Broderick, Kate E

    2013-10-01

    In vivo electroporation (EP) has been shown to be a highly efficient non-viral method for enhancing DNA vaccine delivery and immunogenicity, when the site of immunization is the skin or muscle of animals and humans. However, the route of entry for many microbial pathogens is via the mucosal surfaces of the human body. We have previously reported on minimally invasive, surface and contactless EP devices for enhanced DNA delivery to dermal tissue. Robust antibody responses were induced following vaccine delivery in several tested animal models using these devices. Here, we investigated extending the modality of the surface device to efficiently deliver DNA vaccines to mucosal tissue. Initially, we demonstrated reporter gene expression in the epithelial layer of buccal mucosa in a guinea pig model. There was minimal tissue damage in guinea pig mucosal tissue resulting from EP. Delivery of a DNA vaccine encoding influenza virus nucleoprotein (NP) of influenza H1N1 elicited robust and sustained systemic IgG antibody responses following EP-enhanced delivery in the mucosa. Upon further analysis, IgA antibody responses were detected in vaginal washes and sustained cellular immune responses were detected in animals immunized at the oral mucosa with the surface EP device. This data confirms that DNA delivery and EP targeting mucosal tissue directly results in both robust and sustainable humoral as well as cellular immune responses without tissue damage. These responses are seen both in the mucosa and systemically in the blood. Direct DNA vaccine delivery enhanced by EP in mucosa may have important clinical applications for delivery of prophylactic and therapeutic DNA vaccines against diseases such as HIV, HPV and pneumonia that enter at mucosal sites and require both cellular and humoral immune responses for protection. PMID:23954979

  6. The Chemical Synthesis of DNA/RNA: Our Gift to Science

    PubMed Central

    Caruthers, Marvin H.

    2013-01-01

    It is a great privilege to contribute to the Reflections essays. In my particular case, this essay has allowed me to weave some of my major scientific contributions into a tapestry held together by what I have learned from three colleagues (Robert Letsinger, Gobind Khorana, and George Rathmann) who molded my career at every important junction. To these individuals, I remain eternally grateful, as they always led by example and showed many of us how to break new ground in both science and biotechnology. Relative to my scientific career, I have focused primarily on two related areas. The first is methodologies we developed for chemically synthesizing DNA and RNA. Synthetic DNA and RNA continue to be an essential research tool for biologists, biochemists, and molecular biologists. The second is developing new approaches for solving important biological problems using synthetic DNA, RNA, and their analogs. PMID:23223445

  7. Quantitative Profiling of DNA Damage and Apoptotic Pathways in UV Damaged Cells Using PTMScan Direct

    PubMed Central

    Stokes, Matthew P.; Silva, Jeffrey C.; Jia, Xiaoying; Lee, Kimberly A.; Polakiewicz, Roberto D.; Comb, Michael J.

    2013-01-01

    Traditional methods for analysis of peptides using liquid chromatography and tandem mass spectrometry (LC-MS/MS) lack the specificity to comprehensively monitor specific biological processes due to the inherent duty cycle limitations of the MS instrument and the stochastic nature of the analytical platform. PTMScan Direct is a novel, antibody-based method that allows quantitative LC-MS/MS profiling of specific peptides from proteins that reside in the same signaling pathway. New PTMScan Direct reagents have been produced that target peptides from proteins involved in DNA Damage/Cell Cycle and Apoptosis/Autophagy pathways. Together, the reagents provide access to 438 sites on 237 proteins in these signaling cascades. These reagents have been used to profile the response to UV damage of DNA in human cell lines. UV damage was shown to activate canonical DNA damage response pathways through ATM/ATR-dependent signaling, stress response pathways and induce the initiation of apoptosis, as assessed by an increase in the abundance of peptides corresponding to cleaved, activated caspases. These data demonstrate the utility of PTMScan Direct as a multiplexed assay for profiling specific cellular responses to various stimuli, such as UV damage of DNA. PMID:23344034

  8. Direct amplification of DNA from fresh and preserved ectomycorrhizal root tips.

    PubMed

    Bent, Elizabeth; Taylor, D Lee

    2010-02-01

    Methods are described by which DNA can be amplified directly from ectomycorrhizal root tip homogenates of a variety of plant species (Picea mariana (black spruce), Betula papyrifera (paper birch), Populus tremuloides (trembling aspen) and Alnus sp.(alder)), including root tips that have been preserved in RNA Later (Ambion, Austin, TX). In most cases for extracts and homogenates diluted 10-fold prior to PCR, and in all cases for 100-fold dilutions, direct amplification of DNA from fresh root tip homogenates yielded as many or more ng of PCR amplicon (fungal ITS region) than amplification of DNA extracted from the same tips using a commercial kit or a manual ethanol precipitation-based method. For alder root tip extracts diluted 10-fold, the commercial kit method yielded more ng of PCR amplicon than 10-fold diluted, although direct use of homogenates still resulted in amplification in all tips tested. We also demonstrate consistent amplification of DNA from homogenates of birch, spruce and aspen ectomycorrhizal root tips preserved for 4months in RNA Later. PMID:19963016

  9. Chemical reactions occurring during direct solar reduction of CO2.

    PubMed

    Lyma, J L; Jensen, R J

    2001-09-28

    At high temperatures carbon dioxide may absorb solar radiation and react to form carbon monoxide and molecular oxygen. The CO, so produced, may be converted by well-established means to a combustible fuel, such as methanol. We intend to make a future demonstration of the solar reduction of CO2 based on these processes. This paper, however, addresses only the problem of preserving, or even enhancing, the initial photolytic CO by quenching the hot gas with colder H2O or CO2. We present model calculations with a reaction mechanism used extensively in other calculations. If a CO2 gas stream is heated and photolyzed by intense solar radiation and then allowed to cool slowly, it will react back to the initial CO2 by a series of elementary chemical reactions. The back reaction to CO2 can be terminated with the rapid addition of CO2, water, or a mixture. Calculations show that a three-fold quench with pure CO2 will stop the reactions and preserve over 90% of the initial photolytic CO. We find that water has one of two effects. It can either increase the CO level, or it can catalyze the recombination of O and CO to CO2. The gas temperature is the determining factor. If the quench gas is not sufficient to keep the temperature below approximately 1100 K, a chain-branching reaction dominates and the reaction to CO2 occurs. If the temperature stays below that level a chain terminating reaction dominates and the CO is increased. The former case occurs below approximately a fourfold quench with a water/CO2 mixture. The later case occurs when the quench is greater than fourfold. We conclude that CO2, H2O, or a mixture may quench the hot gas stream photolyzed by solar radiation and preserve the photolytic CO. PMID:11589409

  10. A direct detection of Escherichia coli genomic DNA using gold nanoprobes

    PubMed Central

    2012-01-01

    Background In situation like diagnosis of clinical and forensic samples there exists a need for highly sensitive, rapid and specific DNA detection methods. Though conventional DNA amplification using PCR can provide fast results, it is not widely practised in diagnostic laboratories partially because it requires skilled personnel and expensive equipment. To overcome these limitations nanoparticles have been explored as signalling probes for ultrasensitive DNA detection that can be used in field applications. Among the nanomaterials, gold nanoparticles (AuNPs) have been extensively used mainly because of its optical property and ability to get functionalized with a variety of biomolecules. Results We report a protocol for the use of gold nanoparticles functionalized with single stranded oligonucleotide (AuNP- oligo probe) as visual detection probes for rapid and specific detection of Escherichia coli. The AuNP- oligo probe on hybridization with target DNA containing complementary sequences remains red whereas test samples without complementary DNA sequences to the probe turns purple due to acid induced aggregation of AuNP- oligo probes. The color change of the solution is observed visually by naked eye demonstrating direct and rapid detection of the pathogenic Escherichia coli from its genomic DNA without the need for PCR amplification. The limit of detection was ~54 ng for unamplified genomic DNA. The method requires less than 30 minutes to complete after genomic DNA extraction. However, by using unamplified enzymatic digested genomic DNA, the detection limit of 11.4 ng was attained. Results of UV-Vis spectroscopic measurement and AFM imaging further support the hypothesis of aggregation based visual discrimination. To elucidate its utility in medical diagnostic, the assay was validated on clinical strains of pathogenic Escherichia coli obtained from local hospitals and spiked urine samples. It was found to be 100% sensitive and proves to be highly specific without

  11. Single DNA molecule stretching measures the activity of chemicals that target the HIV-1 nucleocapsid protein

    PubMed Central

    Cruceanu, Margareta; Stephen, Andrew G.; Beuning, Penny J.; Gorelick, Robert J.; Fisher, Robert J.; Williams, Mark C.

    2006-01-01

    We develop a biophysical method for investigating chemical compounds that target the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NCp7). We used an optical tweezers instrument to stretch single λ-DNA molecules through the helix-to-coil transition in the presence of NCp7 and various chemical compounds. The change in the helix-coil transition width induced by wild-type NCp7 and its zinc finger variants correlates with in vitro nucleic acid chaperone activity measurements and in vivo assays. The compound-NC interaction measured here reduces NCp7’s capability to alter the transition width. Purified compounds from the NCI Diversity set, 119889, 119911, and 119913 reduce the chaperone activity of 5 nM NC in aqueous solution at 10 nM, 25 nM, and 100 nM concentration, respectively. Similarly, gallein reduced the activity of 4 nM NC at 100 nM concentration. Further analysis allows us to dissect the impact of each compound on both sequence-specific and non-sequence-specific DNA binding of NC, two of the main components of NC’s nucleic acid chaperone activity. These results suggest that DNA stretching experiments can be used to screen chemical compounds targeting NC proteins, and to further explore the mechanisms by which these compounds interact with NC and alter its nucleic acid chaperone activity. PMID:17034752

  12. Direct Measurement of Single-Molecule DNA Hybridization Dynamics with Single-Base Resolution.

    PubMed

    He, Gen; Li, Jie; Ci, Haina; Qi, Chuanmin; Guo, Xuefeng

    2016-07-25

    Herein, we report label-free detection of single-molecule DNA hybridization dynamics with single-base resolution. By using an electronic circuit based on point-decorated silicon nanowires as electrical probes, we directly record the folding/unfolding process of individual hairpin DNAs with sufficiently high signal-to-noise ratio and bandwidth. These measurements reveal two-level current oscillations with strong temperature dependence, enabling us to determine the thermodynamic and kinetic properties of hairpin DNA hybridization. More importantly, successive, stepwise increases and decreases in device conductance at low temperature on a microsecond timescale are successfully observed, indicating a base-by-base unfolding/folding process. The process demonstrates a kinetic zipper model for DNA hybridization/dehybridization at the single base-pair level. This measurement capability promises a label-free single-molecule approach to probe biomolecular interactions with fast dynamics. PMID:27272178

  13. Brownian dynamics simulation of the nucleosome chirality - the wrapping direction of DNA on the histone octamer

    NASA Astrophysics Data System (ADS)

    Wang, Peng-Ye

    2005-03-01

    In eukaryote nucleosome, DNA wraps around a histone octamer in a left-handed way. We study the process of chirality formation of nucleosome with Brownian dynamics simulation. We model the histone octamer with a quantitatively adjustable chirality: left-handed, right-handed or non-chiral, and simulate the dynamical wrapping process of a DNA molecule on it. We find that the chirality of a nucleosome formed is strongly dependent on that of the histone octamer, and different chiralities of the histone octamer induce its different rotation directions in the wrapping process of DNA. In addition, a very weak chirality of the histone octamer is quite enough for sustaining the correct chirality of the nucleosome formed. We also show that the chirality of a nucleosome may be broken at elevated temperature.

  14. DNA hybridization as a guide to phylogeny: chemical and physical limits.

    PubMed

    Schmid, C W; Marks, J

    1990-03-01

    The technique of forming interspecific DNA heteroduplexes and estimating phylogenetic distances from the depression in their duplex melting temperature has several physical and chemical constraints. These constraints determine the maximum phylogenetic distance that may be estimated by this technique and the most appropriate method of analyzing that distance. Melting curves of self-renatured single copy primate DNAs reveal the presence of components absent from the renaturation products of exactly paired sequences. This observation, which confirms existing literature, challenges a fundamental assumption: that orthologous (i.e., corresponding) DNA sequences in the divergent species are being compared in DNA heteroduplex melting experiments. As a model system, the thermal stabilities of heteroduplexes formed between a human alpha-globin cDNA and four alpha-like globin genes isolated from chimpanzee are qualitatively compared. The results of this comparison show that the cross-hybrids of imperfectly matched gene duplicates from divergent species can contribute to the additional components that are present in renatured single copy DNAs. Single copy DNA, as usually defined, includes sequence duplicates that will obscure phylogenetic comparisons in a mass hybridization of genomes. PMID:2109086

  15. DIRECT-ACTING, DNA-DAMAGING AS (III)-METHYLATED SPECIES: IMPLICATIONS FOR A CARCINOGENIC MECHANISM OF ACTION OF ARSENICALS

    EPA Science Inventory

    Direct-acting, DNA-damaging As (III)-methylated species: implications for a carcinogenic . mechanism of action of arsenicals

    Inorganic arsenic (iAs, arsenite and arsenate) has been thought to act as a carcinogen without reacting directly with DNA; neither iAs nor the As(...

  16. Interaction of monoclonal antibodies directed against bromodeoxyuridine with pyrimidine bases, nucleosides, and DNA

    SciTech Connect

    Miller, M.R.; Heyneman, C.; Walker, S.; Ulrich, R.G.

    1986-03-01

    Although antibodies directed against bromodeoxyuridine (BrdU) are being used in both clinical and basic research laboratories as tools to study and monitor DNA synthesis, little is known about the epitopes with which they react. Four monoclonal antibodies directed against BrdU were produced and were characterized to learn more about the epitopes on BrdU which are important for antibody recognition, to identify compounds other than BrdU which react with the antibodies and which might interfere with immunologic assays for BrdU, and to characterize the reaction of these antibodies with BrdU-containing DNA. By radioimmunoassays, the antibodies generally reacted well with 5-iododeoxyuridine, 5-fluorodeoxyuridine, and 5-nitrouracil. However, none of the antibodies reacted well with uridine - indicating that a substituent on uridine C5 was essential for antibody reactivity - or with 5-bromo or iodo-cytosine, indicating that the region around pyrimidine C4 is important for antibody recognition. Although the antibodies reacted with 5-halogen-substituted uracil bases, the antibodies reacted much better with the corresponding halogenated nucleosides, indicating that the sugar moiety was important for recognition. The presence of a triphosphate group of C'5 of BrdU (i.e., BrdUTP) did not detectably alter antibody recognition. S/sub 1/ nuclease treatment of purified DNA suggested that all four monoclonal antibodies reacted exclusively with single-stranded regions of BrdU-containing DNA. Comparison of detecting DNA synthesis by (/sup 3/H)TdR incorporation followed by autoradiography with that by BrdU incorporation followed by indirect immunofluorescence indicated that the latter technique was both an accurate and a sensitive measure of DNA synthesis.

  17. One-Directional Fluidic Flow Induced by Chemical Wave Propagation in a Microchannel.

    PubMed

    Arai, Miyu; Takahashi, Kazuhiro; Hattori, Mika; Hasegawa, Takahiko; Sato, Mami; Unoura, Kei; Nabika, Hideki

    2016-05-26

    A one-directional flow induced by chemical wave propagation was investigated to understand the origin of its dynamic flow. A cylindrical injection port was connected with a straight propagation channel; the chemical wave was initiated at the injection port. Chemical waves propagated with a constant velocity irrespective of the channel width, indicating that the dynamics of the chemical waves were governed by a geometry-independent interplay between the chemical reaction and diffusion. In contrast, the velocity of the one-directional flow was dependent on the channel width. Furthermore, enlargement of the injection port volume increased the flow velocity and volume flux. These results imply that the one-directional flow in the microchannel is due to a hydrodynamic effect induced in the injection port. Spectroscopic analysis of a pH indicator revealed the simultaneous behavior between the pH increase near the injection port and the one-directional flow. Hence, we can conclude that the one-directional flow in the microchannel with chemical wave propagation was caused by a proton consumption reaction in the injection port, probably through liquid volume expansion by the reaction products and the reaction heat. It is a characteristic feature of the present system that the hydrodynamic flow started from the chemical wave initiation point and not the propagation wavefront, as observed for previous systems. PMID:27167307

  18. Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification

    NASA Astrophysics Data System (ADS)

    Yan, Yurong; Ding, Shijia; Zhao, Dan; Yuan, Rui; Zhang, Yuhong; Cheng, Wei

    2016-01-01

    Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification.

  19. Direct detection of DNA methylation during single-molecule, real-time sequencing

    PubMed Central

    Flusberg, Benjamin A.; Webster, Dale; Lee, Jessa; Travers, Kevin; Olivares, Eric; Clark, Tyson A.; Korlach, Jonas; Turner, Stephen W.

    2010-01-01

    We describe the direct detection of DNA methylation, without bisulfite conversion, through single-molecule real-time (SMRT) sequencing. In SMRT sequencing, DNA polymerases catalyze the incorporation of fluorescently labeled nucleotides into complementary nucleic acid strands. The arrival times and durations of the resulting fluorescence pulses yield information about polymerase kinetics and allow direct detection of modified nucleotides in the DNA template, including N6-methyladenosine, 5-methylcytosine, and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of the primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We utilize these kinetic signatures to identify adenosine methylation in genomic samples and show that, in combination with circular consensus sequencing, they can enable single-molecule identification of epigenetic modifications with base-pair resolution. This method is amenable to long read lengths and will likely enable mapping of methylation patterns within even highly repetitive genomic regions. PMID:20453866

  20. Specific and reversible DNA-directed self-assembly of oil-in-water emulsion droplets

    PubMed Central

    Hadorn, Maik; Boenzli, Eva; Sørensen, Kristian T.; Fellermann, Harold; Eggenberger Hotz, Peter; Hanczyc, Martin M.

    2012-01-01

    Higher-order structures that originate from the specific and reversible DNA-directed self-assembly of microscopic building blocks hold great promise for future technologies. Here, we functionalized biotinylated soft colloid oil-in-water emulsion droplets with biotinylated single-stranded DNA oligonucleotides using streptavidin as an intermediary linker. We show the components of this modular linking system to be stable and to induce sequence-specific aggregation of binary mixtures of emulsion droplets. Three length scales were thereby involved: nanoscale DNA base pairing linking microscopic building blocks resulted in macroscopic aggregates visible to the naked eye. The aggregation process was reversible by changing the temperature and electrolyte concentration and by the addition of competing oligonucleotides. The system was reset and reused by subsequent refunctionalization of the emulsion droplets. DNA-directed self-assembly of oil-in-water emulsion droplets, therefore, offers a solid basis for programmable and recyclable soft materials that undergo structural rearrangements on demand and that range in application from information technology to medicine. PMID:23175791

  1. Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification

    PubMed Central

    Yan, Yurong; Ding, Shijia; Zhao, Dan; Yuan, Rui; Zhang, Yuhong; Cheng, Wei

    2016-01-01

    Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification. PMID:26729209

  2. Efficient processing of TFO-directed psoralen DNA interstrand crosslinks by the UvrABC nuclease.

    PubMed

    Christensen, Laura A; Wang, Hong; Van Houten, Bennett; Vasquez, Karen M

    2008-12-01

    Photoreactive psoralens can form interstrand crosslinks (ICLs) in double-stranded DNA. In eubacteria, the endonuclease UvrABC plays a key role in processing psoralen ICLs. Psoralen-modified triplex-forming oligonucleotides (TFOs) can be used to direct ICLs to specific genomic sites. Previous studies of pyrimidine-rich methoxypsoralen-modified TFOs indicated that the TFO inhibits cleavage by UvrABC. Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease. Using an HMT-modified TFO to direct ICLs to a specific site, we found that UvrABC made incisions on the purine-rich strand of the duplex approximately 3 bases from the 3'-side and approximately 9 bases from the 5'-side of the ICL, within the TFO-binding region. In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone. Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB. As mutagenesis by TFO-directed ICLs requires nucleotide excision repair, the efficient processing of these lesions supports the use of triplex technology to direct DNA damage for genome modification. PMID:18996898

  3. DNA polymerases engineered by directed evolution to incorporate non-standard nucleotides

    PubMed Central

    Laos, Roberto; Thomson, J. Michael; Benner, Steven A.

    2014-01-01

    DNA polymerases have evolved for billions of years to accept natural nucleoside triphosphate substrates with high fidelity and to exclude closely related structures, such as the analogous ribonucleoside triphosphates. However, polymerases that can accept unnatural nucleoside triphosphates are desired for many applications in biotechnology. The focus of this review is on non-standard nucleotides that expand the genetic “alphabet.” This review focuses on experiments that, by directed evolution, have created variants of DNA polymerases that are better able to accept unnatural nucleotides. In many cases, an analysis of past evolution of these polymerases (as inferred by examining multiple sequence alignments) can help explain some of the mutations delivered by directed evolution. PMID:25400626

  4. Homology-directed Fanconi anemia pathway crosslink repair is dependent on DNA replication

    PubMed Central

    Nakanishi, Koji; Cavallo, Francesca; Perrouault, Loïc; Giovannangeli, Carine; Moynahan, Mary Ellen; Barchi, Marco; Brunet, Erika; Jasin, Maria

    2012-01-01

    Homologous recombination (also termed homology-directed repair, HDR) is a major pathway for the repair of DNA interstrand crosslinks (ICLs) in mammalian cells. Cells from Fanconi anemia (FA) patients are characterized by extreme ICL sensitivity, but their reported defect in HDR is mild. Here, we examined ICL-induced HDR using a GFP reporter and observed a profound defect in ICL-induced HDR in FA cells, but only when the reporter could replicate. PMID:21423196

  5. Chase the direct impact of rainfall into groundwater in Mt. Fuji from multiple analyses including microbial DNA

    NASA Astrophysics Data System (ADS)

    Kato, Kenji; Sugiyama, Ayumi; Nagaosa, Kazuyo; Tsujimura, Maki

    2016-04-01

    A huge amount of groundwater is stored in subsurface environment of Mt. Fuji, the largest volcanic mountain in Japan. Based on the concept of piston flow transport of groundwater an apparent residence time was estimated to ca. 30 years by 36Cl/Cl ratio (Tosaki et al., 2011). However, this number represents an averaged value of the residence time of groundwater which had been mixed before it flushes out. We chased signatures of direct impact of rainfall into groundwater to elucidate the routes of groundwater, employing three different tracers; stable isotopic analysis (delta 18O), chemical analysis (concentration of silica) and microbial DNA analysis. Though chemical analysis of groundwater shows an averaged value of the examined water which was blended by various water with different sources and routes in subsurface environment, microbial DNA analysis may suggest the place where they originated, which may give information of the source and transport routes of the water examined. Throughout the in situ observation of four rainfall events showed that stable oxygen isotopic ratio of spring water and shallow groundwater obtained from 726m a.s.l. where the average recharge height of rainfall was between 1500 and 1800 m became higher than the values before a torrential rainfall, and the concentration of silica decreased after this event when rainfall exceeded 300 mm in precipitation of an event. In addition, the density of Prokaryotes in spring water apparently increased. Those changes did not appear when rainfall did not exceed 100 mm per event. Thus, findings shown above indicated a direct impact of rainfall into shallow groundwater, which appeared within a few weeks of torrential rainfall in the studied geological setting. In addition, increase in the density of Archaea observed at deep groundwater after the torrential rainfall suggested an enlargement of the strength of piston flow transport through the penetration of rainfall into deep groundwater. This finding was

  6. The antitumor toxin CD437 is a direct inhibitor of DNA polymerase α.

    PubMed

    Han, Ting; Goralski, Maria; Capota, Emanuela; Padrick, Shae B; Kim, Jiwoong; Xie, Yang; Nijhawan, Deepak

    2016-07-01

    CD437 is a retinoid-like small molecule that selectively induces apoptosis in cancer cells, but not in normal cells, through an unknown mechanism. We used a forward-genetic strategy to discover mutations in POLA1 that coincide with CD437 resistance (POLA1(R)). Introduction of one of these mutations into cancer cells by CRISPR-Cas9 genome editing conferred CD437 resistance, demonstrating causality. POLA1 encodes DNA polymerase α, the enzyme responsible for initiating DNA synthesis during the S phase of the cell cycle. CD437 inhibits DNA replication in cells and recombinant POLA1 activity in vitro. Both effects are abrogated by the identified POLA1 mutations, supporting POLA1 as the direct antitumor target of CD437. In addition, we detected an increase in the total fluorescence intensity and anisotropy of CD437 in the presence of increasing concentrations of POLA1 that is consistent with a direct binding interaction. The discovery of POLA1 as the direct anticancer target for CD437 has the potential to catalyze the development of CD437 into an anticancer therapeutic. PMID:27182663

  7. PE-Swab Direct STR Amplification of Forensic Touch DNA Samples.

    PubMed

    Liu, Jason Y

    2015-05-01

    The PE-Swab direct STR amplification workflow was developed to process low-level "touch DNA" samples. In this workflow, a forensic sample is first collected on a 4-mm PE-Swab (a novel sample collection device); two 2-mm punches containing collected samples are then generated from the PE-Swab and directly amplified for STR typing. Compared to the conventional STR workflow, which involves DNA extraction, purification, and elution volume reduction, the PE-Swab direct STR amplification workflow does not require sample preparation and takes <60 sec before a touch sample is ready for STR amplification. Because there is no DNA loss due to sample preparation, the PE-Swab workflow is more sensitive than the conventional STR workflow. The average peak height per sample obtained by the PE-swab workflow is 3 times higher than that from the conventional workflow with both low-level single source and two-contributor mixture samples tested in this study. PMID:25684449

  8. Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes

    SciTech Connect

    Novicki, D.L.; Rosenberg, M.R.; Michalopoulos, G.

    1985-01-01

    Rat hepatocytes in primary culture can be stimulated to replicate under the influence of rat serum and sparse plating conditions. Higher replication rates are induced by serum from two-thirds partially hepatectomized rats. The effects of carcinogens and noncarcinogens on the ability of hepatocytes to synthesize DNA were examined by measuring the incorporation of (3H)thymidine by liquid scintillation counting and autoradiography. Hepatocyte DNA synthesis was not decreased by ethanol or dimethyl sulfoxide at concentrations less than 0.5%. No effect was observed when 0.1 mM ketamine, Nembutal, hypoxanthine, sucrose, ascorbic acid, or benzo(e)pyrene was added to cultures of replicating hepatocytes. Estrogen, testosterone, tryptophan, and vitamin E inhibited DNA synthesis by approximately 50% at 0.1 mM, a concentration at which toxicity was noticeable. Several carcinogens requiring metabolic activation as well as the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine interfered with DNA synthesis. Aflatoxin B1 inhibited DNA synthesis by 50% (ID50) at concentrations between 1 X 10(-8) and 1 X 10(-7) M. The ID50 for 2-acetylaminofluorene was between 1 X 10(-7) and 1 X 10(-6) M. Benzo(a)pyrene and 3'-methyl-4-dimethylaminoazobenzene inhibited DNA synthesis 50% between 1 X 10(-5) and 1 X 10(-4) M. Diethylnitrosamine and dimethylnitrosamine (ID50 between 1 X 10(-4) and 5 X 10(-4) M) and 1- and 2-naphthylamine (ID50 between 1 X 10(-5) and 5 X 10(-4) M) caused inhibition of DNA synthesis at concentrations which overlapped with concentrations that caused measurable toxicity.

  9. DETECTION OF LOW DOSE RADIATION-AND CHEMICALLY-INDUCED DNA DAMAGE USING TEMPERATURE DIFFERENTIAL FLUORESCENCE ASSAYS

    EPA Science Inventory

    Rapid, sensitive and simple assays for radiation- and chemically-induced DNA damage can be of significant benefit to a number of fields including radiation biology, clinical research, and environmental monitoring. Although temperature-induced DNA strand separation has been use...

  10. Monitoring DNA Contamination in Handled vs. Directly Excavated Ancient Human Skeletal Remains

    PubMed Central

    Pilli, Elena; Modi, Alessandra; Serpico, Ciro; Achilli, Alessandro; Lancioni, Hovirag; Lippi, Barbara; Bertoldi, Francesca; Gelichi, Sauro; Lari, Martina; Caramelli, David

    2013-01-01

    Bones, teeth and hair are often the only physical evidence of human or animal presence at an archaeological site; they are also the most widely used sources of samples for ancient DNA (aDNA) analysis. Unfortunately, the DNA extracted from ancient samples, already scarce and highly degraded, is widely susceptible to exogenous contaminations that can affect the reliability of aDNA studies. We evaluated the molecular effects of sample handling on five human skeletons freshly excavated from a cemetery dated between the 11 to the 14th century. We collected specimens from several skeletal areas (teeth, ribs, femurs and ulnas) from each individual burial. We then divided the samples into two different sets: one labeled as “virgin samples” (i.e. samples that were taken by archaeologists under contamination-controlled conditions and then immediately sent to the laboratory for genetic analyses), and the second called “lab samples”(i.e. samples that were handled without any particular precautions and subject to normal washing, handling and measuring procedures in the osteological lab). Our results show that genetic profiles from “lab samples” are incomplete or ambiguous in the different skeletal areas while a different outcome is observed in the “virgin samples” set. Generally, all specimens from different skeletal areas in the exception of teeth present incongruent results between “lab” and “virgin” samples. Therefore teeth are less prone to contamination than the other skeletal areas we analyzed and may be considered a material of choice for classical aDNA studies. In addition, we showed that bones can also be a good candidate for human aDNA analysis if they come directly from the excavation site and are accompanied by a clear taphonomic history. PMID:23372650

  11. Direct single-molecule observations of local denaturation of a DNA double helix under a negative supercoil state.

    PubMed

    Takahashi, Shunsuke; Motooka, Shinya; Usui, Tomohiro; Kawasaki, Shohei; Miyata, Hidefumi; Kurita, Hirofumi; Mizuno, Takeshi; Matsuura, Shun-ichi; Mizuno, Akira; Oshige, Masahiko; Katsura, Shinji

    2015-03-17

    Effects of a negative supercoil on the local denaturation of the DNA double helix were studied at the single-molecule level. The local denaturation in λDNA and λDNA containing the SV40 origin of DNA replication (SV40ori-λDNA) was directly observed by staining single-stranded DNA regions with a fusion protein comprising the ssDNA binding domain of a 70-kDa subunit of replication protein A and an enhanced yellow fluorescent protein (RPA-YFP) followed by staining the double-stranded DNA regions with YOYO-1. The local denaturation of λDNA and SV40ori-λDNA under a negative supercoil state was observed as single bright spots at the single-stranded regions. When negative supercoil densities were gradually increased to 0, -0.045, and -0.095 for λDNA and 0, -0.047, and -0.1 for SV40ori-λDNA, single bright spots at the single-stranded regions were frequently induced under higher negative supercoil densities of -0.095 for λDNA and -0.1 for SV40ori-λDNA. However, single bright spots of the single-stranded regions were rarely observed below a negative supercoil density of -0.045 and -0.047 for λDNA and SV40ori-λDNA, respectively. The probability of occurrence of the local denaturation increased with negative superhelicity for both λDNA and SV40ori-λDNA. PMID:25697222

  12. Functionalized 2'-amino-alpha-L-LNA: directed positioning of intercalators for DNA targeting.

    PubMed

    Kumar, T Santhosh; Madsen, Andreas S; Østergaard, Michael E; Sau, Sujay P; Wengel, Jesper; Hrdlicka, Patrick J

    2009-02-01

    Chemically modified oligonucleotides are increasingly applied in nucleic acid based therapeutics and diagnostics. LNA (locked nucleic acid) and its diastereomer alpha-L-LNA are two promising examples thereof that exhibit increased thermal and enzymatic stability. Herein, the synthesis, biophysical characterization, and molecular modeling of N2'-functionalized 2'-amino-alpha-L-LNA is described. Chemoselective N2'-functionalization of protected amino alcohol 1 followed by phosphitylation afforded a structurally varied set of target phosphoramidites, which were incorporated into oligodeoxyribonucleotides. Incorporation of pyrene-functionalized building blocks such as 2'-N-(pyren-1-yl)carbonyl-2'-amino-alpha-L-LNA (monomer X) led to extraordinary increases in thermal affinity of up to +19.5 degrees C per modification against DNA targets in particular. In contrast, incorporation of building blocks with small nonaromatic N2'-functionalities such as 2'-N-acetyl-2'-amino-alpha-L-LNA (monomer V) had detrimental effects on thermal affinity toward DNA/RNA complements with decreases of as much as -16.5 degrees C per modification. Extensive thermal DNA selectivity, favorable entropic contributions upon duplex formation, hybridization-induced bathochromic shifts of pyrene absorption maxima and increases in circular dichroism signal intensity, and molecular modeling studies suggest that pyrene-functionalized 2'-amino-alpha-L-LNA monomers W-Y having short linkers between the bicyclic skeleton and the pyrene moiety allow high-affinity hybridization with DNA complements and precise positioning of intercalators in nucleic acid duplexes. This rigorous positional control has been utilized for the development of probes for emerging therapeutic and diagnostic applications focusing on DNA targeting. PMID:19108636

  13. Functionalized 2′-Amino-α-L-LNA - Directed Positioning of Intercalators for DNA Targeting

    PubMed Central

    Kumar, T. Santhosh; Madsen, Andreas S.; Østergaard, Michael E.; Sau, Sujay P.; Wengel, Jesper; Hrdlicka, Patrick J.

    2010-01-01

    Chemically modified oligonucleotides are increasingly applied in nucleic acid based therapeutics and diagnostics. LNA (Locked Nucleic Acid) and its diastereomer α-L-LNA are two promising examples hereof that exhibit increased thermal and enzymatic stability. Herein, the synthesis, biophysical characterization and molecular modeling of N2′-functionalized 2′-amino-α-L-LNA is described. Chemoselective N2′-functionalization of protected amino alcohol 1 followed by phosphitylation afforded a structurally varied set of target phosphoramidites, which were incorporated into oligodeoxyribonucleotides. Incorporation of pyrene-functionalized building blocks such as 2′-N-(pyren-1-yl)carbonyl-2′-amino-α-L-LNA (monomer X) led to extraordinary increases in thermal affinity of up to +19.5 °C per modification against DNA targets in particular. In contrast, incorporation of building blocks with small non-aromatic N2′-functionalities such as 2′-N-acetyl-2′-amino-α-L-LNA (monomer V) had detrimental effects on thermal affinity toward DNA/RNA complements with decreases of as much as −16.5 °C per modification. Extensive thermal DNA selectivity, favorable entropic contributions upon duplex formation, hybridization-induced bathochromic shifts of pyrene absorption maxima and increases of circular dichroism signals, and molecular modeling studies suggest that pyrene functionalized 2′-amino-α-L-LNA monomers W-Y having short linkers between the bicyclic skeleton and the pyrene moiety, allow high-affinity hybridization with DNA complements and precise positioning of intercalators in nucleic acid duplexes. This rigorous positional control has been utilized for the development probes for emerging therapeutic and diagnostic applications focusing on DNA-targeting. PMID:19108636

  14. [DNA and chemical analyses of commercial fly agaric-related products].

    PubMed

    Maruyama, Takuro; Kawahara, Nobuo; Fukiharu, Toshimitsu; Yokoyama, Kazumasa; Makino, Yukiko; Goda, Yukihiro

    2005-04-01

    Since June 6, 2002, psilocin and psilocybin-containing fungi (commonly called "magic mushrooms") have been regulated by the Narcotics and Psychotropics Control Law in Japan. However, various fly agaric-related products are now entering the Japanese market via the internet. In this study, fly agaric-related products available in this way were investigated for raw materials by DNA analysis and for additives by chemical analysis. Nucleotide sequence analysis of the mitochondrial 12S rDNA region suggested that these fly agaric-related products originate from A. muscaria or A. muscaria var. persicina. Furthermore, they were classified into three strains based on the ITS2-LSU nucleotide sequence. Harmine derivatives and/or tryptamine derivatives were detected in some of these products by LC/MS analysis. In accordance with this, the matK gene of Peganum harmala was found in all of the harmine derivative-containing samples. PMID:16018591

  15. Direct electrical and mechanical characterization of in situ generated DNA between the tips of silicon nanotweezers (SNT).

    PubMed

    Karsten, Stanislav L; Kumemura, Momoko; Jalabert, Laurent; Lafitte, Nicolas; Kudo, Lili C; Collard, Dominique; Fujita, Hiroyuki

    2016-05-24

    Previously, we reported the application of micromachined silicon nanotweezers (SNT) integrated with a comb-drive actuator and capacitive sensors for capturing and mechanical characterization of DNA bundles. Here, we demonstrate direct DNA amplification on such a MEMS structure with subsequent electrical and mechanical characterization of a single stranded DNA (ssDNA) bundle generated between the tips of SNT via isothermal rolling circle amplification (RCA) and dielectrophoresis (DEP). An in situ generated ssDNA bundle was visualized and evaluated via electrical conductivity (I-V) and mechanical frequency response measurements. Colloidal gold nanoparticles significantly enhanced (P < 0.01) the electrical properties of thin ssDNA bundles. The proposed technology allows direct in situ synthesis of DNA with a predefined sequence on the tips of a MEMS sensor device, such as SNT, followed by direct DNA electrical and mechanical characterization. In addition, our data provides a "proof-of-principle" for the feasibility of the on-chip label free DNA detection device that can be used for a variety of biomedical applications focused on sequence specific DNA detection. PMID:27161663

  16. Direct observation of the hole protonation state and hole localization site in DNA-oligomers

    PubMed Central

    Adhikary, Amitava; Khanduri, Deepti; Sevilla, Michael D.

    2009-01-01

    In this work, it is shown that the incorporation of an 8-deuteroguanine (G*) moiety in DNA-oligomers allows for direct determination at 77 K of (i) the location of holes (i.e., the radical site) within dsDNA at specific base sites, even within stacks of G, as well as (ii) the protonation state of the hole at that site. These findings are based on our work and demonstrate that selective deuteration at C-8 on guanine moiety in dGuo results in an ESR signal from the guanine cation radical (G*•+) which is easily distinguishable from that of the undeuterated guanine cation radical (G•+). G*•+ is also found to be easily distinguishable from its conjugate base, the N1-deprotonated radical, G*(−H)•. Our ESR results clearly establish that at 77 K (i) one-electron oxidized guanine in double stranded DNA-oligomers exists as the deprotonated neutral radical G(−H)• as a result of facile proton transfer to the hydrogen bonded cytosine, and (ii) the hole is preferentially located at the 5′-end in several ds DNA-oligomers with a GGG sequence. PMID:19469533

  17. Directing folding pathways for multi-component DNA origami nanostructures with complex topology

    NASA Astrophysics Data System (ADS)

    Marras, A. E.; Zhou, L.; Kolliopoulos, V.; Su, H.-J.; Castro, C. E.

    2016-05-01

    Molecular self-assembly has become a well-established technique to design complex nanostructures and hierarchical mesoscale assemblies. The typical approach is to design binding complementarity into nucleotide or amino acid sequences to achieve the desired final geometry. However, with an increasing interest in dynamic nanodevices, the need to design structures with motion has necessitated the development of multi-component structures. While this has been achieved through hierarchical assembly of similar structural units, here we focus on the assembly of topologically complex structures, specifically with concentric components, where post-folding assembly is not feasible. We exploit the ability to direct folding pathways to program the sequence of assembly and present a novel approach of designing the strand topology of intermediate folding states to program the topology of the final structure, in this case a DNA origami slider structure that functions much like a piston-cylinder assembly in an engine. The ability to program the sequence and control orientation and topology of multi-component DNA origami nanostructures provides a foundation for a new class of structures with internal and external moving parts and complex scaffold topology. Furthermore, this work provides critical insight to guide the design of intermediate states along a DNA origami folding pathway and to further understand the details of DNA origami self-assembly to more broadly control folding states and landscapes.

  18. A Single Subunit Directs the Assembly of the Escherichia coli DNA Sliding Clamp Loader

    PubMed Central

    Park, Ah Young; Jergic, Slobodan; Politis, Argyris; Ruotolo, Brandon T.; Hirshberg, Daniel; Jessop, Linda L.; Beck, Jennifer L.; Barsky, Daniel; O’Donnell, Mike; Dixon, Nicholas E.; Robinson, Carol V.

    2016-01-01

    SUMMARY Multi-protein clamp loader complexes are required to load sliding clamps onto DNA. In Escherichia coli the clamp loader contains three DnaX (τ/γ) proteins, δ, and δ′, which together form an asymmetric pentameric ring that also interacts with ψχ. Here we used mass spectrometry to examine the assembly and dynamics of the clamp loader complex. We find that γ exists exclusively as a stable homotetramer, while τ is in a monomer-dimer-trimer-tetramer equilibrium. δ′ plays a direct role in the assembly as a τ/γ oligomer breaker, thereby facilitating incorporation of lower oligomers. With δ′, both δ and ψχ stabilize the trimeric form of DnaX, thus completing the assembly. When τ and γ are present simultaneously, mimicking the situation in vivo, subunit exchange between τ and γ tetramers occurs rapidly to form heterocomplexes but is retarded when δδ′ is present. The implications for intracellular assembly of the DNA polymerase III holoenzyme are discussed. PMID:20223211

  19. A QuikChange-like method to realize efficient blunt-ended DNA directional cloning and site-directed mutagenesis simultaneously.

    PubMed

    An, Yingfeng; Lv, Anguo; Wu, Wenfang

    2010-06-25

    Here we present a QuikChange-like method to efficiently realize blunt-ended DNA cloning and conveniently introduce a site-directed mutation to recombinant plasmid at the same time. After blunt-ended DNA ligation and transformation, the plasmid DNA mixture is extracted from pooled transformants and directly used as template for PCR amplification with a pair of complementary mutagenic primers. With this method, sam1 gene was inserted into pUC19 vector by blunt-end ligation, and a unique restriction site Spe I was introduced to the recombinant plasmid at the same time. The randomly selected transformants were analyzed by DNA sequencing, and most of the clones were found to have correct sequences. However, no correct construct was found from randomly selected transformants after traditional blunt-ended DNA ligation and transformation. PMID:20471367

  20. DNA Concentration-Dependent Dissociation of EcoRI: Direct Transfer or Reaction during Hopping

    PubMed Central

    Sidorova, Nina Y.; Scott, Thomas; Rau, Donald C.

    2013-01-01

    Direct transfer of proteins between DNA helices is a recognized important feature of the recognition site search process. Direct transfer is characterized by a dissociation rate that depends on total DNA concentration. This is taken as evidence for the formation of an intermediate DNA-protein-DNA ternary complex. We find that the dissociation rate of EcoRI-DNA-specific complexes at 80 mM NaCl depends on the concentration of competitor oligonucleotide suggesting that direct transfer contributes to EcoRI dissociation. This dependence on competitor DNA concentration is not seen at 180 mM salt. A careful examination of the salt concentration dependence of the dissociation rate, however, shows that the predictions for the formation of a ternary complex are not observed experimentally. The findings can be rationalized by considering that just after dissociating from a DNA fragment the protein remains in close proximity to that fragment, can reassociate with it, and diffuse back to the recognition site rather than bind to an oligonucleotide in solution, a hopping excursion. The probability that a protein will bind to an oligonucleotide during a hop can be approximately calculated and shown to explain the data. A dependence of the dissociation rate of a DNA-protein complex on competitor DNA concentration does not necessarily mean direct transfer. PMID:23528089

  1. Dictyostelium discoideum, a lower eukaryote model for the study of DNA repair: Implications for the role of DNA-damaging chemicals in the evolution of repair proficient cells

    NASA Astrophysics Data System (ADS)

    Deering, R. A.

    1994-10-01

    The evolution of the ability of living cells to cope with stress is crucial for the maintenance of their genetic integrity. Yet low levels of mutation must remain to allow adaptation to environmental changes. The cellular slime mold D. discoideum is a good system for studying molecular aspects of the repair of lethal and mutagenic damage to DNA by radiation and chemicals. The wild-type strains of this soil microorganism are extremely resistant to DNA damaging agents. In nature the amoeboid cells in their replicative stage feed on soil bacteria and are exposed to numerous DNA-damaging chemicals produced by various soil microorganisms. It is probable that the evolution of repair systems in this organism and perhaps in others is a consequence of the necessity to cope with chemical damage which also confers resistance to radiation.

  2. Dictyostelium discoideum, a lower eukaryote model for the study of DNA repair: implications for the role of DNA-damaging chemicals in the evolution of repair proficient cells.

    PubMed

    Deering, R A

    1994-10-01

    The evolution of the ability of living cells to cope with stress is crucial for the maintenance of their genetic integrity. Yet low levels of mutation must remain to allow adaptation to environmental changes. The cellular slime mold D. discoideum is a good system for studying molecular aspects of the repair of lethal and mutagenic damage to DNA by radiation and chemicals. The wild-type strains of this soil microorganism are extremely resistant to DNA damaging agents. In nature the amoeboid cells in their replicative stage feed on soil bacteria and are exposed to numerous DNA-damaging chemicals produced by various soil microorganisms. It is probable that the evolution of repair systems in this organism and perhaps in others is a consequence of the necessity to cope with chemical damage which also confers resistance to radiation. PMID:11539974

  3. Sequence-specific assembly of FtsK hexamers establishes directional translocation on DNA

    PubMed Central

    Graham, James E.; Sherratt, David J.; Szczelkun, Mark D.

    2010-01-01

    FtsK is a homohexameric, RecA-like dsDNA translocase that plays a key role in bacterial chromosome segregation. The FtsK regulatory γ-subdomain determines directionality of translocation through its interaction with specific 8 base pair chromosomal sequences [(KOPS); FtsK Orienting / Polarizing Sequence(s)] that are cooriented with the direction of replication in the chromosome. We use millisecond-resolution ensemble translocation and ATPase assays to analyze the assembly, initiation, and translocation of FtsK. We show that KOPS are used to initiate new translocation events rather than reorient existing ones. By determining kinetic parameters, we show sigmoidal dependences of translocation and ATPase rates on ATP concentration that indicate sequential cooperative coupling of ATP hydrolysis to DNA motion. We also estimate the ATP coupling efficiency of translocation to be 1.63–2.11 bp of dsDNA translocated/ATP hydrolyzed. The data were used to derive a model for the assembly, initiation, and translocation of FtsK hexamers. PMID:21048089

  4. Electron-beam lithography and molecular liftoff for directed attachment of DNA nanostructures on silicon: top-down meets bottom-up.

    PubMed

    Pillers, Michelle; Goss, Valerie; Lieberman, Marya

    2014-06-17

    CONSPECTUS: Our work on lithographic patterning of DNA nanostructures was inspired by a collaboration on molecular electronic devices known as quantum-dot cellular automata or QCA. QCA is a paradigm for computation in which information is transmitted and processed through the interaction of coupled electrical charges or magnetic dipoles. We began to explore the idea of molecular scale QCA and found that ab initio methods, a thermodynamic Ising model, and larger scale circuit design work suggested that circuits that did computationally interesting things could function at room temperature if made from molecular QCA cells of chemically reasonable design. But how could the QCA cells be patterned to form the complex arrays needed for computationally interesting circuitry, and how could those arrays of molecular circuitry be integrated with conventional electronic inputs and outputs? Top-down methods lacked the spatial resolution and high level of parallelism needed to make molecular circuits. Bottom-up chemical synthesis lacked the ability to fabricate arbitrary and heterogeneous structures tens to hundreds of nanometers in size. Chemical self-assembly at the time could produce structures in the right size scale, but was limited to homogeneous arrays. A potential solution to this conundrum was just being demonstrated in the late 1990s and early 2000s: DNA nanostructures self-assembled from oligonucleotides, whose high information density could handle the creation of arbitrary structures and chemical inhomogeneity. Our group became interested in whether DNA nanostructures could function as self-assembling circuit boards for electrical or magnetic QCA systems. This Account focuses on what we learned about the interactions of DNA nanostructures with silicon substrates and, particularly, on how electron-beam lithography could be used to direct the binding of DNA nanostructures on a variety of functional substrates. PMID:24716716

  5. Connecting the dots of RNA-directed DNA methylation in Arabidopsis thaliana.

    PubMed

    Costa-Nunes, Pedro; Vitins, Alexa; Pontes, Olga

    2014-06-01

    Noncoding RNAs are the rising stars of genome regulation and are crucial to an organism's metabolism, development, and defense. One of their most notable functions is its ability to direct epigenetic modifications through small RNA molecules to specific genomic regions, ensuring transcriptional regulation, proper genome organization, and maintenance of genome integrity. Here, we review the current knowledge of the spatial organization of the Arabidopsis thaliana RNA-directed DNA methylation pathway within the cell nucleus, which, while known to be essential for the proper establishment of epigenetic modifications, remains poorly understood. We will also discuss possible future cytological approaches that have the potential of unveiling functional insights into how small RNA-directed epigenetics is regulated through the spatiotemporal regulation of its major components within the cell. PMID:24846724

  6. Direct mechanical stimulation of tip links in hair cells through DNA tethers

    PubMed Central

    Basu, Aakash; Lagier, Samuel; Vologodskaia, Maria; Fabella, Brian A; Hudspeth, AJ

    2016-01-01

    Mechanoelectrical transduction by hair cells commences with hair-bundle deflection, which is postulated to tense filamentous tip links connected to transduction channels. Because direct mechanical stimulation of tip links has not been experimentally possible, this hypothesis has not been tested. We have engineered DNA tethers that link superparamagnetic beads to tip links and exert mechanical forces on the links when exposed to a magnetic-field gradient. By pulling directly on tip links of the bullfrog's sacculus we have evoked transduction currents from hair cells, confirming the hypothesis that tension in the tip links opens transduction channels. This demonstration of direct mechanical access to tip links additionally lays a foundation for experiments probing the mechanics of individual channels. DOI: http://dx.doi.org/10.7554/eLife.16041.001 PMID:27331611

  7. The possible roles of water in the prebiotic chemical evolution of DNA.

    PubMed

    Cui, Shuxun

    2010-09-21

    There is no doubt that water is pivotal to life. Yet, as the emergence of life is still a big challenge in science, the detailed involvement of water in that process is not well recognized. Following the clues provided by recent single-molecule studies on DNA, we attempt to elucidate the possible roles of water in the prebiotic chemical evolution. Water has long been recognized as an important reactant in the Miller-Urey experiment and then as the only solvent of the primitive soup. Besides that, water also played a vital role in the prebiotic chemical evolution: water is the important criterion in the combinatorial library screening for self-assembling macromolecules. With this notion, the uniformity of biochemistry for all terrestrial life may be explained. A possible roadmap from the inorganic world to the origin of life is also discussed. PMID:20577681

  8. RNA-directed DNA methylation enforces boundaries between heterochromatin and euchromatin in the maize genome

    PubMed Central

    Li, Qing; Gent, Jonathan I.; Zynda, Greg; Song, Jawon; Makarevitch, Irina; Hirsch, Cory D.; Hirsch, Candice N.; Dawe, R. Kelly; Madzima, Thelma F.; McGinnis, Karen M.; Lisch, Damon; Schmitz, Robert J.; Vaughn, Matthew W.; Springer, Nathan M.

    2015-01-01

    The maize genome is relatively large (∼2.3 Gb) and has a complex organization of interspersed genes and transposable elements, which necessitates frequent boundaries between different types of chromatin. The examination of maize genes and conserved noncoding sequences revealed that many of these are flanked by regions of elevated asymmetric CHH (where H is A, C, or T) methylation (termed mCHH islands). These mCHH islands are quite short (∼100 bp), are enriched near active genes, and often occur at the edge of the transposon that is located nearest to genes. The analysis of DNA methylation in other sequence contexts and several chromatin modifications revealed that mCHH islands mark the transition from heterochromatin-associated modifications to euchromatin-associated modifications. The presence of an mCHH island is fairly consistent in several distinct tissues that were surveyed but shows some variation among different haplotypes. The presence of insertion/deletions in promoters often influences the presence and position of an mCHH island. The mCHH islands are dependent upon RNA-directed DNA methylation activities and are lost in mop1 and mop3 mutants, but the nearby genes rarely exhibit altered expression levels. Instead, loss of an mCHH island is often accompanied by additional loss of DNA methylation in CG and CHG contexts associated with heterochromatin in nearby transposons. This suggests that mCHH islands and RNA-directed DNA methylation near maize genes may act to preserve the silencing of transposons from activity of nearby genes. PMID:26553984

  9. The Human Tim-Tipin Complex Interacts Directly with DNA Polymerase ϵ and Stimulates Its Synthetic Activity*

    PubMed Central

    Aria, Valentina; De Felice, Mariarita; Di Perna, Roberta; Uno, Shuji; Masai, Hisao; Syväoja, Juhani E.; van Loon, Barbara; Hübscher, Ulrich; Pisani, Francesca M.

    2013-01-01

    The Tim-Tipin complex plays an important role in the S phase checkpoint and replication fork stability in metazoans, but the molecular mechanism underlying its biological function is poorly understood. Here, we present evidence that the recombinant human Tim-Tipin complex (and Tim alone) markedly enhances the synthetic activity of DNA polymerase ϵ. In contrast, no significant effect on the synthetic ability of human DNA polymerase α and δ by Tim-Tipin was observed. Surface plasmon resonance measurements and co-immunoprecipitation experiments revealed that recombinant DNA polymerase ϵ directly interacts with either Tim or Tipin. In addition, the results of DNA band shift assays suggest that the Tim-Tipin complex (or Tim alone) is able to associate with DNA polymerase ϵ bound to a 40-/80-mer DNA ligand. Our results are discussed in view of the molecular dynamics at the human DNA replication fork. PMID:23511638

  10. The Pseudorabies Virus DNA Polymerase Accessory Subunit UL42 Directs Nuclear Transport of the Holoenzyme

    PubMed Central

    Wang, Yi-Ping; Du, Wen-Juan; Huang, Li-Ping; Wei, Yan-Wu; Wu, Hong-Li; Feng, Li; Liu, Chang-Ming

    2016-01-01

    Pseudorabies virus (PRV) DNA replication occurs in the nuclei of infected cells and requires the viral DNA polymerase. The PRV DNA polymerase comprises a catalytic subunit, UL30, and an accessory subunit, UL42, that confers processivity to the enzyme. Its nuclear localization is a prerequisite for its enzymatic function in the initiation of viral DNA replication. However, the mechanisms by which the PRV DNA polymerase holoenzyme enters the nucleus have not been determined. In this study, we characterized the nuclear import pathways of the PRV DNA polymerase catalytic and accessory subunits. Immunofluorescence analysis showed that UL42 localizes independently in the nucleus, whereas UL30 alone predominantly localizes in the cytoplasm. Intriguingly, the localization of UL30 was completely shifted to the nucleus when it was coexpressed with UL42, demonstrating that nuclear transport of UL30 occurs in an UL42-dependent manner. Deletion analysis and site-directed mutagenesis of the two proteins showed that UL42 contains a functional and transferable bipartite nuclear localization signal (NLS) at amino acids 354–370 and that K354, R355, and K367 are important for the NLS function, whereas UL30 has no NLS. Coimmunoprecipitation assays verified that UL42 interacts with importins α3 and α4 through its NLS. In vitro nuclear import assays demonstrated that nuclear accumulation of UL42 is a temperature- and energy-dependent process and requires both importins α and β, confirming that UL42 utilizes the importin α/β-mediated pathway for nuclear entry. In an UL42 NLS-null mutant, the UL42/UL30 heterodimer was completely confined to the cytoplasm when UL42 was coexpressed with UL30, indicating that UL30 utilizes the NLS function of UL42 for its translocation into the nucleus. Collectively, these findings suggest that UL42 contains an importin α/β-mediated bipartite NLS that transports the viral DNA polymerase holoenzyme into the nucleus in an in vitro expression system

  11. Direct real-time quantitative PCR for measurement of host-cell residual DNA in therapeutic proteins.

    PubMed

    Peper, Grit; Fankhauser, Alexander; Merlin, Thomas; Roscic, Ana; Hofmann, Matthias; Obrdlik, Petr

    2014-11-01

    Real-time quantitative PCR (qPCR) is important for quantification of residual host cell DNA (resDNA) in therapeutic protein preparations. Typical qPCR protocols involve DNA extraction steps complicating sample handling. Here, we describe a "direct qPCR" approach without DNA extraction. To avoid interferences of DNA polymerase with a therapeutic protein, proteins in the samples were digested with proteinase K (PK) in the presence of sodium dodecyl sulfate (SDS). Tween 20 and NaCl were included to minimize precipitation of therapeutic proteins in the PK/SDS mix. After PK treatment, the solution was applied directly for qPCR. Inhibition of DNA polymerase by SDS was prevented by adding 2% (v/v) of Tween 20 to the final qPCR mix. The direct qPCR approach was evaluated for quantification of resDNA in therapeutic proteins manufactured in Chinese hamster ovary (CHO) host cells. First, direct qPCR was compared with qPCR applied on purified DNA ("extraction qPCR"). For both qPCRs, the same CHO-specific primers and probes were used. Comparable residual DNA levels were detected with both PCR approaches in purified and highly concentrated drug proteins as well as in in-process-control samples. Finally, the CHO-specific direct qPCR protocol was validated according to ICH guidelines and applied for 25 different therapeutic proteins. The specific limits of quantification were 0.1-0.8ppb for 24 proteins, and 2.0ppb for one protein. General applicability of the direct qPCR was demonstrated by applying the sample preparation protocol for quantification of resDNA in therapeutic proteins manufactured in other hosts such as Escherichia coli and mouse cells. PMID:25151232

  12. Topoisomerase I-Mediated DNA Cleavage Induced by the Minor Groove-Directed Binding of Bibenzimidazoles to a Distal Site

    PubMed Central

    Khan, Qasim A.; Pilch, Daniel S.

    2007-01-01

    Summary Many agents (e.g., camptothecins, indolocarbazoles, indenoisoquinolines, and dibenzonaphthyridines) stimulate topoisomerase I-mediated DNA cleavage (a behavior termed topoisomerase I poisoning) by interacting with both the DNA and the enzyme at the site of cleavage (typically by intercalation between the −1 and +1 base pairs). The bibenzimidazoles, which include Hoechst 33258 and 33342, are a family of DNA minor groove-directed agents that also stimulate topoisomerase I-mediated DNA cleavage. However, the molecular mechanism by which these ligands poison TOP1 is poorly understood. Toward this goal, we have used a combination of mutational, footprinting, and DNA binding affinity analyses to define the DNA binding site for Hoechst 33258 and a related derivative that results in optimal induction of TOP1-mediated DNA cleavage. We show that this DNA binding site is located downstream from the site of DNA cleavage, encompassing the base pairs from position +4 to +8. The distal nature of this binding site relative to the site of DNA cleavage suggests that minor groove-directed agents like the bibenzimidazoles poison TOP1 via a mechanism distinct from compounds like the camptothecins, which interact at the site of cleavage. PMID:17095016

  13. Directed assembly of hierarchical light-harvesting complexes using virus capsid scaffolds and DNA origami tiles

    NASA Astrophysics Data System (ADS)

    Wang, Debin; Capehart, Stacy; Pal, Suchetan; Liu, Minghui; Lau, Jolene; Yan, Hao; Francis, Matthew; Deyoreo, Jim; LBNL Team; UCB Team; ASU Team

    2013-03-01

    Directed assembly of nanostructures with molecular precision is of great importance to develop an insightful understanding of assembly pathways and dynamics as well as to derive new functionalities. In this work, we explore the use of virus capsids and DNA origami tiles as 3D scaffolds and 2D templates for directed assembly of light-harvesting molecules and plasmonic gold nanoparticles to achieve tunable photoemission. Bacteriophage MS2 virus capsids with well-defined spherical macromolecular structures are genetically modified to provide predictable steric arrangements of light-harvesting molecules. DNA origami tiles act as programmable planar templates to provide higher-order organization of oligonucleotide-functionalized light-harvesting capsids and plasmonic gold nanoparticles. The direct observation of distance dependent photoluminescence emission is carried out by our correlative approach combining atomic force microscopy and confocal fluorescence microscopy, which is in good agreement with our numerical simulation and theoretical calculation. This work will facilitate the construction of multicomponent biological-metal hybrid plasmonic nanostructures for nanophotonics and biosensing applications.

  14. Directed self-assembly of block copolymer films on atomically-thin graphene chemical patterns

    PubMed Central

    Chang, Tzu-Hsuan; Xiong, Shisheng; Jacobberger, Robert M.; Mikael, Solomon; Suh, Hyo Seon; Liu, Chi-Chun; Geng, Dalong; Wang, Xudong; Arnold, Michael S.; Ma, Zhenqiang; Nealey, Paul F.

    2016-01-01

    Directed self-assembly of block copolymers is a scalable method to fabricate well-ordered patterns over the wafer scale with feature sizes below the resolution of conventional lithography. Typically, lithographically-defined prepatterns with varying chemical contrast are used to rationally guide the assembly of block copolymers. The directed self-assembly to obtain accurate registration and alignment is largely influenced by the assembly kinetics. Furthermore, a considerably broad processing window is favored for industrial manufacturing. Using an atomically-thin layer of graphene on germanium, after two simple processing steps, we create a novel chemical pattern to direct the assembly of polystyrene-block-poly(methyl methacrylate). Faster assembly kinetics are observed on graphene/germanium chemical patterns than on conventional chemical patterns based on polymer mats and brushes. This new chemical pattern allows for assembly on a wide range of guiding periods and along designed 90° bending structures. We also achieve density multiplication by a factor of 10, greatly enhancing the pattern resolution. The rapid assembly kinetics, minimal topography, and broad processing window demonstrate the advantages of inorganic chemical patterns composed of hard surfaces. PMID:27528258

  15. Directed self-assembly of block copolymer films on atomically-thin graphene chemical patterns.

    PubMed

    Chang, Tzu-Hsuan; Xiong, Shisheng; Jacobberger, Robert M; Mikael, Solomon; Suh, Hyo Seon; Liu, Chi-Chun; Geng, Dalong; Wang, Xudong; Arnold, Michael S; Ma, Zhenqiang; Nealey, Paul F

    2016-01-01

    Directed self-assembly of block copolymers is a scalable method to fabricate well-ordered patterns over the wafer scale with feature sizes below the resolution of conventional lithography. Typically, lithographically-defined prepatterns with varying chemical contrast are used to rationally guide the assembly of block copolymers. The directed self-assembly to obtain accurate registration and alignment is largely influenced by the assembly kinetics. Furthermore, a considerably broad processing window is favored for industrial manufacturing. Using an atomically-thin layer of graphene on germanium, after two simple processing steps, we create a novel chemical pattern to direct the assembly of polystyrene-block-poly(methyl methacrylate). Faster assembly kinetics are observed on graphene/germanium chemical patterns than on conventional chemical patterns based on polymer mats and brushes. This new chemical pattern allows for assembly on a wide range of guiding periods and along designed 90° bending structures. We also achieve density multiplication by a factor of 10, greatly enhancing the pattern resolution. The rapid assembly kinetics, minimal topography, and broad processing window demonstrate the advantages of inorganic chemical patterns composed of hard surfaces. PMID:27528258

  16. ARSENIC (III) METHYLATED SPECIES REACT WITH DNA DIRECTLY AND COULD BE PROXIMATED/ULTIMATE GENOTOXIC FORMS OF ARSENIC

    EPA Science Inventory


    ARSENIC(III) METHYLATED SPECIES REACT WITH DNA DIRECTL Y AND COULD BE PROXIMATE/ULTIMATE GENOTOXIC FORMS OF ARSENIC


    Arsenite and arsenate (iAs, inorganic arsenic) have been thought to act as genotoxicants without reacting directly with DNA; neither iAs nor As(V) m...

  17. The Influence of Primary and Secondary DNA Structure in Deletion and Duplication between Direct Repeats in Escherichia Coli

    PubMed Central

    Trinh, T. Q.; Sinden, R. R.

    1993-01-01

    We describe a system to measure the frequency of both deletions and duplications between direct repeats. Short 17- and 18-bp palindromic and nonpalindromic DNA sequences were cloned into the EcoRI site within the chloramphenicol acetyltransferase gene of plasmids pBR325 and pJT7. This creates an insert between direct repeated EcoRI sites and results in a chloramphenicol-sensitive phenotype. Selection for chloramphenicol resistance was utilized to select chloramphenicol resistant revertants that included those with precise deletion of the insert from plasmid pBR325 and duplication of the insert in plasmid pJT7. The frequency of deletion or duplication varied more than 500-fold depending on the sequence of the short sequence inserted into the EcoRI site. For the nonpalindromic inserts, multiple internal direct repeats and the length of the direct repeats appear to influence the frequency of deletion. Certain palindromic DNA sequences with the potential to form DNA hairpin structures that might stabilize the misalignment of direct repeats had a high frequency of deletion. Other DNA sequences with the potential to form structures that might destabilize misalignment of direct repeats had a very low frequency of deletion. Duplication mutations occurred at the highest frequency when the DNA between the direct repeats contained no direct or inverted repeats. The presence of inverted repeats dramatically reduced the frequency of duplications. The results support the slippage-misalignment model, suggesting that misalignment occurring during DNA replication leads to deletion and duplication mutations. The results also support the idea that the formation of DNA secondary structures during DNA replication can facilitate and direct specific mutagenic events. PMID:8325478

  18. Linking DNA adduct formation and human cancer risk in chemical carcinogenesis.

    PubMed

    Poirier, Miriam C

    2016-08-01

    Over two centuries ago, Sir Percival Pott, a London surgeon, published a pioneering treatise showing that soot exposure was the cause of high incidences of scrotal cancers occurring in young men who worked as chimney sweeps. Practicing at a time when cellular pathology was not yet recognized, Sir Percival nonetheless observed that the high incidence and short latency of the chimney sweep cancers, was fundamentally different from the rare scrotal cancers typically found in elderly men. Furthermore, his diagnosis that the etiology of these cancers was related to chimney soot exposure, was absolutely accurate, conceptually novel, and initiated the field of "occupational cancer epidemiology." After many intervening years of research focused on mechanisms of chemical carcinogenesis, briefly described here, it is clear that DNA damage, or DNA adduct formation, is "necessary but not sufficient" for tumor induction, and that many additional factors contribute to carcinogenesis. This review includes a synopsis of carcinogen-induced DNA adduct formation in experimental models and in the human population, with particular attention paid to molecular dosimetry and molecular cancer epidemiology. Environ. Mol. Mutagen. 57:499-507, 2016. © 2016 Wiley Periodicals, Inc. PMID:27346877

  19. Directional Regulation of Enzyme Pathways through the Control of Substrate Channeling on a DNA Origami Scaffold.

    PubMed

    Ke, Guoliang; Liu, Minghui; Jiang, Shuoxing; Qi, Xiaodong; Yang, Yuhe Renee; Wootten, Shaun; Zhang, Fei; Zhu, Zhi; Liu, Yan; Yang, Chaoyong James; Yan, Hao

    2016-06-20

    Artificial multi-enzyme systems with precise and dynamic control over the enzyme pathway activity are of great significance in bionanotechnology and synthetic biology. Herein, we exploit a spatially addressable DNA nanoplatform for the directional regulation of two enzyme pathways (G6pDH-MDH and G6pDH-LDH) through the control of NAD(+) substrate channeling by specifically shifting NAD(+) between the two enzyme pairs. We believe that this concept will be useful for the design of regulatory biological circuits for synthetic biology and biomedicine. PMID:27159899

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

    NASA Astrophysics Data System (ADS)

    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

  1. Polycationic ligands of different chemical classes stimulate DNA strand displacement between short oligonucleotides in a protein-free system.

    PubMed

    Volodin, Alexander A; Bocharova, Tatiana N; Smirnova, Elena A

    2016-09-01

    The ability of polycationic ligands to stimulate DNA strand displacement between short oligonucleotides in a protein-free system is demonstrated. We show that two ligands, tetracationic aliphatic amine (spermine) and a dicationic intercalating drug (chloroquine), promote strand displacement in a concentration-dependent manner. At low concentrations both ligands decelerate spontaneous strand displacement because of their impact on the stability of the DNA duplex. At elevated concentrations they accelerate strand displacement via formation of intermediate structures containing three DNA strands. The rate of the last process does not correlate with the thermal dissociation rate of the entire DNA duplex. It indicates that, possibly, the action of these agents cannot be explained by their influence on the stability of the DNA duplex. In general, our results suggest that the ability to stimulate DNA strand displacement appears to be a common feature of polycations of different chemical and structural classes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 633-641, 2016. PMID:27106951

  2. [Chemical and DNA analyses for the products of a psychoactive plant, Voacanga africana].

    PubMed

    Kikura-Hanajiri, Ruri; Maruyama, Takuro; Miyashita, Akinori; Goda, Yukihiro

    2009-08-01

    Voacanga africana (Apocynaceae) is a small tropical African tree. The root bark and seeds of this tree contain a number of alkaloids, including ibogaine (a hallucinogenic/aphrodisiac compound in bark), tabersonine (a major constituteent of seeds) and other voacanga alkaloids, traditionally used in Africa for religious purposes. Recently, some kinds of products containing this plant (root bark and seeds) have been distributed in the drug market in expectation of its hallucinogenic/aphrodisiac effects. There has been no report that has discussed quantitative analyses of these alkaloids in the products and their botanical origins. In this study, to investigate the trend of such a non-controlled psychotropic plant of abuse, a simultaneous analytical method was developed using LC/MS for the voacanga alkaloids including ibogaine and tabersonine in the commercial products of V. africana. Moreover, the botanical origins of these products were investigated by DNA analyses. As a result of the LC/MS analyses, the products were classified into two chemical types; an ibogaine-type and a tabersonine-type. The samples of the ibogaine-type contain ibogaine (0.05-0.6%) and other voacanga alkaloids; voacamine, voacamidine and voacangine, while those of the tabersonine-type mainly contain tabersonine (0.6-1.6%). The sequence analyses of chloroplast DNA, trnL-F region suggested that most of the products were derived from V. africana or closely related plants. They were classified into four genotypes based on nucleotide sequence of the trnL-F IGS region. The proposed methods of chemical and DNA analyses would be useful for investigating the trend in the distribution of the products of V. africana. PMID:19652504

  3. Effect of diffusive direction across the skin on the penetration profile of chemicals in vitro.

    PubMed

    Morofuji, Ryo; Hikima, Tomohiro; Tojo, Kakuji

    2013-01-01

    Skin has various types of transporters and is a biochemically active organ. These aspects of skin influence the distribution of chemicals in skin and their elimination from skin. The biochemical and histological variations of the skin must be taken into account when conducting transdermal penetration research. Here we used hairless mouse skin to investigate the percutaneous absorption of chemicals in vitro from the stratum corneum (SC) side to the viable skin (VS) side (forward direction) and from the VS side to the SC side (backward direction). We examined the effects of molecular weight, lipophilicity (Log Ko/w), electric charge, and the molecular structure of penetrants. The penetration flux of verapamil hydrochloride (VRP) for the backward direction was 3.2 times larger than that for the forward direction. The flux values of benzoic acid (BA) and para-hydroxybenzoic acid (pHBA) for the forward direction were 2.1 and 4.6 times larger than those for the backward direction, respectively. This directional difference was caused by the active transporter for VRP, the histological distribution of BA solubility, and the intermolecular hydrogen bonding between pHBA and skin tissue in the stripped skin. Across intact skin, in contrast, there was no difference in the skin penetration profile between the forward direction and backward directions. PMID:24189420

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

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2010-02-01

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

  6. Trace DNA from insect skins: a comparison of five extraction protocols and direct PCR on chironomid pupal exuviae.

    PubMed

    Kranzfelder, Petra; Ekrem, Torbjørn; Stur, Elisabeth

    2016-01-01

    Insect skins (exuviae) are of extracellular origin and shed during moulting. The skins do not contain cells or DNA themselves, but epithelial cells and other cell-based structures might accidentally attach as they are shed. This source of trace DNA can be sufficient for PCR amplification and sequencing of target genes and aid in species identification through DNA barcoding or association of unknown life stages. Species identification is essential for biomonitoring programs, as species vary in sensitivities to environmental factors. However, it requires a DNA isolation protocol that optimizes the output of target DNA. Here, we compare the relative effectiveness of five different DNA extraction protocols and direct PCR in isolation of DNA from chironomid pupal exuviae. Chironomidae (Diptera) is a species-rich group of aquatic macroinvertebrates widely distributed in freshwater environments and considered a valuable bioindicator of water quality. Genomic DNA was extracted from 61.2% of 570 sampled pupal exuviae. There were significant differences in the methods with regard to cost, handling time, DNA quantity, PCR success, sequence success and the ability to sequence target taxa. The NucleoSpin(®) Tissue XS Kit, DNeasy(®) Blood and Tissue kit, and QuickExtract(™) DNA Extraction Solution provided the best results in isolating DNA from single pupal exuviae. Direct PCR and DTAB/CTAB methods gave poor results. While the observed differences in DNA isolation methods on trace DNA will be relevant to research that focuses on aquatic macroinvertebrate ecology, taxonomy and systematics, they should also be of interest for studies using environmental barcoding and metabarcoding of aquatic environments. PMID:26186122

  7. Direct observation of a 91 bp LacI-mediated, negatively supercoiled DNA loop by atomic force microscope.

    PubMed

    Fulcrand, Geraldine; Chapagain, Prem; Dunlap, David; Leng, Fenfei

    2016-03-01

    Escherichia coli lactose repressor (LacI), a tetrameric protein, is a paradigmatic transcriptional factor that controls the expression of lacZYA in the lac operon. It specifically binds to the O1, O2, and O3 operators of the lac promoter, forms DNA loops, and regulates transcription of the lac operon. In this article, utilizing combined techniques of DNA-nicking assay and AFM imaging, we directly observed a 91 bp LacI-mediated, negatively supercoiled DNA loop mimicking the DNA loop between the O1 and O3 operators in the lac promoter. PMID:26878689

  8. Closed-Loop Control of Chemical Injection Rate for a Direct Nozzle Injection System

    PubMed Central

    Cai, Xiang; Walgenbach, Martin; Doerpmond, Malte; Schulze Lammers, Peter; Sun, Yurui

    2016-01-01

    To realize site-specific and variable-rate application of agricultural pesticides, accurately metering and controlling the chemical injection rate is necessary. This study presents a prototype of a direct nozzle injection system (DNIS) by which chemical concentration transport lag was greatly reduced. In this system, a rapid-reacting solenoid valve (RRV) was utilized for injecting chemicals, driven by a pulse-width modulation (PWM) signal at 100 Hz, so with varying pulse width the chemical injection rate could be adjusted. Meanwhile, a closed-loop control strategy, proportional-integral-derivative (PID) method, was applied for metering and stabilizing the chemical injection rate. In order to measure chemical flow rates and input them into the controller as a feedback in real-time, a thermodynamic flowmeter that was independent of chemical viscosity was used. Laboratory tests were conducted to assess the performance of DNIS and PID control strategy. Due to the nonlinear input–output characteristics of the RRV, a two-phase PID control process obtained better effects as compared with single PID control strategy. Test results also indicated that the set-point chemical flow rate could be achieved within less than 4 s, and the output stability was improved compared to the case without control strategy. PMID:26805833

  9. Closed-Loop Control of Chemical Injection Rate for a Direct Nozzle Injection System.

    PubMed

    Cai, Xiang; Walgenbach, Martin; Doerpmond, Malte; Schulze Lammers, Peter; Sun, Yurui

    2016-01-01

    To realize site-specific and variable-rate application of agricultural pesticides, accurately metering and controlling the chemical injection rate is necessary. This study presents a prototype of a direct nozzle injection system (DNIS) by which chemical concentration transport lag was greatly reduced. In this system, a rapid-reacting solenoid valve (RRV) was utilized for injecting chemicals, driven by a pulse-width modulation (PWM) signal at 100 Hz, so with varying pulse width the chemical injection rate could be adjusted. Meanwhile, a closed-loop control strategy, proportional-integral-derivative (PID) method, was applied for metering and stabilizing the chemical injection rate. In order to measure chemical flow rates and input them into the controller as a feedback in real-time, a thermodynamic flowmeter that was independent of chemical viscosity was used. Laboratory tests were conducted to assess the performance of DNIS and PID control strategy. Due to the nonlinear input-output characteristics of the RRV, a two-phase PID control process obtained better effects as compared with single PID control strategy. Test results also indicated that the set-point chemical flow rate could be achieved within less than 4 s, and the output stability was improved compared to the case without control strategy. PMID:26805833

  10. A high-throughput chemical screen with FDA approved drugs reveals that the antihypertensive drug Spironolactone impairs cancer cell survival by inhibiting homology directed repair

    PubMed Central

    Shahar, Or David; Kalousi, Alkmini; Eini, Lital; Fisher, Benoit; Weiss, Amelie; Darr, Jonatan; Mazina, Olga; Bramson, Shay; Kupiec, Martin; Eden, Amir; Meshorer, Eran; Mazin, Alexander V.; Brino, Laurent; Goldberg, Michal; Soutoglou, Evi

    2014-01-01

    DNA double-strand breaks (DSBs) are the most severe type of DNA damage. DSBs are repaired by non-homologous end-joining or homology directed repair (HDR). Identifying novel small molecules that affect HDR is of great importance both for research use and therapy. Molecules that elevate HDR may improve gene targeting whereas inhibiting molecules can be used for chemotherapy, since some of the cancers are more sensitive to repair impairment. Here, we performed a high-throughput chemical screen for FDA approved drugs, which affect HDR in cancer cells. We found that HDR frequencies are increased by retinoic acid and Idoxuridine and reduced by the antihypertensive drug Spironolactone. We further revealed that Spironolactone impairs Rad51 foci formation, sensitizes cancer cells to DNA damaging agents, to Poly (ADP-ribose) polymerase (PARP) inhibitors and cross-linking agents and inhibits tumor growth in xenografts, in mice. This study suggests Spironolactone as a new candidate for chemotherapy. PMID:24682826

  11. Direct electrochemistry of glucose oxidase and biosensing for glucose based on DNA/chitosan film.

    PubMed

    Gu, Tingting; Zhang, Yang; Deng, Fei; Zhang, Jing; Hasebe, Yasushi

    2011-06-01

    Glucose oxidase (GOD) is widely used in the glucose biosensor industry. The amperometric biosensors based on directly electron transfer (DET) between an electrode and immobilized GOD are especially promising. In this article, GOD was immobilized with a DNA/chitosan bio-material film on GC electrode, and the DET of GOD on DNA/chitosan was studied. The cyclic voltammetric results indicated that the GOD immobilized in the DNA/chitosan film underwent DET reaction, and the cyclic voltammogram displayed a pair of well-defined redox peaks with a formal potential of -0.45 V (vs. Ag/AgCl) at pH 5.5. The response showed a surface-controlled electrode process with an electron transfer rate constant of 0.91 sec(-1) determined in the scan rate range from 10 to 100 mV/sec. The GOD immobilized in DNA/chitosan membrane retained its biocatalytic activity and stability. The immobilized GOD could electrocatalyze the reduction of dissolved oxygen and resulted in a great increase of the reduction peak current. Upon the addition of glucose, the reduction peak current decreased, which could be used for glucose detection with a sensitivity of 0.48 μA/(mmol/L), a linear range from 0.04 to 2.28 mmol/L and a detection limit of 0.04 mmol/L at a signal-to-noise ratio of 3. The sensor could exclude the interference of commonly coexisted uricacid and ascorbic acid. PMID:25084597

  12. Direct Estimation of the Mitochondrial DNA Mutation Rate in Drosophila melanogaster

    PubMed Central

    Haag-Liautard, Cathy; Coffey, Nicole; Houle, David; Lynch, Michael; Charlesworth, Brian; Keightley, Peter D

    2008-01-01

    Mitochondrial DNA (mtDNA) variants are widely used in evolutionary genetics as markers for population history and to estimate divergence times among taxa. Inferences of species history are generally based on phylogenetic comparisons, which assume that molecular evolution is clock-like. Between-species comparisons have also been used to estimate the mutation rate, using sites that are thought to evolve neutrally. We directly estimated the mtDNA mutation rate by scanning the mitochondrial genome of Drosophila melanogaster lines that had undergone approximately 200 generations of spontaneous mutation accumulation (MA). We detected a total of 28 point mutations and eight insertion-deletion (indel) mutations, yielding an estimate for the single-nucleotide mutation rate of 6.2 × 10−8 per site per fly generation. Most mutations were heteroplasmic within a line, and their frequency distribution suggests that the effective number of mitochondrial genomes transmitted per female per generation is about 30. We observed repeated occurrences of some indel mutations, suggesting that indel mutational hotspots are common. Among the point mutations, there is a large excess of G→A mutations on the major strand (the sense strand for the majority of mitochondrial genes). These mutations tend to occur at nonsynonymous sites of protein-coding genes, and they are expected to be deleterious, so do not become fixed between species. The overall mtDNA mutation rate per base pair per fly generation in Drosophila is estimated to be about 10× higher than the nuclear mutation rate, but the mitochondrial major strand G→A mutation rate is about 70× higher than the nuclear rate. Silent sites are substantially more strongly biased towards A and T than nonsynonymous sites, consistent with the extreme mutation bias towards A+T. Strand-asymmetric mutation bias, coupled with selection to maintain specific nonsynonymous bases, therefore provides an explanation for the extreme base composition of

  13. Sensitive and direct electrochemical detection of double-stranded DNA utilizing alkaline phosphatase-labelled zinc finger proteins.

    PubMed

    Noh, Soodong; Ha, Dat Thinh; Yang, Haesik; Kim, Moon-Soo

    2015-06-21

    Direct detection of double-stranded DNA (dsDNA) using zinc finger proteins (ZFPs) is of great importance in biomedical applications such as identifying pathogens and circulating DNAs. However, its sensitivity is still not sufficiently high because limited signalling labels can be conjugated or fused. Herein, we report sensitive and direct detection of dsDNA using (i) alkaline phosphatase (ALP) as a fast catalytic label conjugated to ZFPs along with (ii) electrochemical measurement of an ALP product (l-ascorbic acid) at the indium-tin oxide electrode with a high signal-to-background ratio. ALP is simply conjugated to a ZFP through lysine residues in a ZFP purification tag, a maltose binding protein (MBP). Sandwich-type electrochemical detection of dsDNA allows a detection limit of ca. 100 fM without using DNA amplification. PMID:25969923

  14. [Rapid site-directed mutagenesis on full-length plasmid DNA by using designed restriction enzyme assisted mutagenesis].

    PubMed

    Zhang, Baozhong; Ran, Duoliang; Zhang, Xin; An, Xiaoping; Shan, Yunzhu; Zhou, Yusen; Tong, Yigang

    2009-02-01

    To use the designed restriction enzyme assisted mutagenesis technique to perform rapid site-directed mutagenesis on double-stranded plasmid DNA. The target amino acid sequence was reversely translated into DNA sequences with degenerate codons, resulting in large amount of silently mutated sequences containing various restriction endonucleases (REs). Certain mutated sequence with an appropriate RE was selected as the target DNA sequence for designing mutation primers. The full-length plasmid DNA was amplified with high-fidelity Phusion DNA polymerase and the amplified product was 5' phosphorylated by T4 polynucleotide kinase and then self-ligated. After transformation into an E. coli host the transformants were rapidly screened by cutting with the designed RE. With this strategy we successfully performed the site-directed mutagenesis on an 8 kb plasmid pcDNA3.1-pIgR and recovered the wild-type amino acid sequence of human polymeric immunoglobulin receptor (pIgR). A novel site-directed mutagenesis strategy based on DREAM was developed which exploited RE as a rapid screening measure. The highly efficient, high-fidelity Phusion DNA polymerase was applied to ensure the efficient and faithful amplification of the full-length sequence of a plasmid of up to 8 kb. This rapid mutagenesis strategy avoids using any commercial site-directed mutagenesis kits, special host strains or isotopes. PMID:19459340

  15. Solid-state NMR chemical-shift perturbations indicate domain reorientation of the DnaG primase in the primosome of Helicobacter pylori.

    PubMed

    Gardiennet, Carole; Wiegand, Thomas; Bazin, Alexandre; Cadalbert, Riccardo; Kunert, Britta; Lacabanne, Denis; Gutsche, Irina; Terradot, Laurent; Meier, Beat H; Böckmann, Anja

    2016-03-01

    We here investigate the interactions between the DnaB helicase and the C-terminal domain of the corresponding DnaG primase of Helicobacter pylori using solid-state NMR. The difficult crystallization of this 387 kDa complex, where the two proteins interact in a six to three ratio, is circumvented by simple co-sedimentation of the two proteins directly into the MAS-NMR rotor. While the amount of information that can be extracted from such a large protein is still limited, we can assign a number of amino-acid residues experiencing significant chemical-shift perturbations upon helicase-primase complex formation. The location of these residues is used as a guide to model the interaction interface between the two proteins in the complex. Chemical-shift perturbations also reveal changes at the interaction interfaces of the hexameric HpDnaB assembly on HpDnaG binding. A structural model of the complex that explains the experimental findings is obtained. PMID:26961129

  16. 2′,2′-Ligation Demonstrates the Thermal Dependence of DNA-Directed Positional Control

    PubMed Central

    Liu, Yu; Sha, Ruojie; Wang, Risheng; Ding, Liang; Canary, James W.; Seeman, Nadrian C.

    2008-01-01

    An oligodeoxynucleotide (ODN) containing a 2′-aminoalkylmercaptouridine was ligated chemically with another ODN containing a 2′-carboxyalkylmercaptouridine to produce a 2′,2′-amide linked conjugate. Templation by a DNA hairpin resulted in ligation yields of > 95%. At room temperature, similar yields were obtained for amide ligations between pendent nucleotides located adjacent to each other or separated by three nucleotides. However, at 4 °C, ligation was vastly favored for the case where the pendent amine and carboxylate were attached to adjacent residues (i.e., at the 3′- and 5′-termini of the two ODNs). No reaction was observed to occur either at 4 °C or at room temperature between pendent nucleotides separated by 9 nucleotides. Denaturing gel electrophoresis, MALDI-TOF MS and full nuclease digestion coupled with LC-MS were used to characterize the reaction products. PMID:19122844

  17. Ultrastable-Stealth Large Gold Nanoparticles with DNA Directed Biological Functionality.

    PubMed

    Heo, Jun Hyuk; Kim, Kyung-Il; Cho, Hui Hun; Lee, Jin Woong; Lee, Byoung Sang; Yoon, Seokyoung; Park, Kyung Jin; Lee, Seungwoo; Kim, Jaeyun; Whang, Dongmok; Lee, Jung Heon

    2015-12-29

    The stability of gold nanoparticles (AuNPs) in biological samples is very important for their biomedical applications. Although various molecules such as polystyrenesulfonate (PSS), phosphine, DNA, and polyethylene glycol (PEG) have been used to stabilize AuNPs, it is still very difficult to stabilize large AuNPs. As a result, biomedical applications of large (30-100 nm) AuNPs are limited, even though they possess more favorable optical properties and are easier to be taken up by cells than smaller AuNPs. To overcome this limitation, we herein report a novel method of preparing large (30-100 nm) AuNPs with a high colloidal stability and facile chemical or biological functionality, via surface passivation with an amphiphilic polymer polyvinylpyrrolidone (PVP). This PVP passivation results in an extraordinary colloidal stability for 13, 30, 50, 70, and 100 nm AuNPs to be stabilized in PBS for at least 3 months. More importantly, the PVP capped AuNPs (AuNP-PVP) were also resistant to protein adsorption in the presence of serum containing media and exhibit a negligible cytotoxicity. The AuNP-PVPs functionalized with a DNA aptamer AS1411 remain biologically active, resulting in significant increase in the uptake of the AuNPs (∼12,200 AuNPs per cell) in comparison with AuNPs capped by a control DNA of the same length. The novel method developed in this study to stabilize large AuNPs with high colloidal stability and biological activity will allow much wider applications of these large AuNPs for biomedical applications, such as cellular imaging, molecular diagnosis, and targeted therapy. PMID:26638691

  18. Evaluating legacy contaminants and emerging chemicals in marine environments using adverse outcome pathways and biological effects-directed analysis.

    PubMed

    Hutchinson, Thomas H; Lyons, Brett P; Thain, John E; Law, Robin J

    2013-09-30

    Natural and synthetic chemicals are essential to our daily lives, food supplies, health care, industries and safe sanitation. At the same time protecting marine ecosystems and seafood resources from the adverse effects of chemical contaminants remains an important issue. Since the 1970s, monitoring of persistent, bioaccumulative and toxic (PBT) chemicals using analytical chemistry has provided important spatial and temporal trend data in three important contexts; relating to human health protection from seafood contamination, addressing threats to marine top predators and finally providing essential evidence to better protect the biodiversity of commercial and non-commercial marine species. A number of regional conventions have led to controls on certain PBT chemicals over several years (termed 'legacy contaminants'; e.g. cadmium, lindane, polycyclic aromatic hydrocarbons [PAHs] and polychlorinated biphenyls [PCBs]). Analytical chemistry plays a key role in evaluating to what extent such regulatory steps have been effective in leading to reduced emissions of these legacy contaminants into marine environments. In parallel, the application of biomarkers (e.g. DNA adducts, CYP1A-EROD, vitellogenin) and bioassays integrated with analytical chemistry has strengthened the evidence base to support an ecosystem approach to manage marine pollution problems. In recent years, however,the increased sensitivity of analytical chemistry, toxicity alerts and wider environmental awareness has led to a focus on emerging chemical contaminants (defined as chemicals that have been detected in the environment, but which are currently not included in regulatory monitoring programmes and whose fate and biological impacts are poorly understood). It is also known that natural chemicals (e.g. algal biotoxins) may also pose a threat to marine species and seafood quality. Hence complex mixtures of legacy contaminants, emerging chemicals and natural biotoxins in marine ecosystems represent

  19. Analysis of weblike network structures of directed graphs for chemical reactions in methane plasmas

    NASA Astrophysics Data System (ADS)

    Sakai, Osamu; Nobuto, Kyosuke; Miyagi, Shigeyuki; Tachibana, Kunihide

    2015-10-01

    Chemical reactions of molecular gases like methane are so complicated that a chart of decomposed and/or synthesized species originating from molecules in plasma resembles a weblike network in which we write down species and reactions among them. Here we consider properties of the network structures of chemical reactions in methane plasmas. In the network, atoms/molecules/radical species are assumed to form nodes and chemical reactions correspond to directed edges in the terminology of graph theory. Investigation of the centrality index reveals importance of CH3 in the global chemical reaction, and difference of an index for each radical species between cases with and without electrons clarifies that the electrons are at an influential position to tighten the network structure.

  20. Analysis of weblike network structures of directed graphs for chemical reactions in methane plasmas

    SciTech Connect

    Sakai, Osamu Nobuto, Kyosuke; Miyagi, Shigeyuki; Tachibana, Kunihide

    2015-10-15

    Chemical reactions of molecular gases like methane are so complicated that a chart of decomposed and/or synthesized species originating from molecules in plasma resembles a weblike network in which we write down species and reactions among them. Here we consider properties of the network structures of chemical reactions in methane plasmas. In the network, atoms/molecules/radical species are assumed to form nodes and chemical reactions correspond to directed edges in the terminology of graph theory. Investigation of the centrality index reveals importance of CH{sub 3} in the global chemical reaction, and difference of an index for each radical species between cases with and without electrons clarifies that the electrons are at an influential position to tighten the network structure.

  1. Evaluation of microbial community in hydrothermal field by direct DNA sequencing

    NASA Astrophysics Data System (ADS)

    Kawarabayasi, Y.; Maruyama, A.

    2002-12-01

    Many extremophiles have been discovered from terrestrial and marine hydrothermal fields. Some thermophiles can grow beyond 90°C in culture, while direct microscopic analysis occasionally indicates that microbes may survive in much hotter hydrothermal fluids. However, it is very difficult to isolate and cultivate such microbes from the environments, i.e., over 99% of total microbes remains undiscovered. Based on experiences of entire microbial genome analysis (Y.K.) and microbial community analysis (A.M.), we started to find out unique microbes/genes in hydrothermal fields through direct sequencing of environmental DNA fragments. At first, shotgun plasmid libraries were directly constructed with the DNA molecules prepared from mixed microbes collected by an in situ filtration system from low-temperature fluids at RM24 in the Southern East Pacific Rise (S-EPR). A gene amplification (PCR) technique was not used for preventing mutation in the process. The nucleotide sequences of 285 clones indicated that no sequence had identical data in public databases. Among 27 clones determined entire sequences, no ORF was identified on 14 clones like intron in Eukaryote. On four clones, tetra-nucleotide-long multiple tandem repetitive sequences were identified. This type of sequence was identified in some familiar disease in human. The result indicates that living/dead materials with eukaryotic features may exist in this low temperature field. Secondly, shotgun plasmid libraries were constructed from the environmental DNA prepared from Beppu hot springs. In randomly-selected 143 clones used for sequencing, no known sequence was identified. Unlike the clones in S-EPR library, clear ORFs were identified on all nine clones determined the entire sequence. It was found that one clone, H4052, contained the complete Aspartyl-tRNA synthetase. Phylogenetic analysis using amino acid sequences of this gene indicated that this gene was separated from other Euryarchaea before the

  2. Development of an electrochemical surface-enhanced Raman spectroscopy (EC-SERS) aptasensor for direct detection of DNA hybridization.

    PubMed

    Karaballi, R A; Nel, A; Krishnan, S; Blackburn, J; Brosseau, C L

    2015-09-01

    Rapid detection of disease biomarkers at the patient point-of-care is essential to timely and effective treatment. The research described herein focuses on the development of an electrochemical surface-enhanced Raman spectroscopy (EC-SERS) DNA aptasensor capable of direct detection of tuberculosis (TB) DNA. Specifically, a plausible DNA biomarker present in TB patient urine was chosen as the model target for detection. Cost-effective screen printed electrodes (SPEs) modified with silver nanoparticles (AgNP) were used as the aptasensor platform, onto which the aptamer specific for the target DNA was immobilized. Direct detection of the target DNA was demonstrated through the appearance of SERS peaks characteristic for adenine, present only in the target strand. Modulation of the applied potential allowed for a sizeable increase in the observed SERS response and the use of thiol back-filling prevented non-specific adsorption of non-target DNA. To our knowledge, this work represents the first EC-SERS study of an aptasensor for the direct, label-free detection of DNA hybridization. Such a technology paves the way for rapid detection of disease biomarkers at the patient point-of-care. PMID:25780805

  3. One-pot DNA construction for synthetic biology: the Modular Overlap-Directed Assembly with Linkers (MODAL) strategy.

    PubMed

    Casini, Arturo; MacDonald, James T; De Jonghe, Joachim; Christodoulou, Georgia; Freemont, Paul S; Baldwin, Geoff S; Ellis, Tom

    2014-01-01

    Overlap-directed DNA assembly methods allow multiple DNA parts to be assembled together in one reaction. These methods, which rely on sequence homology between the ends of DNA parts, have become widely adopted in synthetic biology, despite being incompatible with a key principle of engineering: modularity. To answer this, we present MODAL: a Modular Overlap-Directed Assembly with Linkers strategy that brings modularity to overlap-directed methods, allowing assembly of an initial set of DNA parts into a variety of arrangements in one-pot reactions. MODAL is accompanied by a custom software tool that designs overlap linkers to guide assembly, allowing parts to be assembled in any specified order and orientation. The in silico design of synthetic orthogonal overlapping junctions allows for much greater efficiency in DNA assembly for a variety of different methods compared with using non-designed sequence. In tests with three different assembly technologies, the MODAL strategy gives assembly of both yeast and bacterial plasmids, composed of up to five DNA parts in the kilobase range with efficiencies of between 75 and 100%. It also seamlessly allows mutagenesis to be performed on any specified DNA parts during the process, allowing the one-step creation of construct libraries valuable for synthetic biology applications. PMID:24153110

  4. Peptide-Metal Organic Framework Swimmers that Direct the Motion toward Chemical Targets.

    PubMed

    Ikezoe, Yasuhiro; Fang, Justin; Wasik, Tomasz L; Shi, Menglu; Uemura, Takashi; Kitagawa, Susumu; Matsui, Hiroshi

    2015-06-10

    Highly efficient and robust chemical motors are expected for the application in microbots that can selectively swim toward targets and accomplish their tasks in sensing, labeling, and delivering. However, one of major issues for such development is that current artificial swimmers have difficulty controlling their directional motion toward targets like bacterial chemotaxis. To program synthetic motors with sensing capability for the target-directed motion, we need to develop swimmers whose motions are sensitive to chemical gradients in environments. Here we create a new intelligent biochemical swimmer by integrating metal organic frameworks (MOFs) and peptides that can sense toxic heavy metals in solution and swim toward the targets. With the aid of Pb-binding enzymes, the peptide-MOF motor can directionally swim toward PbSe quantum dots (QD) by sensing pH gradient and eventually complete the motion as the swimmer reaches the highest gradient point at the target position in solution. This type of technology could be evolved to miniaturize chemical robotic systems that sense target chemicals and swim toward target locations. PMID:26010172

  5. DNA aptamers for selective identification and separation of flame retardant chemicals.

    PubMed

    Kim, Un-Jung; Kim, Byoung Chan

    2016-09-14

    Polybrominated diphenyl ethers (PBDEs) are group of chemicals which are representative persistent organic pollutants (POPs) and used as brominated flame retardants for many consumer products. PBDEs were phased out since 2009 but are still frequently observed in various environmental matrices and human body. Here, we report ssDNA aptamers which bind to BDE47, one of the PBDE congeners commonly found in various environmental matrices, and show affinity to other major tri-to hepta- BDE congeners. The PBDE specific aptamers were isolated from random library of ssDNA using Mag-SELEX. Two out of 15 sequences, based on their alignment and hairpin loop structures, were chosen to determine dissociation constant with BDE47 and showed from picomolar to nanomolar affinities (200 pM and 1.53 nM). The aptamers displayed high selectivity to the original target, BDE47, and implying general specificity to PBDE backbone with varying affinities to other congeners. Further, we showed that the use of two aptamers together could enhance the separation efficiency of BDE47 and other BDE congeners when dissolved in a solvent compared to use of single aptamer. These aptamers are expected to provide a tool for preliminary screening or quick separation of PBDEs in environmental samples prior to trace quantitative analysis. PMID:27566357

  6. Chemical Elemental Distribution and Soil DNA Fingerprints Provide the Critical Evidence in Murder Case Investigation

    PubMed Central

    Concheri, Giuseppe; Bertoldi, Daniela; Polone, Elisa; Otto, Stefan; Larcher, Roberto; Squartini, Andrea

    2011-01-01

    Background The scientific contribution to the solution of crime cases, or throughout the consequent forensic trials, is a crucial aspect of the justice system. The possibility to extract meaningful information from trace amounts of samples, and to match and validate evidences with robust and unambiguous statistical tests, are the key points of such process. The present report is the authorized disclosure of an investigation, carried out by Attorney General appointment, on a murder case in northern Italy, which yielded the critical supporting evidence for the judicial trial. Methodology/Principal Findings The proportional distribution of 54 chemical elements and the bacterial community DNA fingerprints were used as signature markers to prove the similarity of two soil samples. The first soil was collected on the crime scene, along a corn field, while the second was found in trace amounts on the carpet of a car impounded from the main suspect in a distant location. The matching similarity of the two soils was proven by crossing the results of two independent techniques: a) elemental analysis via inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectrometry (ICP-OES) approaches, and b) amplified ribosomal DNA restriction analysis by gel electrophoresis (ARDRA). Conclusions Besides introducing the novel application of these methods to forensic disciplines, the highly accurate level of resolution observed, opens new possibilities also in the fields of soil typing and tracking, historical analyses, geochemical surveys and global land mapping. PMID:21674041

  7. Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines

    PubMed Central

    Yamamoto, Kimiyo N.; Hirota, Kouji; Kono, Koichi; Takeda, Shunichi; Sakamuru, Srilatha; Xia, Menghang; Huang, Ruili; Austin, Christopher P.; Witt, Kristine L.; Tice, Raymond R.

    2012-01-01

    Included among the quantitative high throughput screens (qHTS) conducted in support of the U.S. Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in 7 isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis. PMID:21538559

  8. Chemical and structural characterization of interstrand cross-links formed between abasic sites and adenine residues in duplex DNA

    PubMed Central

    Price, Nathan E.; Catalano, Michael J.; Liu, Shuo; Wang, Yinsheng; Gates, Kent S.

    2015-01-01

    A new type of interstrand DNA–DNA cross-link between abasic (Ap) sites and 2′-deoxyadenosine (dA) residues was recently reported, but the chemical structure and properties of this lesion were not rigorously established. Here we characterized the nucleoside cross-link remnant released by enzymatic digestion of duplex DNA containing the dA-Ap cross-link. A synthetic standard was prepared for the putative nucleoside cross-link remnant 6 in which the anomeric carbon of the 2-deoxyribose residue was connected to the exocyclic N6-amino group of dA. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the synthetic material 6 matched the authentic cross-link remnant released by enzymatic digestion of cross-linked DNA. These findings establish the chemical structure of the dA-Ap cross-link released from duplex DNA and may provide methods for the detection of this lesion in cellular DNA. Both the nucleoside cross-link remnant 6 and the cross-link in duplex DNA were quite stable at pH 7 and 37°C, suggesting that the dA-Ap cross-link could be a persistent lesion with the potential to block the action of various DNA processing enzymes. PMID:25779045

  9. Fiber optic chemical sensors: The evolution of high- density fiber-optic DNA microarrays

    NASA Astrophysics Data System (ADS)

    Ferguson, Jane A.

    2001-06-01

    Sensors were developed for multianalyte monitoring, fermentation monitoring, lactate analysis, remote oxygen detection for use in bioremediation monitoring and in a fuel spill clean-up project, heavy metal analysis, and high density DNA microarrays. The major focus of this thesis involved creating and improving high-density DNA gene arrays. Fiber optic sensors are created using fluorescent indicators, polymeric supports, and optical fiber substrates. The fluorescent indicator is entrapped in a polymer layer and attached to the tip of the optical fiber. The tip of the fiber bearing the sensing layer (the distal end) is placed in the sample of interest while the other end of the fiber (the proximal end) is connected to an analysis system. Any length of fiber can be used without compromising the integrity or sensitivity of the system. A fiber optic oxygen sensor was designed incorporating an oxygen sensitive fluorescent dye and a gas permeable polymer attached to an optical fiber. The construction simplicity and ruggedness of the sensor enabled its deployment for in situ chemical oxidation and bioremediation studies. Optical fibers were also used as the substrate to detect biomolecules in solution. To monitor bioprocesses, the production of the analyte of interest must be coupled with a species that is optically measurable. For example, oxygen is consumed in many metabolic functions. The fiber optic oxygen sensor is equipped with an additional sensing layer. Upon contact with a specific biochemical in the sample, a reaction occurs in the additional sensing layer that either consumes or produces oxygen. This dual layer system was used to monitor the presence of lactate, an important metabolite for clinical and bioprocess analysis. In many biological and environmental systems, the generation of one species occurs coincidentally with the generation or consumption of another species. A multianalyte sensor was prepared that can monitor the simultaneous activity of pH, CO2

  10. Are mutagenic non D-loop direct repeat motifs in mitochondrial DNA under a negative selection pressure?

    PubMed Central

    Lakshmanan, Lakshmi Narayanan; Gruber, Jan; Halliwell, Barry; Gunawan, Rudiyanto

    2015-01-01

    Non D-loop direct repeats (DRs) in mitochondrial DNA (mtDNA) have been commonly implicated in the mutagenesis of mtDNA deletions associated with neuromuscular disease and ageing. Further, these DRs have been hypothesized to put a constraint on the lifespan of mammals and are under a negative selection pressure. Using a compendium of 294 mammalian mtDNA, we re-examined the relationship between species lifespan and the mutagenicity of such DRs. Contradicting the prevailing hypotheses, we found no significant evidence that long-lived mammals possess fewer mutagenic DRs than short-lived mammals. By comparing DR counts in human mtDNA with those in selectively randomized sequences, we also showed that the number of DRs in human mtDNA is primarily determined by global mtDNA properties, such as the bias in synonymous codon usage (SCU) and nucleotide composition. We found that SCU bias in mtDNA positively correlates with DR counts, where repeated usage of a subset of codons leads to more frequent DR occurrences. While bias in SCU and nucleotide composition has been attributed to nucleotide mutational bias, mammalian mtDNA still exhibit higher SCU bias and DR counts than expected from such mutational bias, suggesting a lack of negative selection against non D-loop DRs. PMID:25855815

  11. Direct Observation of the Hole Carriers in DNA Photoinduced Charge Transport.

    PubMed

    Harris, Michelle A; Mishra, Ashutosh Kumar; Young, Ryan M; Brown, Kristen E; Wasielewski, Michael R; Lewis, Frederick D

    2016-05-01

    The excited state behavior of DNA hairpins possessing a diphenylacetylenedicarboxamide (DPA) linker separated from a single guanine-cytosine (G-C) base pair by zero-to-six adenine-thymine (A-T) base pairs has been investigated. In the case of hairpins with zero or one A-T separating DPA and G, formation of both DPA anion radical (DPA(-•)) and G cation radical (G(+•)) are directly observed and characterized by their transient absorption and stimulated Raman spectra. For hairpins with two or more intervening A-T, the transient absorption spectra of DPA(-•) and the adenine polaron (An(+•)) are observed. In addition to characterization of the hole carriers, the dynamics of each step in the charge separation and charge recombination process as well as the overall efficiency of charge separation have been determined, thus providing a complete account of the mechanism and dynamics of photoinduced charge transport in these DNA hairpins. PMID:27082662

  12. Direct Simulation of the Self-Assembly of a Small DNA Origami.

    PubMed

    Snodin, Benedict E K; Romano, Flavio; Rovigatti, Lorenzo; Ouldridge, Thomas E; Louis, Ard A; Doye, Jonathan P K

    2016-02-23

    By using oxDNA, a coarse-grained nucleotide-level model of DNA, we are able to directly simulate the self-assembly of a small 384-base-pair origami from single-stranded scaffold and staple strands in solution. In general, we see attachment of new staple strands occurring in parallel, but with cooperativity evident for the binding of the second domain of a staple if the adjacent junction is already partially formed. For a system with exactly one copy of each staple strand, we observe a complete assembly pathway in an intermediate temperature window; at low temperatures successful assembly is prevented by misbonding while at higher temperature the free-energy barriers to assembly become too large for assembly on our simulation time scales. For high-concentration systems involving a large staple strand excess, we never see complete assembly because there are invariably instances where two copies of the same staple both bind to the scaffold, creating a kinetic trap that prevents the complete binding of either staple. This mutual staple blocking could also lead to aggregates of partially formed origamis in real systems, and helps to rationalize certain successful origami design strategies. PMID:26766072

  13. An unusual case of Streptococcus anginosus group pyomyositis diagnosed using direct 16S ribosomal DNA sequencing

    PubMed Central

    Walkty, Andrew; Embil, John M; Nichol, Kim; Karlowsky, James

    2014-01-01

    Bacteria belonging to the Streptococcus anginosus group (Streptococcus intermedius, Streptococcus constellatus and Streptococcus anginosus) are capable of causing serious pyogenic infections, with a tendency for abscess formation. The present article reports a case of S anginosus group pyomyositis in a 47-year-old man. The pathogen was recovered from one of two blood cultures obtained from the patient, but speciation was initially not performed because the organism was considered to be a contaminant (viridans streptococci group). The diagnosis was ultimately confirmed using 16S ribosomal DNA sequencing of purulent fluid obtained from a muscle abscess aspirate. The present case serves to emphasize that finding even a single positive blood culture of an organism belonging to the S anginosus group should prompt careful evaluation of the patient for a pyogenic focus of infection. It also highlights the potential utility of 16S ribosomal DNA amplification and sequencing in direct pathogen detection from aspirated fluid in cases of pyomyositis in which antimicrobial therapy was initiated before specimen collection. PMID:24634686

  14. Isolation of an RNA-directed RNA polymerase-specific cDNA clone from tomato.

    PubMed Central

    Schiebel, W; Pélissier, T; Riedel, L; Thalmeir, S; Schiebel, R; Kempe, D; Lottspeich, F; Sänger, H L; Wassenegger, M

    1998-01-01

    A 3600-bp RNA-directed RNA polymerase (RdRP)-specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato. The putative protein encoded by this ORF does not share homology with any characterized proteins. Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP protein. The immunoresponse to the antibodies correlated with the enzymatic activity profile of the RdRP after chromatography on Q-, poly(A)-, and poly(U)-Sepharose, hydroxyapatite, and Sephadex G-200 columns. DNA gel blot analysis revealed a single copy of the RdRP gene in tomato. RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were identified by using polymerase chain reaction. A sequence comparison indicated that sequences homologous to RdRP are also present in the yeast Schizosaccharomyces pombe and in the nematode Caenorhabditis elegans. The previously described induction of RdRP activity upon viroid infection is shown to be correlated with an increased steady state level of the corresponding mRNA. The possible involvement of this heretofore functionally elusive plant RNA polymerase in homology-dependent gene silencing is discussed. PMID:9836747

  15. 53BP1 fosters fidelity of homology-directed DNA repair.

    PubMed

    Ochs, Fena; Somyajit, Kumar; Altmeyer, Matthias; Rask, Maj-Britt; Lukas, Jiri; Lukas, Claudia

    2016-08-01

    Repair of DNA double-strand breaks (DSBs) in mammals is coordinated by the ubiquitin-dependent accumulation of 53BP1 at DSB-flanking chromatin. Owing to its ability to limit DNA-end processing, 53BP1 is thought to promote nonhomologous end-joining (NHEJ) and to suppress homology-directed repair (HDR). Here, we show that silencing 53BP1 or exhausting its capacity to bind damaged chromatin changes limited DSB resection to hyper-resection and results in a switch from error-free gene conversion by RAD51 to mutagenic single-strand annealing by RAD52. Thus, rather than suppressing HDR, 53BP1 fosters its fidelity. These findings illuminate causes and consequences of synthetic viability acquired through 53BP1 silencing in cells lacking the BRCA1 tumor suppressor. We show that such cells survive DSB assaults at the cost of increasing reliance on RAD52-mediated HDR, which may fuel genome instability. However, our findings suggest that when challenged by DSBs, BRCA1- and 53BP1-deficient cells may become hypersensitive to, and be eliminated by, RAD52 inhibition. PMID:27348077

  16. DNA: directional neighborhood analysis for detection of breast masses in screening mammograms

    NASA Astrophysics Data System (ADS)

    Eltonsy, Nevine H.; Tourassi, Georgia D.; Habas, Piotr A.; Elmaghraby, Adel S.

    2005-04-01

    We introduce a computer-assisted detection (CAD) system for the automated detection of breast masses in screening mammograms. The system targets the directional behavior of the neighborhood pixels surrounding a reference image pixel. The underlying hypothesis is that in the presence of a mass the directional properties of the breast tissue surrounding the mass should be altered. The hypothesis was tested using a database of 1,337 mammographic regions of interest (ROIs) extracted from DDSM mammograms. There were 681 ROIs containing a biopsy-proven mass centered in the ROI (340 malignant, 341 benign) and 656 ROIs depicting normal breast parenchyma. Initially, eight main directional propagations were identified and modeled given the center of the ROI as the reference pixel. Subsequently, eight novel morphological features were extracted for each direction. The features were designed to characterize the disturbance occurring in normal breast parenchyma due to the presence of a mass. Finally, the extracted features were merged using a back propagation neural network (BPANN). The network served as a non linear classifier trained to determine the presence of a mass centered at the reference image pixel. The BPANN was trained and tested using a leave-one-out sampling scheme. Its performance was evaluated with Receiver Operating Characteristics (ROC) analysis. Our CAD system showed an ROC area index of Az=0.88+/-0.01 for discriminating mass vs. normal ROIs. Detection performance was robust for both malignant (Az=0.88+/-0.01) and benign masses (Az=0.87+/-0.01). Thus, the proposed directional neighborhood analysis (DNA) can be applied effectively to identify suspicious masses in screening mammograms.

  17. Automated and integrated system for high-throughput DNA genotyping directly from blood.

    PubMed

    Zhang, N; Tan, H; Yeung, E S

    1999-03-15

    An automated and integrated system for DNA typing directly from blood samples has been developed. The multiplexed eight-array system is based on capillary microfluidics and capillary array electrophoresis. Three short-tandem-repeat loci, vWA, THO1, and TPOX, are coamplified simultaneously in a fused-silica capillary by a hot-air thermocycler. Blood is directly used as the sample for polymerase chain reaction (PCR) without any pretreatment. Modifications of standard protocols are necessary for direct PCR from blood. A programmable syringe pump plus a set of multiplexed liquid nitrogen freeze/thaw switching valves are employed for liquid handling in the fluid distribution network. The system fully integrates sample loading, PCR, addition of an absolute standard, on-line injection of sample and standards, separation and detection. The genotypes from blood samples can be clearly identified in eight parallel channels when the electropherograms are compared with that of the standard allelic ladder by itself. Regeneration and cleaning of the entire system prior to subsequent runs are also integrated into the instrument. The instrumentation is compatible with future expansion to hundreds of capillaries to achieve even higher throughput. PMID:10093495

  18. Directly incorporating fluorochromes into DNA probes by PCR increases the efficience of fluorescence in situ hybridization

    SciTech Connect

    Dittmer, Joy

    1996-05-01

    The object of this study was to produce a directly labeled whole chromosome probe in a Degenerative Oligonucleotide Primed-Polymerase Chain Reaction (DOP-PCR) that will identify chromosome breaks, deletions, inversions and translocations caused by radiation damage. In this study we amplified flow sorted chromosome 19 using DOP-PCR. The product was then subjected to a secondary DOP PCR amplification, After the secondary amplification the DOP-PCR product was directly labeled in a tertiary PCR reaction with rhodamine conjugated with dUTP (FluoroRed) to produce a DNA fluorescent probe. The probe was then hybridized to human metaphase lymphocytes on slides, washed and counterstained with 4{prime},6-diamino-2-phenylindole (DAPI). The signal of the FluoroRed probe was then compared to a signal of a probe labeled with biotin and stained with avidin fluorescein isothio cynate (FITC) and anti-avidin FITC. The results show that the probe labeled with FluoroRed gave signals as bright as the probe with biotin labeling. The FluoroRed probe had less noise than the biotin labeled probe. Therefore, a directly labeled probe has been successfully produced in a DOP-PCR reaction. In future a probe labeled with FluoroRed will be produced instead of a probe labeled with biotin to increase efficiency.

  19. Stabilisation of self-assembled DNA crystals by triplex-directed photo-cross-linking.

    PubMed

    Abdallah, Hatem O; Ohayon, Yoel P; Chandrasekaran, Arun Richard; Sha, Ruojie; Fox, Keith R; Brown, Tom; Rusling, David A; Mao, Chengde; Seeman, Nadrian C

    2016-06-28

    The tensegrity triangle is a robust DNA motif that can self-assemble to generate macroscopic three-dimensional crystals. However, the stability of these crystals is dependent on the high ionic conditions used for crystal growth. Here we demonstrate that a triplex-forming oligonucleotide can be used to direct the specific intercalation, and subsequent photo-cross-linking, of 4,5',8-trimethylpsoralen to single or multiple loci within or between the tiles of the crystal. Cross-linking between the tiles of the crystal improves their thermal stability. Such an approach is likely to facilitate the removal of crystals from their mother liquor and may prove useful for applications that require greater crystal stability. PMID:27265774

  20. Ultraviolet light photobiology of the protozoan Tetrahymena pyriformis and chemical reactivation of DNA damage

    SciTech Connect

    Wheeler, J.S.

    1988-01-01

    The tunable dye laser was developed in order to perform UV-B and UV-C (254-320 nm) action spectra studies on several different organisms. Using the laser, action spectra studies have been performed for Escherichia coli, Saccharomyces, Chlamydomonas, Caenorhabditis elegans, Paramecium, and Tetrahymena pyriformis. Studies generally indicate increasing LD{sub 50} values with increasing wavelength. Two notable findings were made: (1) The action spectra does not follow the DNA absorption spectra at 280, 290 and 295 nm; (2) The repair competent/repair defective sensitization factor does not remain constant throughout the wavelength region. In addition it was found that the repair defective strain of E. coli, Bs-1, showed an increase in survival with increasing UV irradiation, at certain dose levels. Further experiments were designed to better characterize the reactivation. Tetrahymena were exposed to UV-C and reactivated with methyl methanesulfonate (MMS) and 4-nitro quinoline oxide (4-NQO). In both cases survival was seen to increase after chemical exposure. Likewise, UV-C was found to reactivate chemical damage (MMS).

  1. Methylation interactions in Arabidopsis hybrids require RNA-directed DNA methylation and are influenced by genetic variation.

    PubMed

    Zhang, Qingzhu; Wang, Dong; Lang, Zhaobo; He, Li; Yang, Lan; Zeng, Liang; Li, Yanqiang; Zhao, Cheng; Huang, Huan; Zhang, Heng; Zhang, Huiming; Zhu, Jian-Kang

    2016-07-19

    DNA methylation is a conserved epigenetic mark in plants and many animals. How parental alleles interact in progeny to influence the epigenome is poorly understood. We analyzed the DNA methylomes of Arabidopsis Col and C24 ecotypes, and their hybrid progeny. Hybrids displayed nonadditive DNA methylation levels, termed methylation interactions, throughout the genome. Approximately 2,500 methylation interactions occurred at regions where parental DNA methylation levels are similar, whereas almost 1,000 were at differentially methylated regions in parents. Methylation interactions were characterized by an abundance of 24-nt small interfering RNAs. Furthermore, dysfunction of the RNA-directed DNA methylation pathway abolished methylation interactions but did not affect the increased biomass observed in hybrid progeny. Methylation interactions correlated with altered genetic variation within the genome, suggesting that they may play a role in genome evolution. PMID:27382183

  2. Direct Measurement of Atmospheric Ammonia from an Airborne Miniature Chemical Ionization Mass Spectrometer (miniCIMS)

    NASA Astrophysics Data System (ADS)

    Casados, K.; Schill, S.; Freeman, S.; Zoerb, M.; Bertram, T. H.; Lefer, B. L.

    2015-12-01

    Ammonia is emitted into the atmosphere from a variety of sources such as trees, ocean, diary fields, biomass burning, and fuel emissions. Previous studies have investigated the environmental impacts of atmospheric ammonia which can include chemical reactivity, nucleation of fine particulate matter 2.5 (PM 2.5 ), and implications for human health, but its chemical nature and relatively short lifetime make direct measurement of atmospheric ammonia difficult. During the 2015 NASA Student Airborne Research Program (SARP) an airborne miniature Chemical Ionization Mass Spectrometer (miniCIMS) was deployed on the NASA DC-8 flying laboratory in the Southern California region. The spatial and temporal variability of measured atmospheric ammonia concentrations will be discussed.

  3. Direct numerical simulations of a reacting mixing layer with chemical heat release

    NASA Technical Reports Server (NTRS)

    Mcmurtry, P. A.; Jou, W.-H.; Metcalfe, R. W.; Riley, J. J.

    1985-01-01

    In order to study the coupling between chemical heat release and fluid dynamics, direct numerical simulations of a chemically reacting mixing layer with heat release are performed. The fully compressible equations as well as an approximate set of equations that is asymptotically valid for low-Mach-number flows are treated. These latter equations have the computational advantage that high-frequency acoustic waves have been filtered out, allowing much larger time steps to be taken in the numerical solution procedure. A detailed derivation of these equations along with an outline of the numerical solution technique is given. Simulation results indicate that the rate of chemical product formed, the thickness of the mixing layer, and the amount of mass entrained into the layer all decrease with increasing rates of heat release.

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

    SciTech Connect

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

    2009-05-11

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

  5. A direct qPCR method for residual DNA quantification in monoclonal antibody drugs produced in CHO cells.

    PubMed

    Hussain, Musaddeq

    2015-11-10

    Chinese hamster ovary (CHO) cells are the host cell of choice for manufacturing of monoclonal antibody (mAb) drugs in the biopharmaceutical industry. Host cell DNA is an impurity of such manufacturing process and must be controlled and monitored in order to ensure drug purity and safety. A conventional method for quantification of host residual DNA in drug requires extraction of DNA from the mAb drug substance with subsequent quantification of the extracted DNA using real-time PCR (qPCR). Here we report a method where the DNA extraction step is eliminated prior to qPCR. In this method, which we have named 'direct resDNA qPCR', the mAb drug substance is digested with a protease called KAPA in a 96-well PCR plate, the protease in the digest is then denatured at high temperature, qPCR reagents are added to the resultant reaction wells in the plate along with standards and controls in other wells of the same plate, and the plate subjected to qPCR for analysis of residual host DNA in the samples. This direct resDNA qPCR method for CHO is sensitive to 5.0fg of DNA with high precision and accuracy and has a wide linear range of determination. The method has been successfully tested with four mAbs drug, two IgG1 and two IgG4. Both the purified drug substance as well as a number of process intermediate samples, e.g., bioreactor harvest, Protein A column eluate and ion-exchange column eluates were tested. This method simplifies the residual DNA quantification protocol, reduces time of analysis and leads to increased assay sensitivity and development of automated high-throughput methods. PMID:25850374

  6. Direct calculation of the sizes of DNA fragments separated by gel electrophoresis using programmes written for a pocket calculator.

    PubMed

    Gough, E J; Gough, N M

    1984-01-11

    In order to facilitate the direct computation of the sizes of DNA fragments separated by gel electrophoresis, we have written and evaluated programmes for the Hewlett-Packard 41C programmable calculator. The sizes estimated for DNA fragments of known length using some of these programmes were found to be more accurate than the estimates obtained by conventional graphical procedures. These programmes should be adaptable to other programmable calculators. PMID:6320110

  7. A direct droplet digital PCR method for quantification of residual DNA in protein drugs produced in yeast cells.

    PubMed

    Hussain, Musaddeq; Fantuzzo, Rebecca; Mercorelli, Suzanne; Cullen, Constance

    2016-05-10

    Yeast cells, in particular Pichia pastoris, are the host cell of choice for manufacturing several protein therapeutic agents in the biopharmaceutical industry. Host cell DNA is an impurity of such manufacturing process and the residual DNA after the purification process of the drug must be monitored to ensure drug purity and safety. Currently, real-time PCR (qPCR) based methods are widely employed for quantification of host residual DNA. At the same time the digital PCR technology is coming into prominence with promise of higher sensitivity. Here we report a method where the protein drug is directly added to the droplet digital PCR (ddPCR) reaction including yeast-specific primers and fluorescent-tagged probe and nanoliter-sized droplets are generated. The droplets are then subjected to PCR followed by analysis for fluorescence. This Pichia residual DNA direct ddPCR method for yeast can be used to test higher amount of drug compared to the corresponding qPCR method thereby increasing sensitivity, retaining high precision and accuracy and has a wide linear range of determination. The method has been successfully tested with three batches of a recombinant human IgG1-Fc-based drug (RP-1) and with commercially available human insulin, both manufactured in yeast cells. This method simplifies the residual DNA quantification protocol by eliminating DNA extraction or protease digestion and eliminates use of DNA standards in day-to-day running of the method. PMID:26896631

  8. Low molecular weight chemicals, hypersensitivity, and direct toxicity: the acid anhydrides.

    PubMed Central

    Venables, K M

    1989-01-01

    The acid anhydrides are a group of reactive chemicals used widely in alkyd and epoxy resins. The major hazards to health are mucosal and skin irritation and sensitisation of the respiratory tract. Most occupational asthma caused by acid anhydrides appears to be immunologically mediated. Immunological mechanisms have been proposed to explain an influenza-like syndrome and pulmonary haemorrhage, but direct toxicity may also be important in the aetiology of these conditions. PMID:2653411

  9. Direct evidence of chemical ordering in the FePt nanostructured alloy by HR-TEM

    NASA Astrophysics Data System (ADS)

    Gupta, Rekha; Medwal, Rohit; Annapoorni, S.

    2015-07-01

    The iron-platinum (FePt) alloy exhibits structural and magnetic phase transformation even at a low temperature of 300 °C with an insignificant grain growth. These transformation studies were understood nano-scopically using high resolution-transmission electron microscopy (HR-TEM). The FePt grains show strain induced structural transformation and adopts polycrystalline behaviour. The chemical ordering of FePt grains is explained using Fast Fourier Transform (FFT) analysis of the TEM image. HR-TEM image shows the hexagonal arrangement of Pt atoms in the [0 0 1] direction in the FePt unit cell which gives the direct evidence of chemical ordering in FePt nanostructured alloy. The filtration and reconstruction method has been employed with the help of inverse Fast Fourier Transformation tool, confirming the formation of L10 FePt phase. The chemical ordering is also confirmed by structural and magnetic measurements revealing an order parameter of 0.875 and coercivity 3 kOe respectively at a low annealing temperature of 300 °C. The chemical ordering at low annealing temperature makes it suitable for media storage applications.

  10. Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA

    PubMed Central

    Bell, Jason C.; Plank, Jody L.; Dombrowski, Christopher C.; Kowalczykowski, Stephen C.

    2012-01-01

    Escherichia coli RecA is the defining member of a ubiquitous class of DNA strand exchange proteins that are essential for homologous recombination, a pathway that maintains genomic integrity by repairing broken DNA1. To function, filaments of RecA must nucleate and grow on single-stranded DNA (ssDNA) in direct competition with ssDNA-binding protein (SSB), which rapidly binds and continuously sequesters ssDNA, kinetically blocking RecA assembly2,3. This dynamic self-assembly on a DNA lattice, in competition with another protein, is unique for the RecA-family relative to other filament-forming proteins such as actin and tubulin. The complexity of this process has hindered our understanding of RecA filament assembly because ensemble measurements cannot reliably distinguish between the nucleation and growth phases, despite extensive and diverse attempts2–5. Previous single-molecule assays have measured nucleation and growth of RecA—and its eukaryotic homolog RAD51—on naked dsDNA and ssDNA6–12; however, the template for RecA self-assembly in vivo is SSB-coated ssDNA3. Using single-molecule microscopy, we directly visualized RecA filament assembly on single molecules of SSB-coated ssDNA, simultaneously measuring nucleation and growth. We establish that a dimer of RecA is required for nucleation, followed by growth of the filament through monomer addition, consistent with the finding that nucleation, but not growth, is modulated by nucleotide and magnesium ion cofactors. Filament growth is bidirectional, albeit faster in the 5′→3′ direction. Both nucleation and growth are repressed at physiological conditions, highlighting the essential role of recombination mediators in potentiating assembly in vivo. We define a two-step kinetic mechanism where RecA nucleates on transiently exposed ssDNA during SSB sliding and/or partial dissociation (i.e., DNA unwrapping) and then grows. We further demonstrate that the recombination mediator protein pair, Rec

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

    SciTech Connect

    Linz, U.; Stoecklin, G.

    1985-02-01

    Doubly labeled (U-/sup 14/C, 5-/sup 125/I)iododeoxycytidine (IdC) triphosphate was synthesized and incorporated enzymatically into defined positions of the plasmid pBR322. After storage under various conditions, the stable end products were analyzed using radio-GC, radio-HPLC, and electron microscopy. In addition, solutions of /sup 14/C-IdC-labeled DNA containing Na/sup 125/I as an internal radiation source were studied to investigate the influence of internal radiolysis. Transmutation of the covalently bound /sup 125/I leads to complete destruction of the labeled nucleotide, giving rise to /sup 14/CO/sub 2/ and /sup 14/CO as major products. Electron microscopy studies reveal that decay-induced double strand breaks (dsb) occur both at the site of decay and in areas as far as hundreds of base pairs apart from that site. Number and distribution of the breaks is strongly dependent on solvent and DNA configuration. A direct correlation exists between the extent of fragmentation of the nucleotide and the mean number of dsb.

  12. Production of thymine glycols in DNA by radiation and chemical carcinogens as detected by a monoclonal antibody.

    PubMed

    Leadon, S A

    1987-06-01

    In order to understand the role in carcinogenesis of damage indirectly induced by chemical carcinogens, it is important to identify the primary DNA lesions. We have measured the formation and repair of one type of DNA modification, 5,6-dihydroxydihydrothymine (thymine glycol), following exposure of cultured human cells to the carcinogens N-hydroxy-2-naphthylamine or benzo(a)pyrene. The efficiency of production of thymine glycols in DNA by these carcinogens was compared to that by ionizing radiation and ultraviolet light. Thymine glycols were detected using a monoclonal antibody against this product in a sensitive immunoassay. We found that thymine glycols were produced in DNA in a dose dependent manner after exposure to the carcinogens and that their production was reduced if either catalase or superoxide dismutase or both were present at the time of treatment. The efficiency of thymine glycol production following exposure to the chemical carcinogens was greater than that following equi-toxic doses of radiation. Thymine glycols were efficiently removed from the DNA of human cells following treatment with either the chemical carcinogens, ionizing radiation or ultraviolet light. PMID:3477281

  13. Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair

    PubMed Central

    Singh, Satyendra K.; Wang, Minli; Staudt, Christian; Iliakis, George

    2011-01-01

    In cells exposed to ionizing radiation (IR), double-strand breaks (DSBs) form within clustered-damage sites from lesions disrupting the DNA sugar–phosphate backbone. It is commonly assumed that these DSBs form promptly and are immediately detected and processed by the cellular DNA damage response (DDR) apparatus. This assumption is questioned by the observation that after irradiation of naked DNA, a fraction of DSBs forms minutes to hours after exposure as a result of temperature dependent, chemical processing of labile sugar lesions. Excess DSBs also form when IR-exposed cells are processed at 50°C, but have been hitherto considered method-related artifact. Thus, it remains unknown whether DSBs actually develop in cells after IR exposure from chemically labile damage. Here, we show that irradiation of ‘naked’ or chromatin-organized mammalian DNA produces lesions, which evolve to DSBs and add to those promptly induced, after 8–24 h in vitro incubation at 37°C or 50°C. The conversion is more efficient in chromatin-associated DNA, completed within 1 h in cells and delayed in a reducing environment. We conclude that IR generates sugar lesions within clustered-damage sites contributing to DSB formation only after chemical processing, which occurs efficiently at 37°C. This subset of delayed DSBs may challenge DDR, may affect the perceived repair kinetics and requires further characterization. PMID:21745815

  14. Direct cloning of isogenic murine DNA in yeast and relevance of isogenicity for targeting in embryonic stem cells.

    PubMed

    Andréasson, Claes; Schick, Anna J; Pfeiffer, Susanne M; Sarov, Mihail; Stewart, Francis; Wurst, Wolfgang; Schick, Joel A

    2013-01-01

    Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors. PMID:24058528

  15. Direct Cloning of Isogenic Murine DNA in Yeast and Relevance of Isogenicity for Targeting in Embryonic Stem Cells

    PubMed Central

    Pfeiffer, Susanne M.; Sarov, Mihail; Stewart, Francis; Wurst, Wolfgang; Schick, Joel A.

    2013-01-01

    Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors. PMID:24058528

  16. Direct detection of spin chemical potential shift through spin filtering effect

    NASA Astrophysics Data System (ADS)

    Miao, Guoxing; Moodera, Jagadeesh

    2010-03-01

    Spin filtering (SF) effect is a unique way to generate highly spin-polarized tunnel currents from nonmagnetic electrodes. Magnetic tunnel junctions based on pure SF effect have been realized recently [1] as a clear demonstration of principle for the spin manipulation through SF effect. The next challenge is the readout of spin information. In this work, we present the direct detection of the spin chemical potential shift in an Al nano cluster sandwiched between two SF EuS tunnel barriers. The spin channels are split by depositing Al directly onto EuS, and the indirect exchange interaction between the Al conduction electrons and the localized Eu 4f electrons gives rise to an effective Zeeman splitting with the strength of a few mV. EuS on the readout side is isolated from the Al clusters with a natural Al2O3 barrier. In a vertical measurement geometry with no transport current, we directly detected the spin dependent voltage levels by aligning the detection SF barrier parallel or antiparallel to the first SF barrier, corresponding to the equilibrium up- and down-spin chemical potentials. A simple analysis treating the barriers as a set of resistors revealed that the observed voltage difference is the actual chemical potential shift modulated by the SF efficiency.[4pt] [1] G.X. Miao, M. Muller, J.S. Moodera,PRL102,076601(2009)

  17. Sensitive immunoassay detection of multiple environmental chemicals on protein microarrays using DNA/dye conjugate as a fluorescent label

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Indirect competitive immunoassays were developed on protein microarrays for the sensitive and simultaneous detection of multiple environmental chemicals in one sample. In this assay, a DNA/SYTOX Orange conjugate was employed as antibody labels to increase the fluorescence signal and sensitivity. Ep...

  18. Tuning the strength of chemical patterns for directed self-assembly of block copolymers

    NASA Astrophysics Data System (ADS)

    Williamson, Lance; Lin, Guanyang; Cao, Yi; Gronheid, Roel; Nealey, Paul

    2014-03-01

    Directed self-assembly (DSA) of block copolymers (BCP) via chemo-epitaxy is a potential lithographic solution to patterns of dense features. The LiNe (Liu-Nealey) flow was used to fabricate the chemical pattern, which guides the BCP due to the different wetting behavior of the materials. Fine control of both the chemical pattern chemistry and geometry are important for DSA of BCP. Furthermore, wetting behavior considerations for DSA extend beyond pattern design and include the surrounding region. BCP DSA would be easier to integrate into device design if the patterned region were isolated with a featureless region (horizontal lamellar BCP assembly) rather than undirected BCP fingerprint structures. This paper addresses two processing steps found to be modifying the guide material. For one, the backfill brush grafts to the cross-linked polystyrene (XPS), albeit at a lower rate than the brush grafts to the exposed substrate. Undersaturating the backfill brush only moderately improves the XPS wetting behavior, but also negatively impacts the background region of the chemical pattern. Replacing the brush grafting functionality so that the brush grafts at lower annealing conditions also did not avoid the side reaction between the brush and the XPS. The other step modifying the XPS is the trim etch. Replacing the trim etch process was effective at generating a chemical pattern that can orient the BCP horizontally on a stripe 11 L0 wide passing through a field of chemical pattern.

  19. Processing of triplex-directed psoralen DNA interstrand crosslinks by recombination mechanisms.

    PubMed

    Liu, Yaobin; Nairn, Rodney S; Vasquez, Karen M

    2008-08-01

    Gene targeting via homologous recombination (HR) is an important application in biotechnology and medicine. However, in mammalian cells HR is much less efficient than random integration. Triplex-forming oligonucleotides (TFOs) linked to DNA damaging agents (e.g. psoralen) can stimulate HR, providing the potential to improve gene therapy applications. To elucidate factors affecting TFO-directed psoralen interstrand crosslink (ICL)-induced recombination, we constructed a series of plasmids with duplicated supF reporter genes, each containing an inactivating deletion, to measure HR frequencies in mammalian cells. Our results indicated that TFO-directed ICL-induced recombination frequencies were higher in the plasmids with larger distances between duplicated supF genes than with a smaller separation distance. However, the position of the ICL relative to the reporter genes did not affect HR frequencies. Recombination spectra were altered by the distance between supF copies. Although single-strand annealing (SSA) recombinants were predominant in all plasmid substrates, the plasmid with the shortest interval (60 bp) revealed a significant proportion of gene conversions (GCs). GCs occurred exclusively in the gene containing the shortest deletion, regardless of the distance between supF genes, ICL position or deletion orientation. Our analyses indicated that SSA is the predominant mechanism of ICL processing of these substrates in mammalian cells. PMID:18628293

  20. Promoter-cDNA-directed heterologous protein expression in Xenopus laevis oocytes.

    PubMed Central

    Swick, A G; Janicot, M; Cheneval-Kastelic, T; McLenithan, J C; Lane, M D

    1992-01-01

    Heterologous proteins can be expressed in Xenopus laevis oocytes by cytoplasmic microinjection of mRNA. To circumvent limitations inherent in this approach we investigate direct nuclear injection of strong viral expression vectors to drive transcription and subsequent translation of cDNAs encoding cytoplasmic, secreted, and plasma membrane proteins. After several viral promoters had been tested, the pMT2 vector was found to be a superior expression vector for X. laevis oocytes capable of directing expression of high levels of functional heterologous proteins. Typically the amount of protein derived from transcription-translation of the microinjected cDNA accounts for approximately 1% of total non-yolk protein. Moreover, the inefficiency usually associated with nuclear injections was overcome by coinjection of pMT2 driving expression of a secreted alkaline phosphatase as an internal control to select positive-expressing oocytes. Using this method, we have successfully expressed high levels of chloramphenicol acetyltransferase, the adipocyte-specific cytosolic 422(aP2) protein, and the membrane-associated glucose transporter GLUT1. The system described should be applicable to a wide variety of proteins for which cDNAs are available. Hence, the cumbersome and often inefficient in vitro synthesis of mRNA for studying ion channels, receptors, and transporters as well as for expression cloning in Xenopus oocytes should no longer be necessary. Images PMID:1542676

  1. Controllable g5p-Protein-Directed Aggregation of ssDNA-Gold Nanoparticles

    SciTech Connect

    Lee, S.; Maye, M; Zhang, Y; Gang, O; van der Lelie, D

    2009-01-01

    We assembled single-stranded DNA (ssDNA) conjugated nanoparticles using the phage M13 gene 5 protein (g5p) as the molecular glue to bind two antiparallel noncomplementary ssDNA strands. The entire process was controlled tightly by the concentration of the g5p protein and the presence of double-stranded DNA. The g5p-ssDNA aggregate was disintegrated by hybridization with complementary ssDNA (C-ssDNA) that triggers the dissociation of the complex. Polyhistidine-tagged g5p was bound to nickel nitrilotriacetic acid (Ni2+-NTA) conjugated nanoparticles and subsequently used to coassemble the ssDNA-conjugated nanoparticles into multiparticle-type aggregates. Our approach offers great promise for designing biologically functional, controllable protein/nanoparticle composites.

  2. NMR chemical shift perturbation mapping of DNA binding by a zinc-finger domain from the yeast transcription factor ADR1.

    PubMed Central

    Schmiedeskamp, M.; Rajagopal, P.; Klevit, R. E.

    1997-01-01

    Mutagenesis studies have revealed that the minimal DNA-binding domain of the yeast transcription factor ADR1 consists of two Cys2-His2 zinc fingers plus an additional 20 residues proximal and N-terminal to the fingers. We have assigned NMR 1H, 15N, and 13C chemical shifts for the entire minimal DNA-binding domain of ADR1 both free and bound to specific DNA. 1H chemical shift values suggest little structural difference between the zinc fingers in this construct and in single-finger constructs, and 13C alpha chemical shift index analysis indicates little change in finger structure upon DNA binding. 1H chemical shift perturbations upon DNA binding are observed, however, and these are mapped to define the protein-DNA interface. The two zinc fingers appear to bind DNA with different orientations, as the entire helix of finger 1 is perturbed, while only the extreme N-terminus of the finger 2 helix is affected. Furthermore, residues N-terminal to the first finger undergo large chemical shift changes upon DNA binding suggesting a role at the protein-DNA interface. A striking correspondence is observed between the protein-DNA interface mapped by chemical shift changes and that previously mapped by mutagenesis. PMID:9300483

  3. Noninvasive Facial Rejuvenation. Part 3: Physician-Directed-Lasers, Chemical Peels, and Other Noninvasive Modalities.

    PubMed

    Meaike, Jesse D; Agrawal, Nikhil; Chang, Daniel; Lee, Edward I; Nigro, Marjory G

    2016-08-01

    A proper knowledge of noninvasive facial rejuvenation is integral to the practice of a cosmetic surgeon. Noninvasive facial rejuvenation can be divided into patient- versus physician-directed modalities. Patient-directed facial rejuvenation combines the use of facial products such as sunscreen, moisturizers, retinoids, α-hydroxy acids, and various antioxidants to both maintain youthful skin and rejuvenate damaged skin. Physicians may recommend and often prescribe certain products, but patients are in control with this type of facial rejuvenation. On the other hand, physician-directed facial rejuvenation entails modalities that require direct physician involvement, such as neuromodulators, filler injections, laser resurfacing, microdermabrasion, and chemical peels. With the successful integration of each of these modalities, a complete facial regimen can be established and patient satisfaction can be maximized. This article is the last in a three-part series describing noninvasive facial rejuvenation. Here the authors review the mechanism, indications, and possible complications of lasers, chemical peels, and other commonly used noninvasive modalities. PMID:27478423

  4. Direct Growth Properties of Graphene Layers on Sapphire Substrate by Alcohol-Chemical Vapor Deposition

    NASA Astrophysics Data System (ADS)

    Nakamura, Atsushi; Miyasaka, Yuta; Temmyo, Jiro

    2012-04-01

    Few nanometers thick graphene layers were directly grown on a-plane (11bar 20) sapphire substrates by alcohol-chemical vapor deposition (alcohol-CVD) using ethanol as a carbon source and without any catalytic metal on the substrate surface. The growth relationship between the graphene layer and substrate was analyzed using a transmission electron microscope (TEM). The growth rate of graphene layers with different growth temperatures revealed that the Al atom act as a catalyst for synthesizing a graphitic material during the decomposition of ethanol. An optical transmittance and a sheet resistance of the graphene sheet directly grown on sapphire substrate were observed. SiO2/Si and n-6H-SiC substrates were also examined for graphene direct growth to discuss the catalytic behavior of Si atoms compared with Al atoms.

  5. Direct observation of λ-DNA molecule reversal movement within microfluidic channels under electric field with single molecule imaging technique

    NASA Astrophysics Data System (ADS)

    Fengyun, Yang; Kaige, Wang; Dan, Sun; Wei, Zhao; Hai-qing, Wang; Xin, He; Gui-ren, Wang; Jin-tao, Bai

    2016-07-01

    The electrodynamic characteristics of single DNA molecules moving within micro-/nano-fluidic channels are important in the design of biomedical chips and bimolecular sensors. In this study, the dynamic properties of λ-DNA molecules transferring along the microchannels driven by the external electrickinetic force were systemically investigated with the single molecule fluorescence imaging technique. The experimental results indicated that the velocity of DNA molecules was strictly dependent on the value of the applied electric field and the diameter of the channel. The larger the external electric field, the larger the velocity, and the more significant deformation of DNA molecules. More meaningfully, it was found that the moving directions of DNA molecules had two completely different directions: (i) along the direction of the external electric field, when the electric field intensity was smaller than a certain threshold value; (ii) opposite to the direction of the external electric field, when the electric field intensity was greater than the threshold electric field intensity. The reversal movement of DNA molecules was mainly determined by the competition between the electrophoresis force and the influence of electro-osmosis flow. These new findings will theoretically guide the practical application of fluidic channel sensors and lab-on-chips for precisely manipulating single DNA molecules. Project supported by the National Natural Science Foundation of China (Grant No. 61378083), the International Cooperation Foundation of the National Science and Technology Major Project of the Ministry of Science and Technology of China (Grant No. 2011DFA12220), the Major Research Plan of National Natural Science Foundation of China (Grant No. 91123030), and the Natural Science Foundation of Shaanxi Province of China (Grant Nos. 2010JS110 and 2013SZS03-Z01).

  6. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  7. Profiling Dose-Dependent Activation of p53-Mediated Signaling Pathways by Chemicals with Distinct Mechanisms of DNA Damage

    PubMed Central

    Clewell, Rebecca A.; Sun, Bin; Adeleye, Yeyejide; Carmichael, Paul; Efremenko, Alina; McMullen, Patrick D.; Pendse, Salil; Trask, O. J.; White, Andy; Andersen, Melvin E.

    2014-01-01

    As part of a larger effort to provide proof-of-concept in vitro-only risk assessments, we have developed a suite of high-throughput assays for key readouts in the p53 DNA damage response toxicity pathway: double-strand break DNA damage (p-H2AX), permanent chromosomal damage (micronuclei), p53 activation, p53 transcriptional activity, and cell fate (cell cycle arrest, apoptosis, micronuclei). Dose-response studies were performed with these protein and cell fate assays, together with whole genome transcriptomics, for three prototype chemicals: etoposide, quercetin, and methyl methanesulfonate. Data were collected in a human cell line expressing wild-type p53 (HT1080) and results were confirmed in a second p53 competent cell line (HCT 116). At chemical concentrations causing similar increases in p53 protein expression, p53-mediated protein expression and cellular processes showed substantial chemical-specific differences. These chemical-specific differences in the p53 transcriptional response appear to be determined by augmentation of the p53 response by co-regulators. More importantly, dose-response data for each of the chemicals indicate that the p53 transcriptional response does not prevent micronuclei induction at low concentrations. In fact, the no observed effect levels and benchmark doses for micronuclei induction were less than or equal to those for p53-mediated gene transcription regardless of the test chemical, indicating that p53's post-translational responses may be more important than transcriptional activation in the response to low dose DNA damage. This effort demonstrates the process of defining key assays required for a pathway-based, in vitro-only risk assessment, using the p53-mediated DNA damage response pathway as a prototype. PMID:25078064

  8. Microdosimetry of DNA conformations: relation between direct effect of (60)Co gamma rays and topology of DNA geometrical models in the calculation of A-, B- and Z-DNA radiation-induced damage yields.

    PubMed

    Semsarha, Farid; Raisali, Gholamreza; Goliaei, Bahram; Khalafi, Hossein

    2016-05-01

    In order to obtain the energy deposition pattern of ionizing radiation in the nanometric scale of genetic material and to investigate the different sensitivities of the DNA conformations, direct effects of (60)Co gamma rays on the three A, B and Z conformations of DNA have been studied. For this purpose, single-strand breaks (SSB), double-strand breaks (DSB), base damage (BD), hit probabilities and three microdosimetry quantities (imparted energy, mean chord length and lineal energy) in the mentioned DNA conformations have been calculated and compared by using GEometry ANd Tracking 4 (Geant4) toolkit. The results show that A-, B- and Z-DNA conformations have the highest yields of DSB (1.2 Gy(-1) Gbp(-1)), SSB (25.2 Gy(-1) Gbp(-1)) and BD (4.81 Gy(-1) Gbp(-1)), respectively. Based on the investigation of direct effects of radiation, it can be concluded that the DSB yield is largely correlated to the topological characteristics of DNA models, although the SSB yield is not. Moreover, according to the comparative results of the present study, a reliable candidate parameter for describing the relationship between DNA damage yields and geometry of DNA models in the theoretical radiation biology research studies would be the mean chord length (4 V/S) of the models. PMID:26984469

  9. Direct Measurement and Chemical Speciation of Top Ring Zone Liquid During Engine Operation

    SciTech Connect

    Splitter, Derek A; Burrows, Barry Clay; Lewis Sr, Samuel Arthur

    2015-01-01

    The present manuscript consists of proof of concept experiments involving direct measurements and detailed chemical speciation from the top ring zone of a running engine. The work uses a naturally aspirated single cylinder utility engine that has been modified to allow direct liquid sample acquisition from behind the top ring. Samples were analyzed and spectated using gas chromatographic techniques. Results show that the liquid mixture in the top ring zone is neither neat lubricant nor fuel but a combination of the two with unique chemical properties. At the tested steady state no-load operating condition, the chemical species of the top ring zone liquid were found to be highly dependent on boiling point, where both low reactivity higher boiling point fuel species and lubricant are observed to be the dominant constituents. The results show that at least for the tested condition, approximately 25% of the top ring zone is comprised of gasoline fuel like molecules, which are dominated by high octane number aromatic species, while the remainder of the liquid is comprised of lubricant like species.

  10. Direct observation of DNA motions into solid state nanopore under applied electrical potentials on conductive surface

    NASA Astrophysics Data System (ADS)

    Hayashi, Yoshitaka; Ando, Genki; Idutsu, Ichiro; Mitsui, Toshiyuki

    2011-03-01

    Solid state nanopore is one of emerging methods for rapid single DNA molecule detection because the translocation of the DNA though nanopore produces ionic current changes. One of issues in this method is clogging long DNA molecules. Once DNA molecules clogged, the molecules are rarely removed by varying or switching the polarity of applied bias voltages across the nanopore. We develop a modified nanopore by 50nm Au coating on top of the nanopore surface to be able to remove the clogged DNA molecules during the DNA translocation experiment. Fluorescence microscopy was implemented for observation of stained DNA molecules. The nanopores with diameters near 100 nm can be used initially. DNA translocation rates changes dramatically by tuning the applied electrical potentials on surface higher or lower than the potentials across the nanopore. Furthermore, the Au potentials modifies IV characteristic of the ionic current across the nanopore which is similar to the gate voltages controlling the SD current in FET. We will discuss the influence of surface potential on DNA motion and translocation and clogged DNA molecules. Finally, we will present the recent results of DNA translocation into the SiN-Au-SiO2 nanopore and discuss the effect of applied voltages on Au.

  11. Direct Binding to Replication Protein A (RPA)-coated Single-stranded DNA Allows Recruitment of the ATR Activator TopBP1 to Sites of DNA Damage.

    PubMed

    Acevedo, Julyana; Yan, Shan; Michael, W Matthew

    2016-06-17

    A critical event for the ability of cells to tolerate DNA damage and replication stress is activation of the ATR kinase. ATR activation is dependent on the BRCT (BRCA1 C terminus) repeat-containing protein TopBP1. Previous work has shown that recruitment of TopBP1 to sites of DNA damage and stalled replication forks is necessary for downstream events in ATR activation; however, the mechanism for this recruitment was not known. Here, we use protein binding assays and functional studies in Xenopus egg extracts to show that TopBP1 makes a direct interaction, via its BRCT2 domain, with RPA-coated single-stranded DNA. We identify a point mutant that abrogates this interaction and show that this mutant fails to accumulate at sites of DNA damage and that the mutant cannot activate ATR. These data thus supply a mechanism for how the critical ATR activator, TopBP1, senses DNA damage and stalled replication forks to initiate assembly of checkpoint signaling complexes. PMID:27129245

  12. Quantum chemical study of TiO{sub 2}/dopamine-DNA triads.

    SciTech Connect

    Vega-Arroyo, M.; LeBreton, P. R.; Zapol, P.; Curtiss, L. A.; Rajh, T.; Univ. Illinois at Chicago

    2007-10-15

    Photoinduced charge separation in triads of DNA covalently linked to an anatase nanoparticle via a dopamine bridge was studied by ab initio calculations of the oxidation potentials of carboxyl-DNA trimers and the TiO2/dopamine complex. Conjugation of dopamine to the TiO2 surface results in a lower oxidation potential of the complex relative to the surface and in localization of photogenerated holes on dopamine, while photogenerated electrons are excited into the conduction band of TiO2. Linking dopamine to the DNA trimers at the 5? end of the oligonucleotide may lead to further hole migration to the DNA. Calculations show that for several different sequences hole migration is favorable in double stranded DNA and unfavorable in single-stranded DNA. This extended charge separation was shown to follow from the redox properties of DNA sequence rather than from the modification of DNA's electron donating properties by the dopamine linker, which explains experimental observations.

  13. Directing assembly of DNA-coated colloids with magnetic fields to generate rigid, semiflexible, and flexible chains.

    PubMed

    Byrom, Julie; Han, Patric; Savory, Michael; Biswal, Sibani Lisa

    2014-08-01

    We report the formation of colloidal macromolecules consisting of chains of micron-sized paramagnetic particles assembled using a magnetic field and linked with DNA. The interparticle spacing and chain flexibility were controlled by varying the magnetic field strength and the linker spring constant. Variations in the DNA lengths allowed for the generation of chains with an improved range of flexibility as compared to previous studies. These chains adopted the rigid-rod, semiflexible, and flexible conformations that are characteristic of linear polymer systems. These assembly techniques were investigated to determine the effects of the nanoscale DNA linker properties on the properties of the microscale colloidal chains. With stiff DNA linkers (564 base pairs) the chains were only stable at moderate to high field strengths and produced rigid chains. For flexible DNA linkers (8000 base pairs), high magnetic field strengths caused the linkers to be excluded from the gap between the particles, leading to a transition from very flexible chains at low field strengths to semiflexible chains at high field strengths. In the intermediate range of linker sizes, the chains exhibited predictable behavior, demonstrating increased flexibility with longer DNA linker length or smaller linking field strengths. This study provides insight into the process of directed assembly using magnetic fields and DNA by precisely tuning the components to generate colloidal analogues of linear macromolecular chains. PMID:25052952

  14. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA.

    PubMed

    Richardson, Christopher D; Ray, Graham J; DeWitt, Mark A; Curie, Gemma L; Corn, Jacob E

    2016-03-01

    Targeted genomic manipulation by Cas9 can efficiently generate knockout cells and organisms via error-prone nonhomologous end joining (NHEJ), but the efficiency of precise sequence replacement by homology-directed repair (HDR) is substantially lower. Here we investigate the interaction of Cas9 with target DNA and use our findings to improve HDR efficiency. We show that dissociation of Cas9 from double-stranded DNA (dsDNA) substrates is slow (lifetime ∼6 h) but that, before complete dissociation, Cas9 asymmetrically releases the 3' end of the cleaved DNA strand that is not complementary to the sgRNA (nontarget strand). By rationally designing single-stranded DNA (ssDNA) donors of the optimal length complementary to the strand that is released first, we increase the rate of HDR in human cells when using Cas9 or nickase variants to up to 60%. We also demonstrate HDR rates of up to 0.7% using a catalytically inactive Cas9 mutant (dCas9), which binds DNA without cleaving it. PMID:26789497

  15. Directional mapping of DNA nicking in ejaculated and cauda epididymidal spermatozoa of the short-beaked echidna (Tachyglossus aculeatus: Monotremata).

    PubMed

    Johnston, S D; López-Fernández, C; Gosálbez, A; Holt, W V; Gosálvez, J

    2009-01-01

    Prototherian spermatozoa are unique amongst the Mammalia in terms of their filiform morphology, tandem arrangement of chromosomes and formation of sperm bundles. In the present study, we provide observations of echidna spermatozoa and note that the superstructure of the bundle is engineered around the shape of the individual sperm head and that this in turn may be a consequence of the unusual circumferential and helicoidal condensation of the DNA during spermiogenesis. Frozen-thawed ejaculated echidna spermatozoa were incubated and examined for the presence of non-typical DNA conformation by means of in situ labelling of DNA breaks using Klenow polymerase and via alkaline single-cell comet assays for detection of fragmented DNA. Both techniques successfully revealed the presence of what appeared to be directional DNA nicking, co-localised with the presence of highly sensitive alkali sites along the length of the sperm nucleus. It was not possible to define whether these alternative DNA configurations were associated with a failure of the sperm nucleus to condense appropriately during spermiogenesis or were evidence of DNA fragmentation following post-thaw incubation or a sequential structural chromatin rearrangement necessary for fertilisation. PMID:19874725

  16. Targeted DNA methylation by homology-directed repair in mammalian cells. Transcription reshapes methylation on the repaired gene

    PubMed Central

    Morano, Annalisa; Angrisano, Tiziana; Russo, Giusi; Landi, Rosaria; Pezone, Antonio; Bartollino, Silvia; Zuchegna, Candida; Babbio, Federica; Bonapace, Ian Marc; Allen, Brittany; Muller, Mark T.; Chiariotti, Lorenzo; Gottesman, Max E.; Porcellini, Antonio; Avvedimento, Enrico V.

    2014-01-01

    We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15–20 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression. PMID:24137009

  17. Template-directed covalent conjugation of DNA to native antibodies, transferrin and other metal-binding proteins

    NASA Astrophysics Data System (ADS)

    Rosen, Christian B.; Kodal, Anne L. B.; Nielsen, Jesper S.; Schaffert, David H.; Scavenius, Carsten; Okholm, Anders H.; Voigt, Niels V.; Enghild, Jan J.; Kjems, Jørgen; Tørring, Thomas; Gothelf, Kurt V.

    2014-09-01

    DNA-protein conjugates are important in bioanalytical chemistry, molecular diagnostics and bionanotechnology, as the DNA provides a unique handle to identify, functionalize or otherwise manipulate proteins. To maintain protein activity, conjugation of a single DNA handle to a specific location on the protein is often needed. However, preparing such high-quality site-specific conjugates often requires genetically engineered proteins, which is a laborious and technically challenging approach. Here we demonstrate a simpler method to create site-selective DNA-protein conjugates. Using a guiding DNA strand modified with a metal-binding functionality, we directed a second DNA strand to the vicinity of a metal-binding site of His6-tagged or wild-type metal-binding proteins, such as serotransferrin, where it subsequently reacted with lysine residues at that site. This method, DNA-templated protein conjugation, facilitates the production of site-selective protein conjugates, and also conjugation to IgG1 antibodies via a histidine cluster in the constant domain.

  18. A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells

    PubMed Central

    Soejima, Takashi; Xiao, Jin-zhong; Abe, Fumiaki

    2016-01-01

    Typically, polymerase chain reaction (PCR) is performed after DNA isolation. Real-time PCR (qPCR), also known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA. However, applying this methodology to prokaryotic cells, which have solid cell walls, in contrast to mammalian cells which immediately burst in water, can result in poor detection. We successfully achieved PCR elongation with the addition of 1.3 cfu of Cronobacter muytjensii to a newly developed direct qPCR master mix without performing any crude DNA extraction (detection limit of 1.6 × 100 cfu/ml for the test sample compared with a detection limit of 1.6 × 103 cfu/ml primarily for crude (boiling) or classical DNA isolation). We revealed that the chromosomal DNA retained in prokaryotic cells can function as a PCR template, similarly to the mechanism in in situ PCR. Elucidating this reaction mechanism may contribute to the development of an innovative master mix for direct qPCR to detect genes in a single bacterium with solid cell walls and might lead to numerous novel findings in prokaryotic genomics research. PMID:27334801

  19. A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells.

    PubMed

    Soejima, Takashi; Xiao, Jin-Zhong; Abe, Fumiaki

    2016-01-01

    Typically, polymerase chain reaction (PCR) is performed after DNA isolation. Real-time PCR (qPCR), also known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA. However, applying this methodology to prokaryotic cells, which have solid cell walls, in contrast to mammalian cells which immediately burst in water, can result in poor detection. We successfully achieved PCR elongation with the addition of 1.3 cfu of Cronobacter muytjensii to a newly developed direct qPCR master mix without performing any crude DNA extraction (detection limit of 1.6 × 10(0) cfu/ml for the test sample compared with a detection limit of 1.6 × 10(3) cfu/ml primarily for crude (boiling) or classical DNA isolation). We revealed that the chromosomal DNA retained in prokaryotic cells can function as a PCR template, similarly to the mechanism in in situ PCR. Elucidating this reaction mechanism may contribute to the development of an innovative master mix for direct qPCR to detect genes in a single bacterium with solid cell walls and might lead to numerous novel findings in prokaryotic genomics research. PMID:27334801

  20. Direct Covalent Biomolecule Immobilization on Plasma-Nanotextured Chemically Stable Substrates.

    PubMed

    Tsougeni, K; Petrou, P S; Awsiuk, K; Marzec, M M; Ioannidis, N; Petrouleas, V; Tserepi, A; Kakabakos, S E; Gogolides, E

    2015-07-15

    A new method for direct covalent immobilization of protein molecules (including antibodies) on organic polymers with plasma-induced random micronanoscale topography and stable-in-time chemical functionality is presented. This is achieved using a short (1-5 min) plasma etching and simultaneous micronanotexturing process, followed by a fast thermal annealing step, which induces accelerated hydrophobic recovery while preserving important chemical functionality created by the plasma. Surface-bound biomolecules resist harsh washing with sodium dodecyl sulfate and other detergents even at elevated temperatures, losing less than 40% of the biomolecules bound even at the harshest washing conditions. X-ray photoelectron spectroscopy, secondary-ion mass spectrometry, and electron paramagnetic resonance are used to unveil the chemical modification of the plasma-treated and stabilized surfaces. The nanotextured and chemically stabilized surfaces are used as substrates for the development of immunochemical assays for the sensitive detection of C-reactive protein and salmonella lipopolysaccharides through immobilization of the respective analyte-specific antibodies onto them. Such substrates are stable for a period of 1 year with ambient storage. PMID:26098201

  1. Large-scale dynamics of directed self-assembly defects on chemically pre-patterned surface

    NASA Astrophysics Data System (ADS)

    Yoshimoto, Kenji; Taniguchi, Takashi

    2013-03-01

    Morphological defects of block copolymers are dynamically formed during the annealing step of directed self­ assembly (DSA) process. Understanding the dynamics of such defects is crucial to manufacture defect-free wafers, however it is not well-understood due to difficulties in in-situ measurements. In order to provide some insights into this problem, we have performed dynamic simulations of symmetric diblock copolymers on chemically pre-patterned surface. A simplified model, so-called the Ohta-Kawasaki (OK) model was employed in this study, whose free energy and chemical potential were expressed as a function of the local order parameters. Time evolution of the local order parameters were calculated numerically and iteratively from the equation of continuity. As a test case, the two-dimensional (2D) dynamic simulations were performed including thermal fluctuations. The time evolution of the lamella defects was successfully characterized as a function of the interactive strength between the diblock copolymers and the chemically pre-patterned surface. In the three­ dimensional (3D) dynamic simulations, some complicated morphologies formed on the chemically pre-patterned surface were found to be similar to those obtained from Monte Carlo simulations. Our preliminary simulation data prove that for small χNs, dynamic simulations of diblock copolymers with OK model could be a powerful method to predict DSA defects with reasonable accuracy and with small computational cost.

  2. Biomolecular Damage Induced by Ionizing Radiation: The Direct and Indirect Effects of Low-Energy Electrons on DNA

    NASA Astrophysics Data System (ADS)

    Alizadeh, Elahe; Orlando, Thomas M.; Sanche, Léon

    2015-04-01

    Many experimental and theoretical advances have recently allowed the study of direct and indirect effects of low-energy electrons (LEEs) on DNA damage. In an effort to explain how LEEs damage the human genome, researchers have focused efforts on LEE interactions with bacterial plasmids, DNA bases, sugar analogs, phosphate groups, and longer DNA moieties. Here, we summarize the current understanding of the fundamental mechanisms involved in LEE-induced damage of DNA and complex biomolecule films. Results obtained by several laboratories on films prepared and analyzed by different methods and irradiated with different electron-beam current densities and fluencies are presented. Despite varied conditions (e.g., film thicknesses and morphologies, intrinsic water content, substrate interactions, and extrinsic atmospheric compositions), comparisons show a striking resemblance in the types of damage produced and their yield functions. The potential of controlling this damage using molecular and nanoparticle targets with high LEE yields in targeted radiation-based cancer therapies is also discussed.

  3. Influence of major-groove chemical modifications of DNA on transcription by bacterial RNA polymerases

    PubMed Central

    Raindlová, Veronika; Janoušková, Martina; Slavíčková, Michaela; Perlíková, Pavla; Boháčová, Soňa; Milisavljevič, Nemanja; Šanderová, Hana; Benda, Martin; Barvík, Ivan; Krásný, Libor; Hocek, Michal

    2016-01-01

    DNA templates containing a set of base modifications in the major groove (5-substituted pyrimidines or 7-substituted 7-deazapurines bearing H, methyl, vinyl, ethynyl or phenyl groups) were prepared by PCR using the corresponding base-modified 2′-deoxyribonucleoside triphosphates (dNTPs). The modified templates were used in an in vitro transcription assay using RNA polymerase from Bacillus subtilis and Escherichia coli. Some modified nucleobases bearing smaller modifications (H, Me in 7-deazapurines) were perfectly tolerated by both enzymes, whereas bulky modifications (Ph at any nucleobase) and, surprisingly, uracil blocked transcription. Some middle-sized modifications (vinyl or ethynyl) were partly tolerated mostly by the E. coli enzyme. In all cases where the transcription proceeded, full length RNA product with correct sequence was obtained indicating that the modifications of the template are not mutagenic and the inhibition is probably at the stage of initiation. The results are promising for the development of bioorthogonal reactions for artificial chemical switching of the transcription. PMID:27001521

  4. TANK VAPOR CHEMICALS OF POTENTIAL CONCERN & EXISTING DIRECT READING INSTRUMENTION & PERSONAL PROTECTIVE EQUIPMENT CONSIDERATIONS

    SciTech Connect

    BUTLER, N.K.

    2004-11-01

    This document takes the newly released Industrial Hygiene Chemical Vapor Technical Basis (RPP-22491) and evaluates the chemicals of potential concern (COPC) identified for selected implementation actions by the industrial hygiene organization. This document is not intended as a hazard analysis with recommended controls for all tank farm activities. Not all of the chemicals listed are present in all tanks; therefore, hazard analyses can and should be tailored as appropriate. Detection of each chemical by current industrial hygiene non-specific instrumentation in use at the tank farms is evaluated. Information gaps are identified and recommendations are made to resolve these needs. Of the 52 COPC, 34 can be detected with existing instrumentation. Three additional chemicals could be detected with a photoionization detector (PID) equipped with a different lamp. Discussion with specific instrument manufacturers is warranted. Consideration should be given to having the SapphIRe XL customized for tank farm applications. Other instruments, sampling or modeling techniques should be evaluated to estimate concentrations of chemicals not detected by direct reading instruments. In addition, relative instrument response needs to be factored in to action levels used for direct reading instruments. These action levels should be correlated to exposures to the COPC and corresponding occupational exposure limits (OELs). The minimum respiratory protection for each of the COPC is evaluated against current options. Recommendations are made for respiratory protection based on each chemical. Until exposures are sufficiently quantified and analyzed, the current use of supplied air respiratory protection is appropriate and protective for the COPC. Use of supplied air respiratory protection should be evaluated once a detailed exposure assessment for the COPC is completed. The established tank farm OELs should be documented in the TFC-PLN-34. For chemicals without an established tank farm OEL

  5. A 3D Model of Double-Helical DNA Showing Variable Chemical Details

    ERIC Educational Resources Information Center

    Cady, Susan G.

    2005-01-01

    Since the first DNA model was created approximately 50 years ago using molecular models, students and teachers have been building simplified DNA models from various practical materials. A 3D double-helical DNA model, made by placing beads on a wire and stringing beads through holes in plastic canvas, is described. Suggestions are given to enhance…

  6. Mitochondrial DNA is a direct target of anti-cancer anthracycline drugs

    SciTech Connect

    Ashley, Neil Poulton, Joanna

    2009-01-16

    The anthracyclines, such as doxorubicin (DXR), are potent anti-cancer drugs but they are limited by their clinical toxicity. The mechanisms involved remain poorly understood partly because of the difficulty in determining sub-cellular drug localisation. Using a novel method utilising the fluorescent DNA dye PicoGreen, we found that anthracyclines intercalated not only into nuclear DNA but also mitochondrial DNA (mtDNA). Intercalation of mtDNA by anthracyclines may thus contribute to the marked mitochondrial toxicity associated with these drugs. By contrast, ethidium bromide intercalated exclusively into mtDNA, without interacting with nuclear DNA, thereby explaining why mtDNA is the main target for ethidium. By exploiting PicoGreen quenching we also developed a novel assay for quantification of mtDNA levels by flow-cytometry, an approach which should be useful for studies of mitochondrial dysfunction. In summary our PicoGreen assay should be useful to study drug/DNA interactions within live cells, and facilitate therapeutic drug monitoring and kinetic studies in cancer patients.

  7. Direct Detection and Quantification of Bacterial Genes Associated with Inflammation in DNA Isolated from Stool

    PubMed Central

    Gómez-Moreno, Ramón; Robledo, Iraida E.; Baerga-Ortiz, Abel

    2014-01-01

    Although predominantly associated with health benefits, the gut microbiota has also been shown to harbor genes that promote inflammation. In this work, we report a method for the direct detection and quantification of these pro-inflammatory bacterial genes by PCR and qPCR in DNA extracted from human stool samples. PCR reactions were performed to detect (i) the pks island genes, (ii) tcpC, which is present in some strains of Escherichia coli and (iii) gelE presented in some strains of Enterococcus faecalis. Additionally, we screened for the presence of the following genes encoding cyclomodulins that disrupted mammalian cell division: (iv) cdt (which encodes the cytolethal distending toxin) and (v) cnf-1 (which encodes the cytotoxic necrotizing factor-1). Our results show that 20% of the samples (N = 41) tested positive for detectable amounts of pks island genes, whereas 10% of individuals were positive for tcpC or gelE and only one individual was found to harbor the cnf-1 gene. Of the 13 individuals that were positive for at least one of the pro-inflammatory genes, 5 were found to harbor more than one. A quantitative version of the assay, which used real-time PCR, revealed the pro-inflammatory genes to be in high copy numbers: up to 1.3 million copies per mg of feces for the pks island genes. Direct detection of specific genes in stool could prove useful toward screening for the presence of pro-inflammatory bacterial genes in individuals with inflammatory bowel diseases or colorectal cancer. PMID:25635239

  8. Direct Visualization of Conformation and Dense Packing of DNA-Based Soft Colloids

    NASA Astrophysics Data System (ADS)

    Zhang, Jing; Lettinga, Paul M.; Dhont, Jan K. G.; Stiakakis, Emmanuel

    2014-12-01

    Soft colloids—such as polymer-coated particles, star polymers, block-copolymer micelles, microgels—constitute a broad class of materials where microscopic properties such as deformability and penetrability of the particle play a key role in tailoring their macroscopic properties which is of interest in many technological areas. The ability to access these microscopic properties is not yet demonstrated despite its great importance. Here we introduce novel DNA-coated colloids with star-shaped architecture that allows accessing the above local structural information by directly visualizing their intramolecular monomer density profile and arm's free-end locations with confocal fluorescent microscopy. Compression experiments on a two-dimensional hexagonal lattice formed by these macromolecular assemblies reveal an exceptional resistance to mutual interpenetration of their charged corona at pressures approaching the MPa range. Furthermore, we find that this lattice, in a close packing configuration, is surprisingly tolerant to particle size variation. We anticipate that these stimuli-responsive materials could aid to get deeper insight in a wide range of problems in soft matter, including the study and design of biomimetic lubricated surfaces.

  9. Direct visualization of conformation and dense packing of DNA-based soft colloids.

    PubMed

    Zhang, Jing; Lettinga, Paul M; Dhont, Jan K G; Stiakakis, Emmanuel

    2014-12-31

    Soft colloids--such as polymer-coated particles, star polymers, block-copolymer micelles, microgels--constitute a broad class of materials where microscopic properties such as deformability and penetrability of the particle play a key role in tailoring their macroscopic properties which is of interest in many technological areas. The ability to access these microscopic properties is not yet demonstrated despite its great importance. Here we introduce novel DNA-coated colloids with star-shaped architecture that allows accessing the above local structural information by directly visualizing their intramolecular monomer density profile and arm's free-end locations with confocal fluorescent microscopy. Compression experiments on a two-dimensional hexagonal lattice formed by these macromolecular assemblies reveal an exceptional resistance to mutual interpenetration of their charged corona at pressures approaching the MPa range. Furthermore, we find that this lattice, in a close packing configuration, is surprisingly tolerant to particle size variation. We anticipate that these stimuli-responsive materials could aid to get deeper insight in a wide range of problems in soft matter, including the study and design of biomimetic lubricated surfaces. PMID:25615395

  10. Ultrasensitive electrochemical detection of breast cancer cells based on DNA-rolling-circle-amplification-directed enzyme-catalyzed polymerization.

    PubMed

    Sheng, Qinglin; Cheng, Ni; Bai, Wushuang; Zheng, Jianbin

    2015-02-01

    An ultrasensitive cytosensor based on DNA-rolling-circle-amplification-directed enzyme-catalyzed polymerization is demonstrated. As a proof of concept, the cytosensor shows excellent sensitivity for MCF-7 cell detection with a lower detection limit of 12 cells per mL. PMID:25536491

  11. Direct Chemical Vapor Deposition-Derived Graphene Glasses Targeting Wide Ranged Applications.

    PubMed

    Sun, Jingyu; Chen, Yubin; Priydarshi, Manish Kr; Chen, Zhang; Bachmatiuk, Alicja; Zou, Zhiyu; Chen, Zhaolong; Song, Xiuju; Gao, Yanfeng; Rümmeli, Mark H; Zhang, Yanfeng; Liu, Zhongfan

    2015-09-01

    Direct growth of graphene on traditional glasses is of great importance for various daily life applications. We report herein the catalyst-free atmospheric-pressure chemical vapor deposition approach to directly synthesizing large-area, uniform graphene films on solid glasses. The optical transparency and sheet resistance of such kinds of graphene glasses can be readily adjusted together with the experimentally tunable layer thickness of graphene. More significantly, these graphene glasses find a broad range of real applications by enabling the low-cost construction of heating devices, transparent electrodes, photocatalytic plates, and smart windows. With a practical scalability, the present work will stimulate various applications of transparent, electrically and thermally conductive graphene glasses in real-life scenarios. PMID:26305883

  12. Continuous directed evolution of DNA-binding proteins to improve TALEN specificity.

    PubMed

    Hubbard, Basil P; Badran, Ahmed H; Zuris, John A; Guilinger, John P; Davis, Kevin M; Chen, Liwei; Tsai, Shengdar Q; Sander, Jeffry D; Joung, J Keith; Liu, David R

    2015-10-01

    Nucleases containing programmable DNA-binding domains can alter the genomes of model organisms and have the potential to become human therapeutics. Here we present DNA-binding phage-assisted continuous evolution (DB-PACE) as a general approach for the laboratory evolution of DNA-binding activity and specificity. We used this system to generate transcription activator-like effectors nucleases (TALENs) with broadly improved DNA cleavage specificity, establishing DB-PACE as a versatile approach for improving the accuracy of genome-editing agents. PMID:26258293

  13. Direct DNA extraction method of an obligate parasitic fungus from infected plant tissue.

    PubMed

    Liu, L; Wang, C L; Peng, W Y; Yang, J; Lan, M Q; Zhang, B; Li, J B; Zhu, Y Y; Li, C Y

    2015-01-01

    Powdery mildew and rust fungi are obligate parasites that cannot live without host organisms. They are difficult to culture in synthetic medium in the laboratory. Genomic DNA extraction is one of the basic molecular techniques used to study the genetic structure of populations. In this study, 2 different DNA extraction methods, Chelex-100 and cetyltrimethylammonium bromide (CTAB), were used to extract DNA from euonymus powdery mildew and Puccinia striiformis f. sp Tritici. Polymerase chain reaction was carried out with a race-specific-marker rDNA-internal transcribed spacer sequence. Both DNA extraction methods were compared and analyzed. The results showed that both Chelex-100 and CTAB were effective for extracting genomic DNA from infected plant tissue. However, less DNA was required for the Chelex-100 method than for the CTAB method, and the Chelex-100 method involved fewer steps, was simpler and safer, and did not require organic solvents compared to the CTAB method. DNA quality was evaluated by polymerase chain reaction, and the results showed that genomic DNA extracted using the Chelex-100 method was better than that using CTAB method, and was sufficient for studying the genetic structure of population. PMID:26782502

  14. Continuous directed evolution of DNA-binding proteins to improve TALEN specificity

    PubMed Central

    Hubbard, Basil P.; Badran, Ahmed H.; Zuris, John A.; Guilinger, John P.; Davis, Kevin M.; Chen, Liwei; Tsai, Shengdar Q.; Sander, Jeffry D.; Joung, J. Keith; Liu, David R.

    2015-01-01

    Nucleases containing programmable DNA-binding domains can alter the genomes of model organisms and have the potential to become human therapeutics. Here we present DNA-binding phage-assisted continuous evolution (DB-PACE) as a general approach for the laboratory evolution of DNA-binding activity and specificity. We used this system to generate TALE nucleases with broadly improved DNA cleavage specificity, establishing DB-PACE as a versatile approach for improving the accuracy of genome-editing agents. PMID:26258293

  15. DNA-directed self-assembly of gold nanoparticles into binary and ternary nanostructures

    NASA Astrophysics Data System (ADS)

    Yao, Hui; Yi, Changqing; Tzang, Chi-Hung; Zhu, Junjie; Yang, Mengsu

    2007-01-01

    The assembly and characterization of gold nanoparticle-based binary and ternary structures are reported. Two strategies were used to assemble gold nanoparticles into ordered nanoscale architectures: in strategy 1, gold nanoparticles were functionalized with single-strand DNA (ssDNA) first, and then hybridized with complementary ssDNA-labelled nanoparticles to assemble designed architectures. In strategy 2, the designed architectures were constructed through hybridization between complementary ssDNA first, then by assembling gold nanoparticles to the scaffolding through gold-sulfur bonds. Both TEM measurements and agarose gel electrophoresis confirmed that the latter strategy is more efficient in generating the designed nanostructures.

  16. Medicinal Plants Recommended by the World Health Organization: DNA Barcode Identification Associated with Chemical Analyses Guarantees Their Quality

    PubMed Central

    Palhares, Rafael Melo; Gonçalves Drummond, Marcela; dos Santos Alves Figueiredo Brasil, Bruno; Pereira Cosenza, Gustavo; das Graças Lins Brandão, Maria; Oliveira, Guilherme

    2015-01-01

    Medicinal plants are used throughout the world, and the regulations defining their proper use, such as identification of the correct species and verification of the presence, purity and concentration of the required chemical compounds, are widely recognized. Herbal medicines are made from vegetal drugs, the processed products of medicinal species. These processed materials present a number of challenges in terms of botanical identification, and according to the World Health Organization (WHO), the use of incorrect species is a threat to consumer safety. The samples used in this study consisted of the dried leaves, flowers and roots of 257 samples from 8 distinct species approved by the WHO for the production of medicinal herbs and sold in Brazilian markets. Identification of the samples in this study using DNA barcoding (matK, rbcL and ITS2 regions) revealed that the level of substitutions may be as high as 71%. Using qualitative and quantitative chemical analyses, this study identified situations in which the correct species was being sold, but the chemical compounds were not present. Even more troubling, some samples identified as substitutions using DNA barcoding contained the chemical compounds from the correct species at the minimum required concentration. This last situation may lead to the use of unknown species or species whose safety for human consumption remains unknown. This study concludes that DNA barcoding should be used in a complementary manner for species identification with chemical analyses to detect and quantify the required chemical compounds, thus improving the quality of this class of medicines. PMID:25978064

  17. Medicinal plants recommended by the world health organization: DNA barcode identification associated with chemical analyses guarantees their quality.

    PubMed

    Palhares, Rafael Melo; Gonçalves Drummond, Marcela; Dos Santos Alves Figueiredo Brasil, Bruno; Pereira Cosenza, Gustavo; das Graças Lins Brandão, Maria; Oliveira, Guilherme

    2015-01-01

    Medicinal plants are used throughout the world, and the regulations defining their proper use, such as identification of the correct species and verification of the presence, purity and concentration of the required chemical compounds, are widely recognized. Herbal medicines are made from vegetal drugs, the processed products of medicinal species. These processed materials present a number of challenges in terms of botanical identification, and according to the World Health Organization (WHO), the use of incorrect species is a threat to consumer safety. The samples used in this study consisted of the dried leaves, flowers and roots of 257 samples from 8 distinct species approved by the WHO for the production of medicinal herbs and sold in Brazilian markets. Identification of the samples in this study using DNA barcoding (matK, rbcL and ITS2 regions) revealed that the level of substitutions may be as high as 71%. Using qualitative and quantitative chemical analyses, this study identified situations in which the correct species was being sold, but the chemical compounds were not present. Even more troubling, some samples identified as substitutions using DNA barcoding contained the chemical compounds from the correct species at the minimum required concentration. This last situation may lead to the use of unknown species or species whose safety for human consumption remains unknown. This study concludes that DNA barcoding should be used in a complementary manner for species identification with chemical analyses to detect and quantify the required chemical compounds, thus improving the quality of this class of medicines. PMID:25978064

  18. Chemical and toxicologic characterization of co-processing and two-stage direct coal liquefaction materials

    SciTech Connect

    Wright, C.W.; Stewart, D.L.; Mahlum, D.D.; Chess, E.K.; Wilson, B.W.

    1986-03-01

    Recent advances in coal liquefaction have included two-stage direct coal liquefaction processes and petroleum resid/coal co-processing technology. Two-stage coal liquefaction processes are generally comprised of a first-stage thermal or liquefaction reactor followed by a second-stage hydrogenation step. Petroleum resids and coal are simultaneously converted to liquefaction products in co-processing technology. The purpose of this paper is to report the prelimianry results of the chemical analysis and toxicological testing of a coal liquefaction co-processing sample set, and to compare these results to those obtained from two-stage coal liquefaction materials. Samples were chemically characterized by chemical class fractionation, gas chromatography, gas chromatography-mass spectrometry, and low-voltage probe-inlet mass spectrometry. Toxicological activity was measured using the standard histidine reversion microbial mutagenicity test and an initiation/promotion assay for mouse skin tumorigenesis. A brief description of these methods are presented and results are discussed. 9 refs., 2 figs., 3 tabs.

  19. In Situ Chemical Characterization of Organic Aerosol Surfaces using Direct Analysis in Real Time

    NASA Astrophysics Data System (ADS)

    Chan, M.; Nah, T.; Wilson, K. R.

    2012-12-01

    Obtaining in situ information on the molecular composition of atmospheric aerosol is important for understanding the sources, formation mechanisms, aging and physiochemical properties of atmospheric aerosol. Most recently, we have used Direct Analysis in Real Time (DART), which is a "soft" atmospheric pressure ionization technique, for in situ chemical characterization of a variety of laboratory generated organic aerosol and heterogeneous processing oleic acid aerosol. A stream of aerosol particles is crossed with a thermal flow of metastable He atoms (produced by the DART source) in front of an inlet of a mass spectrometer. The thermally desorbed analytes are subsequently ionized with minimal fragmentation by reactive species in the DART ionization source (e.g., metastable He atoms). The ion signal scales with the aerosol surface area rather than aerosol volume, suggesting that aerosol particles are not completely vaporized in the ionization region. The DART can thus measure the chemical composition as a function of aerosol depth. Probing aerosol depth is determined by the thermal desorption rates of aerosol particles. Here, we investigate how the experimental parameters (e.g., DART gas temperature and residence time) and the physiochemical properties of aerosol particles (e.g., enthalpy of vaporization) affect the probing aerosol depth and the desorption-ionization mechanism of aerosol particles in the DART using a series of model organic compounds. We also demonstrate the potential application of DART for in situ chemically analyzing wet aerosol particles undergoing oxidation reactions.

  20. Chemically tuned anode with tailored aqueous hydrocarbon binder for direct methanol fuel cells.

    PubMed

    Lee, Chang Hyun; Lee, So Young; Lee, Young Moo; McGrath, James E

    2009-07-21

    An anode for direct methanol fuel cells was chemically tuned by tailoring an aqueous hydrocarbon catalyst (SPI-BT) binder instead of using a conventional perfluorinated sulfonic acid ionomer (PFSI). SPI-BT designed in triethylamine salt form showed lower proton conductivity than PFSI, but it was stable in the catalyst ink forming the aqueous colloids. The aqueous colloidal particle size of SPI-BT was much smaller than that of PFSI. The small SPI-BT colloidal particles contributed to forming small catalyst agglomerates and simultaneously reducing their pore volume. Consequently, the high filling level of binders in the pores, where Pt-Ru catalysts are mainly located on the wall and physically interconnected, resulted in increased electrochemical active surface area of the anode, leading to high catalyst utilization. In addition, the chemical affinity between the SPI-BT binder and the membrane material derived from their similar chemical structure induced a stable interface on the membrane-electrode assembly (MEA) and showed low electric resistance. Upon adding SPI-BT, the synergistic effect of high catalyst utilization, improved mass transfer behavior to Pt-Ru catalyst, and low interfacial resistance of MEA became greater than the influence of reduced proton conductivity in the electrochemical performance of single cells. The electrochemical performance of MEAs with SPI-BT anode was enhanced to almost the same degree or somewhat higher than that with PFSI at 90 degrees C. PMID:19485372

  1. Direct chemical oxidation: a non-thermal technology for the destruction of organic wastes

    SciTech Connect

    Balazs, G.B.; Cooper, J. F.; Lewis, P. R.; Adamson, M. G.

    1998-02-01

    Direct Chemical Oxidation (DCO) is a non-thermal, ambient pressure, aqueous-based technology for the oxidative destruction of the organic components of hazardous or mixed waste streams. The process has been developed for applications in waste treatment and chemical demilitarization and decontamination at LLNL since 1992, and is applicable to the destruction of virtually all solid or liquid organics, including: chlorosolvents, oils and greases, detergents, organic-contaminated soils or sludges, explosives, chemical and biological warfare agents, and PCB's. [1-15] The process normally operates at 80-100 C, a heating requirement which increases the difficulty of surface decontamination of large objects or, for example, treatment of a wide area contaminated soil site. The driver for DCO work in FY98 was thus to investigate the use of catalysts to demonstrate the effectiveness of the technology for organics destruction at temperatures closer to ambient. In addition, DCO is at a sufficiently mature stage of development that technology transfer to a commercial entity was a logical next step, and was thus included in FY98 tasks.

  2. Synthesis of Bulk BC8 Silicon Allotrope by Direct Transformation and Reduced-Pressure Chemical Pathways.

    PubMed

    Kurakevych, Oleksandr O; Le Godec, Yann; Crichton, Wilson A; Guignard, Jérémy; Strobel, Timothy A; Zhang, Haidong; Liu, Hanyu; Coelho Diogo, Cristina; Polian, Alain; Menguy, Nicolas; Juhl, Stephen J; Gervais, Christel

    2016-09-01

    Phase-pure samples of a metastable allotrope of silicon, Si-III or BC8, were synthesized by direct elemental transformation at 14 GPa and ∼900 K and also at significantly reduced pressure in the Na-Si system at 9.5 GPa by quenching from high temperatures ∼1000 K. Pure sintered polycrystalline ingots with dimensions ranging from 0.5 to 2 mm can be easily recovered at ambient conditions. The chemical route also allowed us to decrease the synthetic pressures to as low as 7 GPa, while pressures required for direct phase transition in elemental silicon are significantly higher. In situ control of the synthetic protocol, using synchrotron radiation, allowed us to observe the underlying mechanism of chemical interactions and phase transformations in the Na-Si system. Detailed characterization of Si-III using X-ray diffraction, Raman spectroscopy, (29)Si NMR spectroscopy, and transmission electron microscopy are discussed. These large-volume syntheses at significantly reduced pressures extend the range of possible future bulk characterization methods and applications. PMID:27532223

  3. Low Temperature Direct Growth of Graphene Films on Transparent Substrates by Chemical Vapor Deposition

    NASA Astrophysics Data System (ADS)

    Antoine, Geoffrey Sandosh Jeffy

    Graphene, two dimensional sheet of carbon atoms has recently gained attention as some of its properties are very useful for electronics, optoelectronics and photovoltaic applications. Its high mobility makes it useful in radio-frequency applications and its transparency makes it useful as transparent electrodes in photovoltaics. It is known that chemical vapor deposition (CVD) is one of the techniques that can be used to synthesize graphene. A lot of work has been done on selecting appropriate substrates and hydrocarbon sources. Nickel, having a high solubility at high temperatures has been in focus lately. Ethylene which has a lower breaking point compared to other hydrocarbons has a good efficiency in the synthesis of graphene. Complexity associated with graphene synthesis and transfers onto transparent substrates constitute the major obstacles to using this material for photovoltaics and optoelectronics applications. Here we show a novel method of obtaining graphene directly on glass via chemical vapor deposition (CVD) using ethylene as the hydrocarbon source and nickel as the catalyst. The low cracking temperature of ethylene which is 542.8 °C permits us to use glass substrates directly in the CVD furnace. To improve the thickness of graphene, a good manipulation of pressure and hydrogen during the growth process will be useful. We introduce a novel catalyst etching technique after the growth results in graphene settling down on the glass substrate in a transfer-free process. Raman spectroscopy indicated good uniformity and high quality before and after the etching process.

  4. Identification of Lotus rhizobia by direct DNA hybridization of crushed root nodules

    SciTech Connect

    Cooper, J.E.; Bjourson, A.J.; Thompson, J.K.

    1987-07-01

    Hybridization of crushed Lotus pedunculatus root nodules with /sup 32/P-labeled total genomic DNA probes was used to identify Rhizobium loti and Bradyrhizobium sp. (Lotus rhizobia). Probes always hybridized with homologous target DNA and frequency with DNAs of other strains from the same genus. Intergeneric hybridization did not occur. Results were comparable to those from colony hybridization.

  5. Direct observation of ultrafast-electron-transfer reactions unravels high effectiveness of reductive DNA damage.

    PubMed

    Nguyen, Jenny; Ma, Yuhan; Luo, Ting; Bristow, Robert G; Jaffray, David A; Lu, Qing-Bin

    2011-07-19

    Both water and electron-transfer reactions play important roles in chemistry, physics, biology, and the environment. Oxidative DNA damage is a well-known mechanism, whereas the relative role of reductive DNA damage is unknown. The prehydrated electron (e(pre)-), a novel species of electrons in water, is a fascinating species due to its fundamental importance in chemistry, biology, and the environment. e(pre)- is an ideal agent to observe reductive DNA damage. Here, we report both the first in situ femtosecond time-resolved laser spectroscopy measurements of ultrafast-electron-transfer (UET) reactions of e(pre)- with various scavengers (KNO(3), isopropanol, and dimethyl sulfoxide) and the first gel electrophoresis measurements of DNA strand breaks induced by e(pre)- and OH(•) radicals co-produced by two-UV-photon photolysis of water. We strikingly found that the yield of reductive DNA strand breaks induced by each e(pre)- is twice the yield of oxidative DNA strand breaks induced by each OH(•) radical. Our results not only unravel the long-standing mystery about the relative role of radicals in inducing DNA damage under ionizing radiation, but also challenge the conventional notion that oxidative damage is the main pathway for DNA damage. The results also show the potential of femtomedicine as a new transdisciplinary frontier and the broad significance of UET reactions of e(pre)- in many processes in chemistry, physics, biology, and the environment. PMID:21730183

  6. Direct detection of synthetic and biologically generated double-stranded DNA by MALDI-TOF MS

    NASA Astrophysics Data System (ADS)

    Little, Daniel P.; Jacob, Anette; Becker, Thomas; Braun, Andreas; Darnhofer-Demar, Brigitte; Jurinke, Christian; van den Boom, Dirk; Koster, Hubert

    1997-12-01

    A synthetic 50-mer DNA was mixed at room temperature with a non-complementary or a complementary 27-mer and analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. The former resulted in negligible signals from a 27-/50- mer heterodimer, for the latter this was a dominant peak, consistent with maintaining Watson-Crick interactions in the gas phase. Double stranded DNA derived from an enzymatic digestion of a 252-base-pair polymerase chain reaction product when prepared at room temperature yielded very little specific double stranded DNA, but sample preparation at reduced temperature resulted in spectra dominated by the expected heterodimer signals with no random (i.e. non-Watson-Crick) dimers. As a DNA diagnostics genotyping application, apolipoprotein-E alleles were easily distinguished considering only the masses of their double stranded DNA digest fragments.

  7. New insights into the QuikChange™ process guide the use of Phusion DNA polymerase for site-directed mutagenesis.

    PubMed

    Xia, Yongzhen; Chu, Wenqiao; Qi, Qingsheng; Xun, Luying

    2015-01-01

    The QuikChange™ site-directed mutagenesis method is popular but imperfect. An improvement by using partially overlapping primers has been reported several times; however, it is incompatible with the proposed mechanism. The QuikChange™ method using complementary primers is proposed to linearly amplify a target plasmid with the products annealing to produce double-stranded DNA molecules with 5'-overhangs. The overhang annealing is supposed to form circular plasmids with staggered breaks, which can be repaired in Escherichia coli after transformation. Here, we demonstrated that the PCR enzyme fills the 5'-overhangs in the early cycles, and the product is then used as the template for exponential amplification. The linear DNA molecules with homologous ends are joined to generate the plasmid with the desired mutations through homologous recombination in E. coli. The correct understanding is important to method improvements, guiding us to use partially overlapping primers and Phusion DNA polymerase for site-directed mutagenesis. Phusion did not amplify a plasmid with complementary primers but used partially overlapping primers to amplify the plasmid, producing linear DNA molecules with homologous ends for site-directed mutagenesis. PMID:25399421

  8. Multicopy single-stranded DNA directs intestinal colonization of enteric pathogens

    SciTech Connect

    Elfenbein, Johanna R.; Knodler, Leigh A.; Nakayasu, Ernesto S.; Ansong, Charles; Brewer, Heather M.; Bogomolnaya, Lydia; Adams, L. Garry; McClelland, Michael; Adkins, Joshua N.; Andrews-Polymenis, Helene L.; Fang, Ferric C.

    2015-09-14

    Multicopy single-stranded DNAs (msDNAs) are hybrid RNA-DNA molecules encoded on retroelements called retrons and produced by the action of retron reverse transcriptases. Retrons are widespread in bacteria but the natural function of msDNA has remained elusive despite 30 years of study. The major roadblock to elucidation of the function of these unique molecules has been the lack of any identifiable phenotypes for mutants unable to make msDNA. We report that msDNA of the zoonotic pathogen Salmonella Typhimurium is necessary for colonization of the intestine. Similarly, we observed a defect in intestinal persistence in an enteropathogenic E. coli mutant lacking its retron reverse transcriptase. Under anaerobic conditions in the absence of msDNA, proteins of central anaerobic metabolism needed for Salmonella colonization of the intestine are dysregulated. We show that the msDNA-deficient mutant can utilize nitrate, but not other alternate electron acceptors in anaerobic conditions. Consistent with the availability of nitrate in the inflamed gut, a neutrophilic inflammatory response partially rescued the ability of a mutant lacking msDNA to colonize the intestine. These findings together indicate that the mechanistic basis of msDNA function during Salmonella colonization of the intestine is proper production of proteins needed for anaerobic metabolism. We further conclude that a natural function of msDNA is to regulate protein abundance, the first attributable function for any msDNA. Our data provide novel insight into the function of this mysterious molecule that likely represents a new class of regulatory molecules.

  9. Multicopy Single-Stranded DNA Directs Intestinal Colonization of Enteric Pathogens

    PubMed Central

    Elfenbein, Johanna R.; Knodler, Leigh A.; Nakayasu, Ernesto S.; Ansong, Charles; Brewer, Heather M.; Bogomolnaya, Lydia; Adams, L. Garry; McClelland, Michael; Adkins, Joshua N.; Andrews-Polymenis, Helene L.

    2015-01-01

    Multicopy single-stranded DNAs (msDNAs) are hybrid RNA-DNA molecules encoded on retroelements called retrons and produced by the action of retron reverse transcriptases. Retrons are widespread in bacteria but the natural function of msDNA has remained elusive despite 30 years of study. The major roadblock to elucidation of the function of these unique molecules has been the lack of any identifiable phenotypes for mutants unable to make msDNA. We report that msDNA of the zoonotic pathogen Salmonella Typhimurium is necessary for colonization of the intestine. Similarly, we observed a defect in intestinal persistence in an enteropathogenic E. coli mutant lacking its retron reverse transcriptase. Under anaerobic conditions in the absence of msDNA, proteins of central anaerobic metabolism needed for Salmonella colonization of the intestine are dysregulated. We show that the msDNA-deficient mutant can utilize nitrate, but not other alternate electron acceptors in anaerobic conditions. Consistent with the availability of nitrate in the inflamed gut, a neutrophilic inflammatory response partially rescued the ability of a mutant lacking msDNA to colonize the intestine. These findings together indicate that the mechanistic basis of msDNA function during Salmonella colonization of the intestine is proper production of proteins needed for anaerobic metabolism. We further conclude that a natural function of msDNA is to regulate protein abundance, the first attributable function for any msDNA. Our data provide novel insight into the function of this mysterious molecule that likely represents a new class of regulatory molecules. PMID:26367458

  10. Multicopy single-stranded DNA directs intestinal colonization of enteric pathogens

    SciTech Connect

    Elfenbein, Johanna R.; Knodler, Leigh A.; Nakayasu, Ernesto S.; Ansong, Charles; Brewer, Heather M.; Bogomolnaya, Lydia; Adams, L. G.; McClelland, Michael; Adkins, Joshua N.; Andrews-Polymenis, Helene L.

    2015-09-14

    Multicopy single-stranded DNAs (msDNAs) are hybrid RNA-DNA molecules encoded on retroelements called retrons and produced by the action of retron reverse transcriptases. Retrons are widespread in bacteria but the natural function of msDNA has remained elusive despite 30 years of study. The major roadblock to elucidation of the function of these unique molecules has been the lack of any identifiable phenotypes for mutants unable to make msDNA. We report that msDNA of the zoonotic pathogen Salmonella Typhimurium is necessary for colonization of the intestine. Similarly, we observed a defect in intestinal persistence in an enteropathogenic E. coli mutant lacking its retron reverse transcriptase. Under anaerobic conditions in the absence of msDNA, proteins of central anaerobic metabolism needed for Salmonella colonization of the intestine are dysregulated. We show that the msDNA-deficient mutant can utilize nitrate but not other alternate electron acceptors in anaerobic conditions. Consistent with the availability of nitrate in the inflamed gut, a neutrophilic inflammatory response partially rescued the ability of a mutant lacking msDNA to colonize the intestine. These findings together indicate that the mechanistic basis of msDNA function during Salmonella colonization of the intestine is proper production of proteins needed for anaerobic metabolism. We further conclude that a natural function of msDNA is to regulate protein abundance, the first attributable function for any msDNA. Our data provide novel insight into the function of this mysterious molecule that likely represents a new class of regulatory molecules.

  11. Multicopy single-stranded DNA directs intestinal colonization of enteric pathogens

    DOE PAGESBeta

    Elfenbein, Johanna R.; Knodler, Leigh A.; Nakayasu, Ernesto S.; Ansong, Charles; Brewer, Heather M.; Bogomolnaya, Lydia; Adams, L. Garry; McClelland, Michael; Adkins, Joshua N.; Andrews-Polymenis, Helene L.; et al

    2015-09-14

    Multicopy single-stranded DNAs (msDNAs) are hybrid RNA-DNA molecules encoded on retroelements called retrons and produced by the action of retron reverse transcriptases. Retrons are widespread in bacteria but the natural function of msDNA has remained elusive despite 30 years of study. The major roadblock to elucidation of the function of these unique molecules has been the lack of any identifiable phenotypes for mutants unable to make msDNA. We report that msDNA of the zoonotic pathogen Salmonella Typhimurium is necessary for colonization of the intestine. Similarly, we observed a defect in intestinal persistence in an enteropathogenic E. coli mutant lacking itsmore » retron reverse transcriptase. Under anaerobic conditions in the absence of msDNA, proteins of central anaerobic metabolism needed for Salmonella colonization of the intestine are dysregulated. We show that the msDNA-deficient mutant can utilize nitrate, but not other alternate electron acceptors in anaerobic conditions. Consistent with the availability of nitrate in the inflamed gut, a neutrophilic inflammatory response partially rescued the ability of a mutant lacking msDNA to colonize the intestine. These findings together indicate that the mechanistic basis of msDNA function during Salmonella colonization of the intestine is proper production of proteins needed for anaerobic metabolism. We further conclude that a natural function of msDNA is to regulate protein abundance, the first attributable function for any msDNA. Our data provide novel insight into the function of this mysterious molecule that likely represents a new class of regulatory molecules.« less

  12. Chemical Shifts to Metabolic Pathways: Identifying Metabolic Pathways Directly from a Single 2D NMR Spectrum.

    PubMed

    Dubey, Abhinav; Rangarajan, Annapoorni; Pal, Debnath; Atreya, Hanudatta S

    2015-12-15

    Identifying cellular processes in terms of metabolic pathways is one of the avowed goals of metabolomics studies. Currently, this is done after relevant metabolites are identified to allow their mapping onto specific pathways. This task is daunting due to the complex nature of cellular processes and the difficulty in establishing the identity of individual metabolites. We propose here a new method: ChemSMP (Chemical Shifts to Metabolic Pathways), which facilitates rapid analysis by identifying the active metabolic pathways directly from chemical shifts obtained from a single two-dimensional (2D) [(13)C-(1)H] correlation NMR spectrum without the need for identification and assignment of individual metabolites. ChemSMP uses a novel indexing and scoring system comprised of a "uniqueness score" and a "coverage score". Our method is demonstrated on metabolic pathways data from the Small Molecule Pathway Database (SMPDB) and chemical shifts from the Human Metabolome Database (HMDB). Benchmarks show that ChemSMP has a positive prediction rate of >90% in the presence of decluttered data and can sustain the same at 60-70% even in the presence of noise, such as deletions of peaks and chemical shift deviations. The method tested on NMR data acquired for a mixture of 20 amino acids shows a success rate of 93% in correct recovery of pathways. When used on data obtained from the cell lysate of an unexplored oncogenic cell line, it revealed active metabolic pathways responsible for regulating energy homeostasis of cancer cells. Our unique tool is thus expected to significantly enhance analysis of NMR-based metabolomics data by reducing existing impediments. PMID:26556218

  13. RNA-directed DNA methylation efficiency depends on trigger and target sequence identity.

    PubMed

    Dalakouras, Athanasios; Dadami, Elena; Wassenegger, Michèle; Krczal, Gabi; Wassenegger, Michael

    2016-07-01

    RNA-directed DNA methylation (RdDM) in plants has been extensively studied, but the RNA molecules guiding the RdDM machinery to their targets are still to be characterized. It is unclear whether these molecules require full complementarity with their target. In this study, we have generated Nicotiana tabacum (Nt) plants carrying an infectious tomato apical stunt viroid (TASVd) transgene (Nt-TASVd) and a non-infectious potato spindle tuber viroid (PSTVd) transgene (Nt-SB2). The two viroid sequences exhibit 81% sequence identity. Nt-TASVd and Nt-SB2 plants were genetically crossed. In the progeny plants (Nt-SB2/TASVd), deep sequencing of small RNAs (sRNAs) showed that TASVd infection was associated with the accumulation of abundant small interfering RNAs (siRNAs) that mapped along the entire TASVd but only partially matched the SB2 transgene. TASVd siRNAs efficiently targeted SB2 RNA for degradation, but no transitivity was detectable. Bisulfite sequencing in the Nt-SB2/TASVd plants revealed that the TASVd transgene was targeted for dense cis-RdDM along its entire sequence. In the same plants, the SB2 transgene was targeted for trans-RdDM. The SB2 methylation pattern, however, was weak and heterogeneous, pointing to a positive correlation between trigger-target sequence identity and RdDM efficiency. Importantly, trans-RdDM on SB2 was also detected at sites where no homologous siRNAs were detected. Our data indicate that RdDM efficiency depends on the trigger-target sequence identity, and is not restricted to siRNA occupancy. These findings support recent data suggesting that RNAs with sizes longer than 24 nt (>24-nt RNAs) trigger RdDM. PMID:27121647

  14. Detection of the thermostable direct hemolysin gene and related DNA sequences in Vibrio parahaemolyticus and other vibrio species by the DNA colony hybridization test.

    PubMed Central

    Nishibuchi, M; Ishibashi, M; Takeda, Y; Kaper, J B

    1985-01-01

    A specific gene probe for the Vibrio parahaemolyticus thermostable direct hemolysin gene was constructed and used to examine the presence or absence of the thermostable direct hemolysin gene or related DNA sequences in V. parahaemolyticus and other vibrios by the DNA colony hybridization method. The gene probe consisted of a 406-base-pair, completely internal fragment covering 71% of the structural gene with PstI linkers added to the ends. Six copies of this 415-base-pair PstI fragment were cloned into plasmid pBR322, which yielded large amounts of the probe DNA. One hundred forty-one V. parahaemolyticus strains were tested with the gene probe, and the results were compared with those of phenotypic assays for the thermostable direct hemolysin. All Kanagawa phenomenon-positive strains were gene positive. However, 86% of the strains that exhibited weak Kanagawa phenomenon and 16% of Kanagawa phenomenon-negative strains also reacted with the gene probe. Immunological methods for the detection of the thermostable direct hemolysin (modified Elek test, enzyme-linked immunosorbent assay) showed better correlation with gene probe results. All gene-positive strains produced hemolysin detectable in the enzyme-linked immunosorbent assay, although occasional strains showed weak reaction. The modified Elek test was slightly less sensitive than the enzyme-linked immunosorbent assay. All gene-negative strains were also negative in these immunological assays. One hundred twenty-one strains of Vibrio spp. other than V. parahaemolyticus were tested with the gene probe; only Vibrio hollisae strains reacted with the probe under stringent conditions. PMID:4030087

  15. Combination of cascade chemical reactions with graphene-DNA interaction to develop new strategy for biosensor fabrication.

    PubMed

    Zhu, Xiaoli; Sun, Liya; Chen, Yangyang; Ye, Zonghuang; Shen, Zhongming; Li, Genxi

    2013-09-15

    Graphene, a single atom thick and two dimensional carbon nano-material, has been proven to possess many unique properties, one of which is the recent discovery that it can interact with single-stranded DNA through noncovalent π-π stacking. In this work, we demonstrate that a new strategy to fabricate many kinds of biosensors can be developed by combining this property with cascade chemical reactions. Taking the fabrication of glucose sensor as an example, while the detection target, glucose, may regulate the graphene-DNA interaction through three cascade chemical reactions, electrochemical techniques are employed to detect the target-regulated graphene-DNA interaction. Experimental results show that in a range from 5μM to 20mM, the glucose concentration is in a natural logarithm with the logarithm of the amperometric response, suggesting a best detection limit and detection range. The proposed biosensor also shows favorable selectivity, and it has the advantage of no need for labeling. What is more, by controlling the cascade chemical reactions, detection of a variety of other targets may be achieved, thus the strategy proposed in this work may have a wide application potential in the future. PMID:23542067

  16. Enzymatic Treatment of Specimens before DNA Extraction Directly Influences Molecular Detection of Infectious Agents

    PubMed Central

    Goldschmidt, Pablo; Degorge, Sandrine; Merabet, Lilia; Chaumeil, Christine

    2014-01-01

    Introduction Biological samples, pharmaceuticals or food contain proteins, lipids, polymers, ammoniums and macromolecules that alter the detection of infectious agents by DNA amplification techniques (PCR). Moreover the targeted DNA has to be released from the complex cell walls and the compact nucleoprotein matrixes and cleared from potential inhibitors. The goal of the present work was to assess the efficiency of enzymatic pretreatments on infectious agents to make DNA available for further extraction and amplification. Methods Staphylococcus epidermidis, Streptococcus mitis, Propionibacterium acnes, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger and Fusarium solani were mixed with an internal control virus and treated with: 1) proteinase K; 2) lyticase and 3) lyticase followed by proteinase K. DNAs was manually extracted using the QIAmp DNA Mini kit or the MagNA Pure Compact automate. DNA extraction yields and the inhibitors were assessed with a phocid Herpesvirus. Bacterial detection was performed using TaqMan real-time PCR and yeasts and filamentous Fungi with HRM (real-time PCR followed by high-resolution melting analysis). Results Viral DNA was released, extracted and detected using manual and automatic methods without pre enzymatic treatments. Either the manual or the automatic DNA extraction systems did not meet the sensitivity expectations if enzymatic treatments were not performed before: lyticase for Fungi and Proteinase K for Bacteria. The addition of lyticase and proteinase K did not improve results. For Fungi the detection after lyticase was higher than for Proteinase K, for which melting analysis did not allow fungal specification. Discussion Columns and magnetic beads allowed collecting DNA and separate PCR inhibitors. Detection rates cannot be related to DNA-avidity of beads or to elution but to the lack of proteolysis. PMID:24936792

  17. DNA Content in Extracellular Vesicles Isolated from Porcine Coronary Venous Blood Directly after Myocardial Ischemic Preconditioning

    PubMed Central

    Rodsand, Pouria; Hellman, Urban; Waldenström, Anders; Lundholm, Marie; Ahrén, Dag; Biber, Björn; Ronquist, Gunnar; Haney, Michael

    2016-01-01

    Background Extracellular vesicles (EV) are nano-sized membranous structures released from most cells. They have the capacity to carry bioactive molecules and gene expression signals between cells, thus mediating intercellular communication. It is believed that EV confer protection after ischemic preconditioning (IPC). We hypothesize that myocardial ischemic preconditioning will lead to rapid alteration of EV DNA content in EV collected from coronary venous effluent. Materials and Methods In a porcine myocardial ischemic preconditioning model, EV were isolated from coronary venous blood before and after IPC by differential centrifugation steps culminating in preparative ultracentrifugation combined with density gradient ultracentrifugation. The EV preparation was validated, the DNA was extracted and further characterized by DNA sequencing followed by bioinformatics analysis. Results Porcine genomic DNA fragments representing each chromosome, including mitochondrial DNA sequences, were detected in EV isolated before and after IPC. There was no difference detected in the number of sequenced gene fragments (reads) or in the genomic coverage of the sequenced DNA fragments in EV isolated before and after IPC. Gene ontology analysis showed an enrichment of genes coding for ion channels, enzymes and proteins for basal metabolism and vesicle biogenesis and specific cardiac proteins. Conclusions This study demonstrates that porcine EV isolated from coronary venous blood plasma contain fragments of DNA from the entire genome, including the mitochondria. In this model we did not find specific qualitative or quantitative changes of the DNA content in EV collected immediately after an in vivo myocardial IPC provocation. This does not rule out the possibility that EV DNA content changes in response to myocardial IPC which could occur in a later time frame. PMID:27434143

  18. Self-assembly of multiferroic core-shell particulate nanocomposites through DNA-DNA hybridization and magnetic field directed assembly of superstructures

    NASA Astrophysics Data System (ADS)

    Sreenivasulu, Gollapudi; Lochbiler, Thomas A.; Panda, Manashi; Srinivasan, Gopalan; Chavez, Ferman A.

    2016-04-01

    Multiferroic composites of ferromagnetic and ferroelectric phases are of importance for studies on mechanical strain mediated coupling between the magnetic and electric subsystems. This work is on DNA-assisted self-assembly of superstructures of such composites with nanometer periodicity. The synthesis involved oligomeric DNA-functionalized ferroelectric and ferromagnetic nanoparticles, 600 nm BaTiO3 (BTO) and 200 nm NiFe2O4 (NFO), respectively. Mixing BTO and NFO particles, possessing complementary DNA sequences, resulted in the formation of ordered core-shell heteronanocomposites held together by DNA hybridization. The composites were imaged by scanning electron microscopy and scanning microwave microscopy. The presence of heteroassemblies along with core-shell architecture is clearly observed. The reversible nature of the DNA hybridization allows for restructuring the composites into mm-long linear chains and 2D-arrays in the presence of a static magnetic field and ring-like structures in a rotating-magnetic field. Strong magneto-electric (ME) coupling in as-assembled composites is evident from static magnetic field H induced polarization and low-frequency magnetoelectric voltage coefficient measurements. Upon annealing the nanocomposites at high temperatures, evidence for the formation of bulk composites with excellent cross-coupling between the electric and magnetic subsystems is obtained by H-induced polarization and low-frequency ME voltage coefficient. The ME coupling strength in the self-assembled composites is measured to be much stronger than in bulk composites with randomly distributed NFO and BTO prepared by direct mixing and sintering.

  19. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  20. Direct and site-specific quantification of RNA 2'-O-methylation by PCR with an engineered DNA polymerase.

    PubMed

    Aschenbrenner, Joos; Marx, Andreas

    2016-05-01

    Methylation of the 2'-hydroxyl-group of ribonucleotides is found in all major classes of RNA in eukaryotes and is one of the most abundant posttranscriptional modifications of stable RNAs. In spite of intense studies, the multiple functions of RNA 2'-O-methylation are still not understood. One major obstacle in the field are the technical demanding detection methods, which are typically laborious and do not always deliver unambiguous results. We present a thermostable KlenTaq DNA polymerase variant with significant reverse transcription activity that is able to discriminate 2'-O-methylated from unmethylated RNAs. The engineered enzyme catalyzes DNA synthesis from DNA as well as RNA templates and enables expeditious quantification of 2'-O-methylation of individual nucleotides directly from total RNA extracts by a simple qRT-PCR. PMID:27016740

  1. Direct and site-specific quantification of RNA 2′-O-methylation by PCR with an engineered DNA polymerase

    PubMed Central

    Aschenbrenner, Joos; Marx, Andreas

    2016-01-01

    Methylation of the 2′-hydroxyl-group of ribonucleotides is found in all major classes of RNA in eukaryotes and is one of the most abundant posttranscriptional modifications of stable RNAs. In spite of intense studies, the multiple functions of RNA 2′-O-methylation are still not understood. One major obstacle in the field are the technical demanding detection methods, which are typically laborious and do not always deliver unambiguous results. We present a thermostable KlenTaq DNA polymerase variant with significant reverse transcription activity that is able to discriminate 2′-O-methylated from unmethylated RNAs. The engineered enzyme catalyzes DNA synthesis from DNA as well as RNA templates and enables expeditious quantification of 2′-O-methylation of individual nucleotides directly from total RNA extracts by a simple qRT-PCR. PMID:27016740

  2. All-in-One Nanowire-Decorated Multifunctional Membrane for Rapid Cell Lysis and Direct DNA Isolation

    PubMed Central

    2015-01-01

    This paper describes a handheld device that uses an all-in-one membrane for continuous mechanical cell lysis and rapid DNA isolation without the assistance of power sources, lysis reagents, and routine centrifugation. This nanowire-decorated multifunctional membrane was fabricated to isolate DNA by selective adsorption to silica surface immediately after disruption of nucleus membranes by ultrasharp tips of nanowires for a rapid cell lysis, and it can be directly assembled with commercial syringe filter holders. The membrane was fabricated by photoelectrochemical etching to create microchannel arrays followed by hydrothermal synthesis of nanowires and deposition of silica. The proposed membrane successfully purifies high-quality DNA within 5 min, whereas a commercial purification kit needs more than an hour. PMID:25420232

  3. Biophysical and chemical handles to control the size of DNA nanoparticles produced by rolling circle amplification.

    PubMed

    Lee, So Yeon; Kim, Kyoung-Ran; Bang, Duhee; Bae, Se Won; Kim, Hak Joong; Ahn, Dae-Ro

    2016-08-16

    Although rolling circle amplification (RCA) is an efficient method to produce DNA materials for biomedical applications, it does not yield nano-sized products suitable for intracellular delivery. We here provide the ways to control the size of RCA products and show a potential application of the size-controlled DNA nanoparticles. PMID:27464359

  4. A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE INDUCED BY RADIATION, CHEMICAL MUTAGENS AND ENZYMES

    EPA Science Inventory

    A simple and rapid assay to detect DNA damage is reported. This novel assay is based on changes in melting/annealing behavior and facilitated using certain dyes that increase their fluorescence upon association with double stranded (ds)DNA. Damage caused by ultraviolet (UV) ra...

  5. Directed alteration of Saccharomyces cerevisiae mitochondrial DNA by biolistic transformation and homologous recombination

    PubMed Central

    Bonnefoy, Nathalie; Fox, Thomas D.

    2009-01-01

    Saccharomyces cerevisiae is currently the only species in which genetic transformation of mitochondria can be used to generate a wide variety of defined alterations in mtDNA. DNA sequences can be delivered into yeast mitochondria by microprojectile bombardment (biolistic transformation) and subsequently incorporated into mtDNA by the highly active homologous recombination machinery present in the organelle. While transformation frequencies are relatively low, the availability of strong mitochondrial selectable markers for the yeast system, both natural and synthetic, makes the isolation of transformants routine. The strategies and procedures reviewed here allow the researcher to insert defined mutations into endogenous mitochondrial genes, and to insert new genes into mtDNA. These methods provide powerful in vivo tools for the study of mitochondrial biology. PMID:18314724

  6. Self-assembled catalytic DNA nanostructures for synthesis of para-directed polyaniline.

    PubMed

    Wang, Zhen-Gang; Zhan, Pengfei; Ding, Baoquan

    2013-02-26

    Templated synthesis has been considered as an efficient approach to produce polyaniline (PANI) nanostructures. The features of DNA molecules enable a DNA template to be an intriguing template for fabrication of emeraldine PANI. In this work, we assembled HRP-mimicking DNAzyme with different artificial DNA nanostructures, aiming to manipulate the molecular structures and morphologies of PANI nanostructures through the controlled DNA self-assembly. UV-vis absorption spectra were used to investigate the molecular structures of PANI and monitor kinetic growth of PANI. It was found that PANI was well-doped at neutral pH and the redox behaviors of the resultant PANI were dependent on the charge density of the template, which was controlled by the template configurations. CD spectra indicated that the PANI threaded tightly around the helical DNA backbone, resulting in the right handedness of PANI. These reveal the formation of the emeraldine form of PANI that was doped by the DNA. The morphologies of the resultant PANI were studied by AFM and SEM. It was concluded from the imaging and spectroscopic kinetic results that PANI grew preferably from the DNAzyme sites and then expanded over the template to form 1D PANI nanostructures. The strategy of the DNAzyme-DNA template assembly brings several advantages in the synthesis of para-coupling PANI, including the region-selective growth of PANI, facilitating the formation of a para-coupling structure and facile regulation. We believe this study contributes significantly to the fabrication of doped PANI nanopatterns with controlled complexity, and the development of DNA nanotechnology. PMID:23272944

  7. UVA-induced damage to DNA and proteins: direct versus indirect photochemical processes

    NASA Astrophysics Data System (ADS)

    Girard, P. M.; Francesconi, S.; Pozzebon, M.; Graindorge, D.; Rochette, P.; Drouin, R.; Sage, E.

    2011-01-01

    UVA has long been known for generating an oxidative stress in cells. In this paper we review the different types of DNA damage induced by UVA, i.e. strand breaks, bipyrimidine photoproducts, and oxidatively damaged bases. Emphasis is given to the mechanism of formation that is further illustrated by the presentation of new in vitro data. Examples of oxidation of proteins involved in DNA metabolism are also given.

  8. Direct evidence of mitochondrial G-quadruplex DNA by using fluorescent anti-cancer agents.

    PubMed

    Huang, Wei-Chun; Tseng, Ting-Yuan; Chen, Ying-Ting; Chang, Cheng-Chung; Wang, Zi-Fu; Wang, Chiung-Lin; Hsu, Tsu-Ning; Li, Pei-Tzu; Chen, Chin-Tin; Lin, Jing-Jer; Lou, Pei-Jen; Chang, Ta-Chau

    2015-12-01

    G-quadruplex (G4) is a promising target for anti-cancer treatment. In this paper, we provide the first evidence supporting the presence of G4 in the mitochondrial DNA (mtDNA) of live cells. The molecular engineering of a fluorescent G4 ligand, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC), can change its major cellular localization from the nucleus to the mitochondria in cancer cells, while remaining primarily in the cytoplasm of normal cells. A number of BMVC derivatives with sufficient mitochondrial uptake can induce cancer cell death without damaging normal cells. Fluorescence studies of these anti-cancer agents in live cells and in isolated mitochondria from HeLa cells have demonstrated that their major target is mtDNA. In this study, we use fluorescence lifetime imaging microscopy to verify the existence of mtDNA G4s in live cells. Bioactivity studies indicate that interactions between these anti-cancer agents and mtDNA G4 can suppress mitochondrial gene expression. This work underlines the importance of fluorescence in the monitoring of drug-target interactions in cells and illustrates the emerging development of drugs in which mtDNA G4 is the primary target. PMID:26487635

  9. Direct zonal liquid chromatographic method for the kinetic study of actinomycin-DNA binding.

    PubMed

    Vidal-Madjar, Claire; Florentina, Cañada-Cañada; Gherghi, Ioanna; Jaulmes, Alain; Pantazaki, Anastasia; Taverna, Myriam

    2004-07-01

    The binding of an anticancer drug (actinomycin D or ACTD) to double-stranded DNA (dsDNA) was studied by means of high-performance liquid chromatography (HPLC). ACTD is an antitumor antibiotic containing one chromophore group and two pentapeptidic lactone cycles that binds dsDNA. Incubations of ACTD with DNA were performed at physiological pH. The complexed and free ligand concentrations of the mixture were quantified at 440 nm from their separation on a size-exclusion chromatographic (SEC) column using the same buffer for the elution and the sample incubation. The DNA and the ACTD-DNA complexes were eluted at the column exclusion volume while the ligand was retained on the support. An apparent binding curve was obtained by plotting the amount emerging at the exclusion column volume against that eluted at free ACTD retention volume. A dissociating effect was evidenced and the binding parameters were significantly different from those obtained at equilibrium by visible absorbance titration. The equilibrium binding parameters determined by absorption spectroscopy were used as starting data in the numerical simulations of the chromatographic process. The results showed a strong dependency of the apparent binding parameters on the reaction kinetics. Finally the comparison of the apparent binding curve obtained from the HPLC experiments and from the numerical simulations permitted an evaluation of the dissociation rate constant (kd = 0.004 s(-1)). PMID:15296384

  10. Triplex-directed recognition of a DNA nanostructure assembled by crossover strand exchange.

    PubMed

    Rusling, David A; Nandhakumar, Iris S; Brown, Tom; Fox, Keith R

    2012-04-24

    DNA has been widely exploited for the self-assembly of nanosized objects and arrays that offer the potential to act as scaffolds for the spatial positioning of molecular components with nanometer precision. Methods that allow the targeting of components to specific locations within these structures are therefore highly sought after. Here we report that the triplex approach to DNA recognition, which relies on the specific binding of an oligonucleotide within the major groove of double-helical DNA, can be exploited to recognize specific loci within a DNA double-crossover tile and array, a nanostructure assembled by crossover strand exchange. The oligonucleotide can be targeted to both crossover and non-crossover strands and, surprisingly, across the region spanning the crossover junction itself. Moreover, by attaching biotin to the end of the oligonucleotide, we show that streptavidin molecules can be recruited to precise locations within a DX array, with an average spacing of 31.9 (±1.3) nm. This is a promising approach that could be exploited to introduce other components compatible with oligonucleotide synthesis into the wide variety of DNA nanostructures assembled by crossover strand exchange, such as those generated by DNA origami. PMID:22443318

  11. Electric Field Directed Collection and Metering of DNA in Microfluidic Devices

    NASA Astrophysics Data System (ADS)

    Shaikh, Faisal; Ugaz, Victor

    2004-03-01

    Microfluidic technology is a key component in the development of microfabricated lab-on-a-chip systems for use in bioanalytical and biosensing applications. These devices continue to be developed to perform a variety of DNA analysis assays, however many of these applications deal with such minute amounts of DNA that it must first be pre-concentrated to a detectable level. On the macroscale, this pre-concentration is typically performed using centrifugation processes which are difficult to miniaturize and interface with other microfluidic components. In order to address this issue, we have developed microfluidic devices incorporating arrays of on-chip electrodes to locally increase the concentration of DNA in solution. By applying a low voltage between neighboring electrodes positioned inside a microfluidic channel, the negatively charged DNA fragments are induced to migrate toward and collect the anode, thereby allowing the quantity of accumulated DNA to be precisely metered. We demonstrate the application of this technique in electrophoresis microchips to inject a narrow and well-defined DNA plug into an electrophoresis gel. This loading scheme both increases the concentration of the sample to be separated and significantly reduces the degradation in separation resolution due the size of the injected sample plug.

  12. Lsh Is Essential for Maintaining Global DNA Methylation Levels in Amphibia and Fish and Interacts Directly with Dnmt1

    PubMed Central

    Dunican, Donncha S.; Pennings, Sari; Meehan, Richard R.

    2015-01-01

    Eukaryotic genomes are methylated at cytosine bases in the context of CpG dinucleotides, a pattern which is maintained through cell division by the DNA methyltransferase Dnmt1. Dramatic methylation losses are observed in plant and mouse cells lacking Lsh (lymphoid specific helicase), predominantly at repetitive sequences and gene promoters. However, the mechanism by which Lsh contributes to the maintenance of DNA methylation is unknown. Here we show that DNA methylation is lost in Lsh depleted frog and fish embryos, both of which exhibit developmental delay. Additionally, we show that both Lsh and Dnmt1 are associated with chromatin and that Lsh knockdown leads to a decreased Dnmt1-chromatin association. Coimmunoprecipitation experiments reveal that Lsh and Dnmt1 are found in the same protein complex, and pulldowns show this interaction is direct. Our data indicate that Lsh is usually diffuse in the nucleus but can be recruited to heterochromatin in a HP1α-dependent manner. These data together (a) show that the role of Lsh in DNA methylation is conserved in plants, amphibian, fish, and mice and (b) support a model in which Lsh contributes to Dnmt1 binding to chromatin, explaining how its loss can potentially lead to perturbations in DNA methylation maintenance. PMID:26491684

  13. Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae)

    PubMed Central

    Valencia, Laura Carolina; García, Adriana; Ramírez-Pinilla, Martha Patricia; Fuentes, Jorge Luis

    2011-01-01

    The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies. PMID:22215974

  14. Direct chemical conversion of graphene to boron- and nitrogen- and carbon-containing atomic layers

    NASA Astrophysics Data System (ADS)

    Gong, Yongji; Shi, Gang; Zhang, Zhuhua; Zhou, Wu; Jung, Jeil; Gao, Weilu; Ma, Lulu; Yang, Yang; Yang, Shubin; You, Ge; Vajtai, Robert; Xu, Qianfan; MacDonald, Allan H.; Yakobson, Boris I.; Lou, Jun; Liu, Zheng; Ajayan, Pulickel M.

    2014-01-01

    Graphene and hexagonal boron nitride are typical conductor and insulator, respectively, while their hybrids hexagonal boron carbonitride are promising as a semiconductor. Here we demonstrate a direct chemical conversion reaction, which systematically converts the hexagonal carbon lattice of graphene to boron nitride, making it possible to produce uniform boron nitride and boron carbonitride structures without disrupting the structural integrity of the original graphene templates. We synthesize high-quality atomic layer films with boron-, nitrogen- and carbon-containing atomic layers with full range of compositions. Using this approach, the electrical resistance, carrier mobilities and bandgaps of these atomic layers can be tuned from conductor to semiconductor to insulator. Combining this technique with lithography, local conversion could be realized at the nanometre scale, enabling the fabrication of in-plane atomic layer structures consisting of graphene, boron nitride and boron carbonitride. This is a step towards scalable synthesis of atomically thin two-dimensional integrated circuits.

  15. Potential for geothermal direct use in the greenhouse, lumber, chemical, and potato and onion processing industries

    SciTech Connect

    Bressler, S.E.

    1980-09-01

    It has generally been assumed that rising energy costs in industries with high energy needs for low-temperature process heat will induce increasingly widespread geothermal direct use, so long as technical feasibility and cost advantage can be demonstrated. However, few systematic attempts have been made to determine how industry management and technical personnel within these industries view this possibility in light of factors they deem important to their own firms' energy supply choices. This paper discusses that subject in relation to potential commercial geothermal use in the greenhouse, lumber, chemical, and potato and onion processing industries. It is based upon extensive interviews with decision-makers in over 50 firms representing various segments of these industries and is a selected synthesis of material compiled into reports on each industry.

  16. Fluid modeling for plasma-enhanced direct current chemical vapor deposition

    NASA Astrophysics Data System (ADS)

    Ismagilov, Rinat R.; Khamidullin, Ildar R.; Kleshch, Victor I.; Malykhin, Sergei A.; Alexeev, Andrey M.; Obraztsov, Alexander N.

    2016-01-01

    A self-consistent continuum (fluid) model for a direct current discharge used in a chemical vapor deposition system is developed. The model is built for a two-dimensional axisymmetric system and incorporates an electron energy balance for low-pressure Ar gas. The underlying physics of the fluid model is briefly discussed. The fluid and Poisson equations for plasma species are used as the model background. The plasma species considered in the model include electrons, Ar+ ions, and Ar atoms in ground and excited states. Nine reactions between these species are taken into account, including surface reactions. The densities of various plasma species as well as the relative contributions of generation and annihilation processes for electrons, ions, and atoms are calculated. The concentrations for electrons and Ar+ ions on the order of 1020 m-3 are obtained for the plasma in the computer simulations.

  17. Correlation of Chemical Bond Directions and Functional Group Orientations in Solids by Two-Dimensional NMR

    NASA Astrophysics Data System (ADS)

    Weliky, D. P.; Dabbagh, G.; Tycko, R.

    We describe a new two-dimensional NMR technique for structural studies of polycrystalline and noncrystalline solids. The technique is a variant of 2D exchange spectroscopy applicable to organic molecules, macromolecules, or molecular complexes that are doubly 13C-labeled at a specific carboncarbon bond and singly 13C labeled at a specific functional group. A Carr-Purcell sequence is used to obtain dipolar spectra in the t1 dimension. Spectra in the t2 dimension are determined primarily by the chemical-shift anisotropy. With spin diffusion among the labeled sites between the t1 and t2 periods, the resulting 2D spectra reveal correlations between the direction of the labeled bond and the orientation of the labeled functional group. Experimental spectra of two polycrystalline model compounds, dimethyl succinate and diammonium succinate, are presented and compared with simulations to illustrate the structural information contained in the 2D spectra.

  18. Direct computation of general chemical energy differences: Application to ionization potentials, excitation, and bond energies

    SciTech Connect

    Beste, Ariana; Harrison, Robert J; Yanai, Takeshi

    2006-01-01

    Chemists are mainly interested in energy differences. In contrast, most quantum chemical methods yield the total energy which is a large number compared to the difference and has therefore to be computed to a higher relative precision than would be necessary for the difference alone. Hence, it is desirable to compute energy differences directly, thereby avoiding the precision problem. Whenever it is possible to find a parameter which transforms smoothly from an initial to a final state, the energy difference can be obtained by integrating the energy derivative with respect to that parameter (c.f., thermodynamic integration or adiabatic connection methods). If the dependence on the parameter is predominantly linear, accurate results can be obtained by single-point integration. In density functional theory (DFT) and Hartree-Fock, we applied the formalism to ionization potentials, excitation energies, and chemical bond breaking. Example calculations for ionization potentials and excitation energies showed that accurate results could be obtained with a linear estimate. For breaking bonds, we introduce a non-geometrical parameter which gradually turns the interaction between two fragments of a molecule on. The interaction changes the potentials used to determine the orbitals as well as constraining the orbitals to be orthogonal.

  19. Bio- chemical and physical characterizations of mesenchymal stromal cells along the time course of directed differentiation

    PubMed Central

    Chen, Yin-Quan; Liu, Yi-Shiuan; Liu, Yu-An; Wu, Yi-Chang; del Álamo, Juan C.; Chiou, Arthur; Lee, Oscar K.

    2016-01-01

    Cellular biophysical properties are novel biomarkers of cell phenotypes which may reflect the status of differentiating stem cells. Accurate characterizations of cellular biophysical properties, in conjunction with the corresponding biochemical properties could help to distinguish stem cells from primary cells, cancer cells, and differentiated cells. However, the correlated evolution of these properties in the course of directed stem cells differentiation has not been well characterized. In this study, we applied video particle tracking microrheology (VPTM) to measure intracellular viscoelasticity of differentiating human mesenchymal stromal/stem cells (hMSCs). Our results showed that osteogenesis not only increased both elastic and viscous moduli, but also converted the intracellular viscoelasticity of differentiating hMSCs from viscous-like to elastic-like. In contrast, adipogenesis decreased both elastic and viscous moduli while hMSCs remained viscous-like during the differentiation. In conjunction with bio- chemical and physical parameters, such as gene expression profiles, cell morphology, and cytoskeleton arrangement, we demonstrated that VPTM is a unique approach to quantify, with high data throughput, the maturation level of differentiating hMSCs and to anticipate their fate decisions. This approach is well suited for time-lapsed study of the mechanobiology of differentiating stem cells especially in three dimensional physico-chemical biomimetic environments including porous scaffolds. PMID:27526936

  20. Direct chemical grafted curcumin on halloysite nanotubes as dual-responsive prodrug for pharmacological applications.

    PubMed

    Massaro, M; Amorati, R; Cavallaro, G; Guernelli, S; Lazzara, G; Milioto, S; Noto, R; Poma, P; Riela, S

    2016-04-01

    Covalently functionalized halloysite nanotubes (HNTs) were successfully employed as dual-responsive nanocarriers for curcumin (Cur). Particularly, we synthesized HNT-Cur prodrug with a controlled curcumin release on dependence of both intracellular glutathione (GSH) and pH conditions. In order to obtain HNT-Cur produgs, halloysite was firstly functionalized with cysteamine through disulphide linkage. Afterwards, curcumin molecules were chemically conjugated to the amino end groups of halloysite via Schiff's base formation. The successful functionalization of halloysite was proved by thermogravimetric analysis, FT-IR spectroscopy, dynamic light scattering and scanning electron microscopy. Experimental data confirmed the presence of curcumin on HNT external surface. Moreover, we investigated the kinetics of curcumin release by UV-vis spectroscopy, which highlighted that HNT-Cur prodrug possesses dual stimuli-responsive ability upon exposure to GSH-rich or acidic environment. In vitro antiproliferative and antioxidant properties of HNT-Cur prodrug were studied with the aim to explore their potential applications in pharmaceutics. This work puts forward an efficient strategy to prepare halloysite based nanocarriers with controlled drug delivery capacity through direct chemical grafting with stimuli-responsive linkage. PMID:26812638

  1. Reaction ensemble molecular dynamics: Direct simulation of the dynamic equilibrium properties of chemically reacting mixtures

    NASA Astrophysics Data System (ADS)

    Brennan, John K.; Lísal, Martin; Gubbins, Keith E.; Rice, Betsy M.

    2004-12-01

    A molecular simulation method to study the dynamics of chemically reacting mixtures is presented. The method uses a combination of stochastic and dynamic simulation steps, allowing for the simulation of both thermodynamic and transport properties. The method couples a molecular dynamics simulation cell (termed dynamic cell) to a reaction mixture simulation cell (termed control cell) that is formulated upon the reaction ensemble Monte Carlo (RxMC) method, hence the term reaction ensemble molecular dynamics. Thermodynamic and transport properties are calculated in the dynamic cell by using a constant-temperature molecular dynamics simulation method. RxMC forward and reverse reaction steps are performed in the control cell only, while molecular dynamics steps are performed in both the dynamic cell and the control cell. The control cell, which acts as a sink and source reservoir, is maintained at reaction equilibrium conditions via the RxMC algorithm. The reaction ensemble molecular dynamics method is analogous to the grand canonical ensemble molecular dynamics technique, while using some elements of the osmotic molecular dynamics method, and so simulates conditions that directly relate to real, open systems. The accuracy and stability of the method is assessed by considering the ammonia synthesis reaction N2+3H2⇔2NH3 . It is shown to be a viable method for predicting the effects of nonideal environments on the dynamic properties (particularly diffusion) as well as reaction equilibria for chemically reacting mixtures.

  2. Bio- chemical and physical characterizations of mesenchymal stromal cells along the time course of directed differentiation.

    PubMed

    Chen, Yin-Quan; Liu, Yi-Shiuan; Liu, Yu-An; Wu, Yi-Chang; Del Álamo, Juan C; Chiou, Arthur; Lee, Oscar K

    2016-01-01

    Cellular biophysical properties are novel biomarkers of cell phenotypes which may reflect the status of differentiating stem cells. Accurate characterizations of cellular biophysical properties, in conjunction with the corresponding biochemical properties could help to distinguish stem cells from primary cells, cancer cells, and differentiated cells. However, the correlated evolution of these properties in the course of directed stem cells differentiation has not been well characterized. In this study, we applied video particle tracking microrheology (VPTM) to measure intracellular viscoelasticity of differentiating human mesenchymal stromal/stem cells (hMSCs). Our results showed that osteogenesis not only increased both elastic and viscous moduli, but also converted the intracellular viscoelasticity of differentiating hMSCs from viscous-like to elastic-like. In contrast, adipogenesis decreased both elastic and viscous moduli while hMSCs remained viscous-like during the differentiation. In conjunction with bio- chemical and physical parameters, such as gene expression profiles, cell morphology, and cytoskeleton arrangement, we demonstrated that VPTM is a unique approach to quantify, with high data throughput, the maturation level of differentiating hMSCs and to anticipate their fate decisions. This approach is well suited for time-lapsed study of the mechanobiology of differentiating stem cells especially in three dimensional physico-chemical biomimetic environments including porous scaffolds. PMID:27526936

  3. Direct computation of general chemical energy differences: Application to ionization potentials, excitation, and bond energies

    NASA Astrophysics Data System (ADS)

    Beste, A.; Harrison, R. J.; Yanai, T.

    2006-08-01

    Chemists are mainly interested in energy differences. In contrast, most quantum chemical methods yield the total energy which is a large number compared to the difference and has therefore to be computed to a higher relative precision than would be necessary for the difference alone. Hence, it is desirable to compute energy differences directly, thereby avoiding the precision problem. Whenever it is possible to find a parameter which transforms smoothly from an initial to a final state, the energy difference can be obtained by integrating the energy derivative with respect to that parameter (cf. thermodynamic integration or adiabatic connection methods). If the dependence on the parameter is predominantly linear, accurate results can be obtained by single-point integration. In density functional theory and Hartree-Fock, we applied the formalism to ionization potentials, excitation energies, and chemical bond breaking. Example calculations for ionization potentials and excitation energies showed that accurate results could be obtained with a linear estimate. For breaking bonds, we introduce a nongeometrical parameter which gradually turns the interaction between two fragments of a molecule on. The interaction changes the potentials used to determine the orbitals as well as the constraint on the orbitals to be orthogonal.

  4. Chemical form of selenium differentially influences DNA repair pathways following exposure to lead nitrate.

    PubMed

    McKelvey, Shauna M; Horgan, Karina A; Murphy, Richard A

    2015-01-01

    Lead, an environmental toxin is known to induce a broad range of physiological and biochemical dysfunctions in humans through a number of mechanisms including the deactivation of antioxidants thus leading to generation of reactive oxygen species (ROS) and subsequent DNA damage. Selenium on the other hand has been proven to play an important role in the protection of cells from free radical damage and oxidative stress, though its effects are thought to be form and dose dependent. As the liver is the primary organ required for metabolite detoxification, HepG2 cells were chosen to assess the protective effects of various selenium compounds following exposure to the genotoxic agent lead nitrate. Initially DNA damage was quantified using a comet assay, gene expression patterns associated with DNA damage and signalling were also examined using PCR arrays and the biological pathways which were most significantly affected by selenium were identified. Interestingly, the organic type selenium compounds (selenium yeast and selenomethionine) conferred protection against lead induced DNA damage in HepG2 cells; this is evident by reduction in the quantity of DNA present in the comet tail of cells cultured in their presence with lead. This trend also followed through the gene expression changes noted in DNA damage pathways analysed. These results were in contrast with those of inorganic sodium selenite which promoted lead induced DNA damage evident in both the comet assay results and the gene expression analysis. Over all this study provided valuable insights into the effects which various selenium compounds had on the DNA damage and signalling pathway indicating the potential for using organic forms of selenium such as selenium enriched yeast to protect against DNA damaging agents. PMID:25023848

  5. Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes

    PubMed Central

    2009-01-01

    Background One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive. These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels. The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. Results An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other patients). Methods and assay

  6. Grade 12 French Students' use of a Thermodynamic Model for Predicting the Direction of Incomplete Chemical Changes

    NASA Astrophysics Data System (ADS)

    Kermen, Isabelle; Méheut, Martine

    2011-09-01

    The authors of the current chemistry curriculum-implemented in Grade 12 in France-provided a criterion of change allowing prediction of direction of chemical changes and pointed out the difference to be made between experimental facts and models. A study analysing part of the curriculum content and the effects of teaching this content on students' reasoning was conducted. The content analysis presents the functioning of the thermodynamic model, which highlights the links to be made between the experimental situation and the model when predicting the direction of a chemical change. This functioning specifies the role of the chemical equation and that of the criterion of change (comparing the reaction quotient to the equilibrium constant) and stresses the crucial points that may lead to misunderstandings. Written tests were administered to students after teaching them to determine how they predicted the direction of a chemical change, and whether they made a relevant choice between using the chemical equation and using the criterion of change and a clear distinction between the experimental situation and the thermodynamic model. Few students had a good understanding of the respective roles of the criterion and the chemical equation. A majority used the criterion to predict the direction of chemical changes relevantly, but correct answers were not widespread. Two particular mistakes, the modification of the expression of the reaction quotient and the prediction of a change despite a missing reactant, revealed that students do not properly understand the difference and the relationship between the experimental situation and the thermodynamic model.

  7. Viral terminal protein directs early organization of phage DNA replication at the bacterial nucleoid

    PubMed Central

    Muñoz-Espín, Daniel; Holguera, Isabel; Ballesteros-Plaza, David; Carballido-López, Rut; Salas, Margarita

    2010-01-01

    The mechanism leading to protein-primed DNA replication has been studied extensively in vitro. However, little is known about the in vivo organization of the proteins involved in this fundamental process. Here we show that the terminal proteins (TPs) of phages ϕ29 and PRD1, infecting the distantly related bacteria Bacillus subtilis and Escherichia coli, respectively, associate with the host bacterial nucleoid independently of other viral-encoded proteins. Analyses of phage ϕ29 revealed that the TP N-terminal domain (residues 1–73) possesses sequence-independent DNA-binding capacity and is responsible for its nucleoid association. Importantly, we show that in the absence of the TP N-terminal domain the efficiency of ϕ29 DNA replication is severely affected. Moreover, the TP recruits the phage DNA polymerase to the bacterial nucleoid, and both proteins later are redistributed to enlarged helix-like structures in an MreB cytoskeleton-dependent way. These data disclose a key function for the TP in vivo: organizing the early viral DNA replication machinery at the cell nucleoid. PMID:20823229

  8. The role of DNA methylation in directing the functional organization of the cancer epigenome

    PubMed Central

    Lay, Fides D.; Liu, Yaping; Kelly, Theresa K.; Witt, Heather; Farnham, Peggy J.

    2015-01-01

    The holistic role of DNA methylation in the organization of the cancer epigenome is not well understood. Here we perform a comprehensive, high-resolution analysis of chromatin structure to compare the landscapes of HCT116 colon cancer cells and a DNA methylation-deficient derivative. The NOMe-seq accessibility assay unexpectedly revealed symmetrical and transcription-independent nucleosomal phasing across active, poised, and inactive genomic elements. DNA methylation abolished this phasing primarily at enhancers and CpG island (CGI) promoters, with little effect on insulators and non-CGI promoters. Abolishment of DNA methylation led to the context-specific reestablishment of the poised and active states of normal colon cells, which were marked in methylation-deficient cells by distinct H3K27 modifications and the presence of either well-phased nucleosomes or nucleosome-depleted regions, respectively. At higher-order genomic scales, we found that long, H3K9me3-marked domains had lower accessibility, consistent with a more compact chromatin structure. Taken together, our results demonstrate the nuanced and context-dependent role of DNA methylation in the functional, multiscale organization of cancer epigenomes. PMID:25747664

  9. PEA15 Regulates the DNA Damage-Induced Cell Cycle Checkpoint and Oncogene-Directed Transformation

    PubMed Central

    Nagarajan, Arvindhan; Dogra, Shaillay Kumar; Liu, Alex Y.; Green, Michael R.

    2014-01-01

    Regulation of the DNA damage response and cell cycle progression is critical for maintaining genome integrity. Here, we report that in response to DNA damage, COPS5 deubiquitinates and stabilizes PEA15 in an ATM kinase-dependent manner. PEA15 expression oscillates throughout the cell cycle, and the loss of PEA15 accelerates cell cycle progression by activating CDK6 expression via the c-JUN transcription factor. Cells lacking PEA15 exhibit a DNA damage-induced G2/M checkpoint defect due to increased CDC25C activity and, consequentially, higher cyclin-dependent kinase 1 (CDK1)/cyclin B activity, and accordingly they have an increased rate of spontaneous mutagenesis. We find that oncogenic RAS inhibits PEA15 expression and that ectopic PEA15 expression blocks RAS-mediated transformation, which can be partially rescued by ectopic expression of CDK6. Finally, we show that PEA15 expression is downregulated in colon, breast, and lung cancer samples. Collectively, our results demonstrate that tumor suppressor PEA15 is a regulator of genome integrity and is an integral component of the DNA damage response pathway that regulates cell cycle progression, the DNA-damage-induced G2/M checkpoint, and cellular transformation. PMID:24710276

  10. PEA15 regulates the DNA damage-induced cell cycle checkpoint and oncogene-directed transformation.

    PubMed

    Nagarajan, Arvindhan; Dogra, Shaillay Kumar; Liu, Alex Y; Green, Michael R; Wajapeyee, Narendra

    2014-06-01

    Regulation of the DNA damage response and cell cycle progression is critical for maintaining genome integrity. Here, we report that in response to DNA damage, COPS5 deubiquitinates and stabilizes PEA15 in an ATM kinase-dependent manner. PEA15 expression oscillates throughout the cell cycle, and the loss of PEA15 accelerates cell cycle progression by activating CDK6 expression via the c-JUN transcription factor. Cells lacking PEA15 exhibit a DNA damage-induced G2/M checkpoint defect due to increased CDC25C activity and, consequentially, higher cyclin-dependent kinase 1 (CDK1)/cyclin B activity, and accordingly they have an increased rate of spontaneous mutagenesis. We find that oncogenic RAS inhibits PEA15 expression and that ectopic PEA15 expression blocks RAS-mediated transformation, which can be partially rescued by ectopic expression of CDK6. Finally, we show that PEA15 expression is downregulated in colon, breast, and lung cancer samples. Collectively, our results demonstrate that tumor suppressor PEA15 is a regulator of genome integrity and is an integral component of the DNA damage response pathway that regulates cell cycle progression, the DNA-damage-induced G2/M checkpoint, and cellular transformation. PMID:24710276

  11. Comparison of two freshwater turtle species as monitors of radionuclide and chemical contamination: DNA damage and residue analysis

    SciTech Connect

    Meyers-Schoene, L. ); Shugart, L.R.; Beauchamp, J.J.; Walton, B.T. )

    1993-08-01

    Two species of turtles that occupy different ecological niches were compared for their usefulness as monitors of freshwater ecosystems where both low-level radioactive and nonradioactive contaminants are present. The pond slider (Trachemys scripta) and common snapping turtle (Chelydra serpentina) were analyzed for the presence of [sup 90]Sr, [sup 137]Cs, [sup 60]Co, and Hg, radionuclides and chemicals known to be present at the contaminated site, and single-strand breaks in liver DNA. The integrity of the DNA was examined by the alkaline unwinding assay, a technique that detects strand breaks as a biological marker of possible exposure to genotoxic agents. This measure of DNA damage was significantly increased in both species of turtles at the contaminated site compared with turtles of the same species at a reference site, and shows that contaminant-exposed populations were under more severe genotoxic stress than those at the reference site. The level of strand breaks observed at the contaminated site was high and in the range reported for other aquatic species exposed to deleterious concentrations of genotoxic agents such as chemicals and ionizing radiation. Statistically significantly higher concentrations of radionuclides and Hg were detected in the turtles from the contaminated area. Mercury concentrations were significantly higher in the more carnivorous snapping turtle compared with the slider; however, both species were effective monitors of the contaminants.

  12. Induced mutagenesis of plasmid and chromosomal genes inserted into the plasmid DNA. II. Mutagenic action of chemical factors

    SciTech Connect

    Esipova, V.V.; Vedunova, S.L.; Kriviskii, A.S.

    1986-02-01

    Following the study of the mutagenic action of UV and ..gamma..-radiation on plasmid DNA in vitro, they investigated the induction of mutations under the influence of chemical mutagens on the same DNA of plasmid RSF2124, determining the synthesis of colicine E1 and resistance to ampicillin. The inactivating action of the mutagen was assessed from the yield of transformants resistant to the antibiotic and the mutagenic effect from the loss by colonies of transformants that were capable of releasing colicine into the external medium. In these experiments they mainly used chemical compounds whose mutagenic effect if well known in other systems (transforming and transfecting DNA, microbial viruses). As a result all mutagens tested for their activity were divided into four groups: first group, those exceeding the level of mutagenesis by more than 100-fold above the spontaneous background (hydroxylamine, O-methylhydroxylamine); second group, those exceeding it by a factor of 10 (UV radiation (lambda = 254 nm), W-mutagenesis, ionizing radiation, nitrous acid, mitomycin C); third group, those exceeding it by a factor of <10 (indirect UV mutagenesis, nitrous acid, ..beta..-chloroethyldiethylamine hydrochloride, nitrosoguanidine); fourth group, no mutagenic effect (acridine orange, ethyl methane sulfonate, sodium azide, 0-..beta..-diethylaminoethylhydroxylamine).

  13. Synthetic Polymer Hybridization with DNA and RNA Directs Nanoparticle Loading, Silencing Delivery, and Aptamer Function

    PubMed Central

    Zhou, Zhun; Xia, Xin; Bong, Dennis

    2015-01-01

    We report herein discrete triplex hybridization of DNA and RNA with polyacrylates. Length-monodisperse triazine-derivatized polymers were prepared on gram-scale by reversible addition–fragmentation chain-transfer polymerization. Despite stereoregio backbone heterogeneity, the triazine polymers bind T/U-rich DNA or RNA with nanomolar affinity upon mixing in a 1:1 ratio, as judged by thermal melts, circular dichroism, gel-shift assays, and fluorescence quenching. We call these polyacrylates “bifacial polymer nucleic acids” (bPoNAs). Nucleic acid hybridization with bPoNA enables DNA loading onto polymer nanoparticles, siRNA silencing delivery, and can further serve as an allosteric trigger of RNA aptamer function. Thus, bPoNAs can serve as tools for both non-covalent bioconjugation and structure–function nucleation. It is anticipated that bPoNAs will have utility in both bio- and nanotechnology. PMID:26138550

  14. Synthetic Polymer Hybridization with DNA and RNA Directs Nanoparticle Loading, Silencing Delivery, and Aptamer Function.

    PubMed

    Zhou, Zhun; Xia, Xin; Bong, Dennis

    2015-07-22

    We report herein discrete triplex hybridization of DNA and RNA with polyacrylates. Length-monodisperse triazine-derivatized polymers were prepared on gram-scale by reversible addition-fragmentation chain-transfer polymerization. Despite stereoregio backbone heterogeneity, the triazine polymers bind T/U-rich DNA or RNA with nanomolar affinity upon mixing in a 1:1 ratio, as judged by thermal melts, circular dichroism, gel-shift assays, and fluorescence quenching. We call these polyacrylates "bifacial polymer nucleic acids" (bPoNAs). Nucleic acid hybridization with bPoNA enables DNA loading onto polymer nanoparticles, siRNA silencing delivery, and can further serve as an allosteric trigger of RNA aptamer function. Thus, bPoNAs can serve as tools for both non-covalent bioconjugation and structure-function nucleation. It is anticipated that bPoNAs will have utility in both bio- and nanotechnology. PMID:26138550

  15. Directed evolution of novel polymerase activities: Mutation of a DNA polymerase into an efficient RNA polymerase

    PubMed Central

    Xia, Gang; Chen, Liangjing; Sera, Takashi; Fa, Ming; Schultz, Peter G.; Romesberg, Floyd E.

    2002-01-01

    The creation of novel enzymatic function is of great interest, but remains a challenge because of the large sequence space of proteins. We have developed an activity-based selection method to evolve DNA polymerases with RNA polymerase activity. The Stoffel fragment (SF) of Thermus aquaticus DNA polymerase I is displayed on a filamentous phage by fusing it to a pIII coat protein, and the substrate DNA template/primer duplexes are attached to other adjacent pIII coat proteins. Phage particles displaying SF polymerases, which are able to extend the attached oligonucleotide primer by incorporating ribonucleoside triphosphates and biotinylated UTP, are immobilized to streptavidin-coated magnetic beads and subsequently recovered. After four rounds of screening an SF library, three SF mutants were isolated and shown to incorporate ribonucleoside triphosphates virtually as efficiently as the wild-type enzyme incorporates dNTP substrates. PMID:12011423

  16. A sensitive spectrofluorometric method for detection of berberine hydrochloride using Ag nanoclusters directed by natural fish sperm DNA.

    PubMed

    Liang, Sheng; Kuang, Yangfang; Ma, Fangfang; Chen, Shu; Long, Yunfei

    2016-11-15

    A novel DNA-directed AgNCs (DNA-AgNCs) was synthesized with economical raw material (natural fish sperm DNA) through a simple and rapid approach, and it first showed high and stable fluorescence emission as a AgNCs stabilized by natural DNA at about 635nm. Moreover, its emission intensity could be enhanced tremendously in acetic acid (HAc) medium. Whereas, when berberine hydrochloride (BRH) entered the solution system, it would interact and combine efficiently with DNA on the surface of AgNCs, which could lead to subtle change of charge distribution on its surface, and make it more lyophobic, inducing aggregation of DNA-AgNCs. As a result, fluorescence of the system was quenched visually; the process represented a color variance from yellow to hot pink under HAc medium, then back to yellowish-brown when BRH worked. Based on above phenomenon, a selective and accurate spectrofluorometric method for BRH detection was established. It can be applied to detect trace amounts of BRH in aqueous solution in the linear range from 1.0nM to 2000.0nM; and the detection limit (3σ/k) was 0.3nM, which is pretty lower compared to most reported spectral methods. Simultaneously, a semi-quantitative determination by visual evaluation from 5.0nM to 2000.0nM was also achieved. This method provided excellent selectivity for the detection of BRH in the presence of ten kinds of common natural amino acids and nine kinds of common mental ions. Furthermore, the BRH content in compound berberine tablets from drugstore was successfully investigated by this method and the results showed high accuracy. PMID:27266661

  17. Direct Quantification of DNA Base Composition by Surface-Enhanced Raman Scattering Spectroscopy.

    PubMed

    Morla-Folch, Judit; Alvarez-Puebla, Ramon A; Guerrini, Luca

    2016-08-01

    Design of ultrasensitive DNA sensors based on the unique physical properties of plasmonic nanostructures has become one of the most exciting areas in nanomedicine. However, despite the vast number of proposed applications, the determination of the base composition in nucleic acids, a fundamental parameter in genomic analyses and taxonomic classification, is still restricted to time-consuming and poorly sensitive conventional methods. Herein, we demonstrate the possibility of determining the base composition in single- and double-stranded DNA by using a simple, low-cost, high-throughput, and label-free surface-enhanced Raman scattering (SERS) method in combination with cationic nanoparticles. PMID:27441814

  18. Direct Visualization of Etching Trajectories in Metal-Assisted Chemical Etching of Si by the Chemical Oxidation of Porous Sidewalls.

    PubMed

    Yoon, Sung-Soo; Khang, Dahl-Young

    2015-09-29

    We demonstrate a simple method for the visualization of trajectories traced by noble metal nanoparticles during metal-assisted chemical etching (MaCE) of Si. The nanoporous Si layer formed around drilled pores is converted into SiO2 by simple chemical oxidation. Etch removal of the remaining Si using alkali hydroxide leaves SiO2 nanostructures that are the exact replica of those drilled pores or etching trajectories. The differences in etching characteristics between Ag and Au have been investigated using the proposed visualization method. The shape and chemical stability of metal nanoparticles used for MaCE have been found to be critical in determining etching paths. The proposed method would be very helpful in studying the fundamental mechanism of MaCE as well as in micro/nanostructuring of the Si surface for various applications. This approach can also be used for the generation of straight or helical SiO2 nanotubes. PMID:26352208

  19. Direct formation of the C5'-radical in the sugar-phosphate backbone of DNA by high-energy radiation.

    PubMed

    Adhikary, Amitava; Becker, David; Palmer, Brian J; Heizer, Alicia N; Sevilla, Michael D

    2012-05-24

    Neutral sugar radicals formed in DNA sugar-phosphate backbone are well-established as precursors of biologically important damage such as DNA strand scission and cross-linking. In this work, we present electron spin resonance (ESR) evidence showing that the sugar radical at C5' (C5'(•)) is one of the most abundant (ca. 30%) sugar radicals formed by γ- and Ar ion-beam irradiated hydrated DNA samples. Taking dimethyl phosphate as a model of sugar-phosphate backbone, ESR and theoretical (DFT) studies of γ-irradiated dimethyl phosphate were carried out. CH(3)OP(O(2)(-))OCH(2)(•) is formed via deprotonation from the methyl group of directly ionized dimethyl phosphate at 77 K. The formation of CH(3)OP(O(2)(-))OCH(2)(•) is independent of dimethyl phosphate concentration (neat or in aqueous solution) or pH. ESR spectra of C5'(•) found in DNA and of CH(3)OP(O(2)(-))OCH(2)(•) do not show an observable β-phosphorus hyperfine coupling (HFC). Furthermore, C5'(•) found in DNA does not show a significant C4'-H β-proton HFC. Applying the DFT/B3LYP/6-31G(d) method, a study of conformational dependence of the phosphorus HFC in CH(3)OP(O(2)(-))OCH(2)(•) shows that in its minimum energy conformation, CH(3)OP(O(2)(-))OCH(2)(•), has a negligible β-phosphorus HFC. On the basis of these results, the formation of radiation-induced C5'(•) is proposed to occur via a very rapid deprotonation from the directly ionized sugar-phosphate backbone, and the rate of this deprotonation must be faster than that of energetically downhill transfer of the unpaired spin (hole) from ionized sugar-phosphate backbone to the DNA bases. Moreover, C5'(•) in irradiated DNA is found to be in a conformation that does not exhibit β-proton or β-phosphorus HFCs. PMID:22553971

  20. DNA-Origami-Directed Self-Assembly of Discrete Silver-Nanoparticle Architectures

    SciTech Connect

    Pal, Suchetan; Deng, Zhengtao; Ding, Baoquan; Yan, Hao; Liu, Yan

    2010-03-16

    We report a bottom-up method for the fabrication of discrete, well-ordered AgNP nanoarchitectures on self-assembled DNA origami structures of triangular shape by using AgNPs (20 nm in diameter) conjugated with chimeric phosphorothioated DNA (ps-po DNA) as building blocks. Discrete monomeric, dimeric, and trimeric AgNP structures and a AgNP–AuNP hybrid structure could be constructed reliably in high yield. We demonstrate that the center-to-center distance between adjacent AgNPs can be precisely tuned from 94 to 29 nm, whereby the distance distribution is limited by the size distribution of the nanoparticles. The self-assembly of discrete AgNP and AgNP–AuNP nanoarchitectures by using rationally designed DNA templates enabled us to control some of the properties that are essential for hierarchical nanoparticle assembly. These properties include but are not limited to the spatial relationship between the particles and the identity of the particles. The system described herein could potentially be used to gain better insight into particle–particle interactions. Systematic studies with this objective are underway. Although more systematic investigations (e.g. spectroscopic studies combined with theoretical simulation of the assembled structures) are needed to identify the photonic properties of the spatially controlled AgNP architectures, we see no fundamental limitation now to the assembly of target structures.

  1. DNA abasic site-directed formation of fluorescent silver nanoclusters for selective nucleobase recognition

    NASA Astrophysics Data System (ADS)

    Ma, Kun; Cui, Qinghua; Liu, Guiying; Wu, Fei; Xu, Shujuan; Shao, Yong

    2011-07-01

    DNA single-nucleotide polymorphism (SNP) detection has attracted much attention due to mutation related diseases. Various methods for SNP detection have been proposed and many are already in use. Here, we find that the abasic site (AP site) in the DNA duplex can be developed as a capping scaffold for the generation of fluorescent silver nanoclusters (Ag NCs). As a proof of concept, the DNA sequences from fragments near codon 177 of cancer supression gene p53 were used as a model for SNP detection by in situ formed Ag NCs. The formation of fluorescent Ag NCs in the AP site-containing DNA duplex is highly selective for cytosine facing the AP site and guanines flanking the site and can be employed in situ as readout for SNP detection. The fluorescent signal-on sensing for SNP based on this inorganic fluorophore is substantially advantageous over the previously reported signal-off responses using low-molecular-weight organic ligands. The strong dependence of fluorescent Ag NC formation on the sequences surrounding the AP site was successfully used to identify mutations in codon 177 of cancer supression gene p53. We anticipate that this approach will be employed to develop a practical SNP detection method by locating an AP site toward the midway cytosine in a target strand containing more than three consecutive cytosines.

  2. Information transfer from DNA to peptide nucleic acids by template-directed syntheses

    NASA Technical Reports Server (NTRS)

    Schmidt, J. G.; Christensen, L.; Nielsen, P. E.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1997-01-01

    Peptide nucleic acids (PNAs) are analogs of nucleic acids in which the ribose-phosphate backbone is replaced by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. Oligocytidylates have been shown to act as templates for formation of longer oligomers of G from PNA G2 dimers. In this paper we show that information can be transferred from DNA to PNA. DNA C4T2C4 is an efficient template for synthesis of PNA G4A2G4 using G2 and A2 units as substrates. The corresponding synthesis of PNA G4C2G4 on DNA C4G2C4 is less efficient. Incorporation of PNA T2 into PNA products on DNA C4A2C4 is the least efficient of the three reactions. These results, obtained using PNA dimers as substrates, parallel those obtained using monomeric activated nucleotides.

  3. Characterization of Heme-DNA Complexes Composed of Some Chemically Modified Hemes and Parallel G-Quadruplex DNAs.

    PubMed

    Yamamoto, Yasuhiko; Kinoshita, Masashi; Katahira, Yuya; Shimizu, Haruna; Di, Yue; Shibata, Tomokazu; Tai, Hulin; Suzuki, Akihiro; Neya, Saburo

    2015-12-15

    Heme {Fe(II)- or Fe(III)-protoporphyrin IX complex [heme(Fe(2+)) or heme(Fe(3+)), respectively]} binds selectively to the 3'-terminal G-quartet of a parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), through a π-π stacking interaction between the porphyrin moiety of the heme and the G-quartet. The binding affinities of some chemically modified hemes(Fe(3+)) for DNA and the structures of complexes between the modified hemes(Fe(2+)) and DNA, with carbon monoxide (CO) coordinated to the heme Fe atom on the side of the heme opposite the G6 G-quartet, have been characterized to elucidate the interaction between the heme and G-quartet in the complexes through analysis of the effects of the heme modification on the structural properties of the complex. The study revealed that the binding affinities and structures of the complexes were barely affected by the heme modification performed in the study. Such plasticity in the binding of heme to the G-quartet is useful for the versatile design of the complex through heme chemical modification and DNA sequence alteration. Furthermore, exchangeable proton signals exhibiting two-proton intensity were observed at approximately -3.5 ppm in the (1)H nuclear magnetic resonance (NMR) spectra of the CO adducts of the complexes. Through analysis of the NMR results, together with theoretical consideration, we concluded that the heme(Fe(2+)) axial ligand trans to CO in the complex is a water molecule (H2O). Identification of the Fe-bound H2O accommodated between the heme and G-quartet planes in the complex provides new insights into the structure-function relationship of the complex. PMID:26595799

  4. Direct real-time molecular scale visualisation of the degradation of condensed DNA complexes exposed to DNase I

    PubMed Central

    Abdelhady, Hosam G.; Allen, Stephanie; Davies, Martyn C.; Roberts, Clive J.; Tendler, Saul J. B.; Williams, Philip M.

    2003-01-01

    The need to protect DNA from in vivo degradation is one of the basic tenets of therapeutic gene delivery and a standard test for any proposed delivery vector. The currently employed in vitro tests, however, presently provide no direct link between the molecular structure of the vector complexes and their success in this role, thus hindering the rational design of successful gene delivery agents. Here we apply atomic force microscopy (AFM) in liquid to visualise at the molecular scale and in real time, the effect of DNase I on generation 4 polyamidoamine dendrimers (G4) complexed with DNA. These complexes are revealed to be dynamic in nature showing a degree of mobility, in some cases revealing the addition and loss of dendrimers to individual complexes. The formation of the G4–DNA complexes is observed to provide a degree of protection to the DNA. This protection is related to the structural morphology of the formed complex, which is itself shown to be dependent on the dendrimer loading and the time allowed for complex formation. PMID:12853616

  5. HDA6 Directly Interacts with DNA Methyltransferase MET1 and Maintains Transposable Element Silencing in Arabidopsis1[W][OA

    PubMed Central

    Liu, Xuncheng; Yu, Chun-Wei; Duan, Jun; Luo, Ming; Wang, Koching; Tian, Gang; Cui, Yuhai; Wu, Keqiang

    2012-01-01

    The molecular mechanism of how the histone deacetylase HDA6 participates in maintaining transposable element (TE) silencing in Arabidopsis (Arabidopsis thaliana) is not yet defined. In this study, we show that a subset of TEs was transcriptionally reactivated and that TE reactivation was associated with elevated histone H3 and H4 acetylation as well as increased H3K4Me3 and H3K4Me2 in hda6 mutants. Decreased DNA methylation of the TEs was also detected in hda6 mutants, suggesting that HDA6 silences the TEs by regulating histone acetylation and methylation as well as the DNA methylation status of the TEs. Similarly, transcripts of some of these TEs were also increased in the methyltransferase1 (met1) mutant, with decreased DNA methylation. Furthermore, H4 acetylation, H3K4Me3, H3K4Me2, and H3K36Me2 were enriched at the coregulated TEs in the met1 and hda6 met1 mutants. Protein-protein interaction analysis indicated that HDA6 physically interacts with MET1 in vitro and in vivo, and further deletion analysis demonstrated that the carboxyl-terminal region of HDA6 and the bromo-adjacent homology domain of MET1 were responsible for the interaction. These results suggested that HDA6 and MET1 interact directly and act together to silence TEs by modulating DNA methylation, histone acetylation, and histone methylation status. PMID:21994348

  6. Nanotechnology in plant disease management: DNA-directed silver nanoparticles on graphene oxide as an antibacterial against Xanthomonas perforans.

    PubMed

    Ocsoy, Ismail; Paret, Mathews L; Ocsoy, Muserref Arslan; Kunwar, Sanju; Chen, Tao; You, Mingxu; Tan, Weihong

    2013-10-22

    Bacterial spot caused by Xanthomonas perforans is a major disease of tomatoes, leading to reduction in production by 10-50%. While copper (Cu)-based bactericides have been used for disease management, most of the X. perforans strains isolated from tomatoes in Florida and other locations worldwide are Cu-resistant. We have developed DNA-directed silver (Ag) nanoparticles (NPs) grown on graphene oxide (GO). These Ag@dsDNA@GO composites effectively decrease X. perforans cell viability in culture and on plants. At the very low concentration of 16 ppm of Ag@dsDNA@GO, composites show excellent antibacterial capability in culture with significant advantages in improved stability, enhanced antibacterial activity, and stronger adsorption properties. Application of Ag@dsDNA@GO at 100 ppm on tomato transplants in a greenhouse experiment significantly reduced the severity of bacterial spot disease compared to untreated plants, giving results similar to those of the current grower standard treatment, with no phytotoxicity. PMID:24016217

  7. Direct extraction of genomic DNA from maize with aqueous ionic liquid buffer systems for applications in genetically modified organisms analysis.

    PubMed

    Gonzalez García, Eric; Ressmann, Anna K; Gaertner, Peter; Zirbs, Ronald; Mach, Robert L; Krska, Rudolf; Bica, Katharina; Brunner, Kurt

    2014-12-01

    To date, the extraction of genomic DNA is considered a bottleneck in the process of genetically modified organisms (GMOs) detection. Conventional DNA isolation methods are associated with long extraction times and multiple pipetting and centrifugation steps, which makes the entire procedure not only tedious and complicated but also prone to sample cross-contamination. In recent times, ionic liquids have emerged as innovative solvents for biomass processing, due to their outstanding properties for dissolution of biomass and biopolymers. In this study, a novel, easily applicable, and time-efficient method for the direct extraction of genomic DNA from biomass based on aqueous-ionic liquid solutions was developed. The straightforward protocol relies on extraction of maize in a 10 % solution of ionic liquids in aqueous phosphate buffer for 5 min at room temperature, followed by a denaturation step at 95 °C for 10 min and a simple filtration to remove residual biopolymers. A set of 22 ionic liquids was tested in a buffer system and 1-ethyl-3-methylimidazolium dimethylphosphate, as well as the environmentally benign choline formate, were identified as ideal candidates. With this strategy, the quality of the genomic DNA extracted was significantly improved and the extraction protocol was notably simplified compared with a well-established method. PMID:25381609

  8. Nanotechnology in Plant Disease Management: DNA-Directed Silver Nanoparticles on Graphene Oxide as an Antibacterial Against Xanthomonas Perforans

    PubMed Central

    Ocsoy, Ismail; Paret, Mathews L.; Ocsoy, Muserref Arslan; Kunwar, Sanju; Chen, Tao; You, Mingxu; Tan, Weihong

    2013-01-01

    Bacterial spot caused by Xanthomonas perforans is a major disease of tomatoes, leading to reduction in production by 10–50%. While copper (Cu)-based bactericides have been used for disease management, most of the X. perforans strains isolated from tomatoes in Florida and other locations worldwide are Cu-resistant. We have developed DNA-directed silver (Ag) nanoparticles (NPs) grown on graphene oxide (GO). These Ag@dsDNA@GO composites effectively decrease X. perforans cell viability in culture and on plants. At the very low concentration of 16 ppm of Ag@dsDNA@GO, composites show excellent antibacterial capability in culture with significant advantages in improved stability, enhanced antibacterial activity and stronger adsorption properties. Application of Ag@dsDNA@GO at 100 ppm on tomato transplants in a greenhouse experiment significantly reduced the severity of bacterial spot disease compared to untreated plants, giving results similar to those of the current grower standard treatment, with no phytotoxicity. PMID:24016217

  9. Directed evolution of DNA polymerase, RNA polymerase and reverse transcriptase activity in a single polypeptide.

    PubMed

    Ong, Jennifer L; Loakes, David; Jaroslawski, Szymon; Too, Kathleen; Holliger, Philipp

    2006-08-18

    DNA polymerases enable key technologies in modern biology but for many applications, native polymerases are limited by their stringent substrate recognition. Here we describe short-patch compartmentalized self-replication (spCSR), a novel strategy to expand the substrate spectrum of polymerases in a targeted way. spCSR is based on the previously described CSR, but unlike CSR only a short region (a "patch") of the gene under investigation is diversified and replicated. This allows the selection of polymerases under conditions where catalytic activity and processivity are compromised to the extent that full self-replication is inefficient. We targeted two specific motifs involved in substrate recognition in the active site of DNA polymerase I from Thermus aquaticus (Taq) and selected for incorporation of both ribonucleotide- (NTP) and deoxyribonucleotide-triphosphates (dNTPs) using spCSR. This allowed the isolation of multiple variants of Taq with apparent dual substrate specificity. They were able to synthesize RNA, while still retaining essentially wild-type (wt) DNA polymerase activity as judged by PCR. One such mutant (AA40: E602V, A608V, I614M, E615G) was able to incorporate both NTPs and dNTPs with the same catalytic efficiency as the wt enzyme incorporates dNTPs. AA40 allowed the generation of mixed RNA-DNA amplification products in PCR demonstrating DNA polymerase, RNA polymerase as well as reverse transcriptase activity within the same polypeptide. Furthermore, AA40 displayed an expanded substrate spectrum towards other 2'-substituted nucleotides and was able to synthesize nucleic acid polymers in which each base bore a different 2'-substituent. Our results suggest that spCSR will be a powerful strategy for the generation of polymerases with altered substrate specificity for applications in nano- and biotechnology and in the enzymatic synthesis of antisense and RNAi probes. PMID:16859707

  10. Chemically selective polymer substrate based direct isotope dilution alpha spectrometry of Pu.

    PubMed

    Paul, Sumana; Pandey, Ashok K; Shah, R V; Aggarwal, S K

    2015-06-01

    Quantification of actinides in the complex environmental, biological, process and waste streams samples requires multiple steps like selective preconcentration and matrix elimination, solid source preparations generally by evaporation or electrodeposition, and finally alpha spectrometry. To minimize the sample manipulation steps, a membrane based isotope dilution alpha spectrometry method was developed for the determination of plutonium concentrations in the complex aqueous solutions. The advantages of this method are that it is Pu(IV) selective at 3M HNO3, high preconcentration factor can be achieved, and obviates the need of solid source preparation. For this, a thin phosphate-sulfate bifunctional polymer layer was anchored on the surface of microporous poly(ethersulfone) membrane by UV induced surface grafting. The thickness of the bifunctional layer on one surface of the poly(ethersulfone) membrane was optimized. The thickness, physical and chemical structures of the bifunctional layer were studied by secondary ionization mass spectrometry (SIMS), scanning electron microscopy (SEM) and SEM-EDS (energy-dispersive spectroscopy). The optimized membrane was used for preconcentration of Pu(IV) from aqueous solutions having 3-4M HNO3, followed by direct quantification of the preconcentrated Pu(IV) by isotope dilution alpha spectrometry using (238)Pu spike. The chemical recovery efficiency of Pu(IV) was found to be 86±3% below Pu(IV) loading capacity (1.08 μg in 2×1 cm(2)) of the membrane sample. The experiments with single representative actinides indicated that Am(III) did not sorb to significant extent (7%) but U(VI) sorbed with 78±3% efficiency from the solutions having 3M HNO3 concentration. However, Pu(IV) chemical recovery in the membrane remained unaffected from the solution containing 1:1000 wt. proportion of Pu(IV) to U(VI). Pu concentrations in the (U, Pu)C samples and in the irradiated fuel dissolver solutions were determined. The results thus obtained

  11. Protocol: a beginner’s guide to the analysis of RNA-directed DNA methylation in plants

    PubMed Central

    2014-01-01

    acids that are core components of RNA-directed DNA methylation. Our results not only demonstrated the efficacy of this procedure, but also provide lists of methylation-sensitive restriction enzymes, novel DNA methylation marker loci, and related siRNA sequences, all of which can be valuable for future epigenetic studies. Importantly, step-by-step protocols are provided in details such that the approaches can be easily followed by biologists with little experience in epigenetics. PMID:24955108

  12. Silver colloids as plasmonic substrates for direct label-free surface-enhanced Raman scattering analysis of DNA.

    PubMed

    Torres-Nuñez, A; Faulds, K; Graham, D; Alvarez-Puebla, R A; Guerrini, L

    2016-08-15

    Ultrasensitive direct SERS analysis offers a powerful analytical tool for the structural characterization and classification of nucleic acids. However, acquisition of reliable spectral fingerprints of such complex biomolecules poses important challenges. In recent years, many efforts have been devoted to overcome these limitations, mainly implementing silver colloids as plasmonic substrates. However, a reliable cross-comparison of results reported in the recent literature is extremely hard to achieve, mostly due to the broad set of different surface properties of the plasmonic nanoparticles. Herein, we perform a thorough investigation of the role played by the metal/liquid interface composition of silver colloids in the direct label-free SERS analysis of DNA. Target molecules of increasing complexity, from short homopolymeric strands to long genomic duplexes, were used as probes. We demonstrate how apparently subtle changes in the colloidal surface chemistry can dramatically modify the affinity and the final SERS spectral profile of DNA. This has significant implications for the future design of new analytical strategies for the detection of DNA using SERS without labels. PMID:27213770

  13. New reagents for the introduction of reactive functional groups into chemically synthesized DNA probes.

    PubMed

    Skrzypczynski, Zbigniew; Wayland, Sarah

    2003-01-01

    An efficient and versatile preparative approach is described, allowing for the preparation of DNA probes modified with an aldehyde group at the 3'- or 5'-end. The developed synthetic strategy allows for the preparation of a new family of phosphoramidites and solid supports compatible with the automated synthesis of modified oligonucleotide probes. These new reagents were prepared from intermediates 3 and 3a, obtained from the commercially available aleuritic acid 1. It was demonstrated that the new phosphoramidite reagents also could be used as new types of cleavable linkers. A new and efficient method for the production of 5' aldehyde-labeled DNA probes was developed. PMID:12757390

  14. Tandem virtual screening targeting the SRA domain of UHRF1 identifies a novel chemical tool modulating DNA methylation.

    PubMed

    Myrianthopoulos, Vassilios; Cartron, Pierre Francois; Liutkevičiūtė, Zita; Klimašauskas, Saulius; Matulis, Daumantas; Bronner, Christian; Martinet, Nadine; Mikros, Emmanuel

    2016-05-23

    Ubiquitin-like protein UHRF1 that contains PHD and RING finger domain 1 is a key epigenetic protein enabling maintenance of the DNA methylation status through replication. A tandem virtual screening approach was implemented for identifying small molecules able to bind the 5-methylcytosine pocket of UHRF1 and inhibit its functionality. The NCI/DTP small molecules Repository was screened in silico by a combined protocol implementing structure-based and ligand-based methodologies. Consensus ranking was utilized to select a set of 27 top-ranked compounds that were subsequently evaluated experimentally in a stepwise manner for their ability to demethylate DNA in cellulo using PCR-MS and HPLC-MS/MS. The most active molecules were further assessed in a cell-based setting by the Proximity Ligation In Situ Assay and the ApoTome technology. Both evaluations confirmed that the DNMT1/UHRF1 interactions were significantly reduced after 4 h of incubation of U251 glioma cells with the most potent compound NSC232003, showing a 50% interaction inhibition at 15 μM as well as induction of global DNA cytosine demethylation as measured by ELISA. This is the first report of a chemical tool that targets UHRF1 and modulates DNA methylation in a cell context by potentially disrupting DNMT1/UHRF1 interactions. Compound NSC232003, a uracil derivative freely available by the NCI/DTP Repository, provides a versatile lead for developing highly potent and cell-permeable UHRF1 inhibitors that will enable dissection of DNA methylation inheritance. PMID:27049577

  15. Solid-State and Biological Nanopore for Real-Time Sensing of Single Chemical and Sequencing of DNA.

    PubMed

    Haque, Farzin; Li, Jinghong; Wu, Hai-Chen; Liang, Xing-Jie; Guo, Peixuan

    2013-02-01

    Sensitivity and specificity are two most important factors to take into account for molecule sensing, chemical detection and disease diagnosis. A perfect sensitivity is to reach the level where a single molecule can be detected. An ideal specificity is to reach the level where the substance can be detected in the presence of many contaminants. The rapidly progressing nanopore technology is approaching this threshold. A wide assortment of biomotors and cellular pores in living organisms perform diverse biological functions. The elegant design of these transportation machineries has inspired the development of single molecule detection based on modulations of the individual current blockage events. The dynamic growth of nanotechnology and nanobiotechnology has stimulated rapid advances in the study of nanopore based instrumentation over the last decade, and inspired great interest in sensing of single molecules including ions, nucleotides, enantiomers, drugs, and polymers such as PEG, RNA, DNA, and polypeptides. This sensing technology has been extended to medical diagnostics and third generation high throughput DNA sequencing. This review covers current nanopore detection platforms including both biological pores and solid state counterparts. Several biological nanopores have been studied over the years, but this review will focus on the three best characterized systems including α-hemolysin and MspA, both containing a smaller channel for the detection of single-strand DNA, as well as bacteriophage phi29 DNA packaging motor connector that contains a larger channel for the passing of double stranded DNA. The advantage and disadvantage of each system are compared; their current and potential applications in nanomedicine, biotechnology, and nanotechnology are discussed. PMID:23504223

  16. Solid-State and Biological Nanopore for Real-Time Sensing of Single Chemical and Sequencing of DNA

    PubMed Central

    Haque, Farzin; Li, Jinghong; Wu, Hai-Chen; Liang, Xing-Jie; Guo, Peixuan

    2013-01-01

    Sensitivity and specificity are two most important factors to take into account for molecule sensing, chemical detection and disease diagnosis. A perfect sensitivity is to reach the level where a single molecule can be detected. An ideal specificity is to reach the level where the substance can be detected in the presence of many contaminants. The rapidly progressing nanopore technology is approaching this threshold. A wide assortment of biomotors and cellular pores in living organisms perform diverse biological functions. The elegant design of these transportation machineries has inspired the development of single molecule detection based on modulations of the individual current blockage events. The dynamic growth of nanotechnology and nanobiotechnology has stimulated rapid advances in the study of nanopore based instrumentation over the last decade, and inspired great interest in sensing of single molecules including ions, nucleotides, enantiomers, drugs, and polymers such as PEG, RNA, DNA, and polypeptides. This sensing technology has been extended to medical diagnostics and third generation high throughput DNA sequencing. This review covers current nanopore detection platforms including both biological pores and solid state counterparts. Several biological nanopores have been studied over the years, but this review will focus on the three best characterized systems including α-hemolysin and MspA, both containing a smaller channel for the detection of single-strand DNA, as well as bacteriophage phi29 DNA packaging motor connector that contains a larger channel for the passing of double stranded DNA. The advantage and disadvantage of each system are compared; their current and potential applications in nanomedicine, biotechnology, and nanotechnology are discussed. PMID:23504223

  17. Direct Solution of the Chemical Master Equation Using Quantized Tensor Trains

    PubMed Central

    Kazeev, Vladimir; Khammash, Mustafa; Nip, Michael; Schwab, Christoph

    2014-01-01

    The Chemical Master Equation (CME) is a cornerstone of stochastic analysis and simulation of models of biochemical reaction networks. Yet direct solutions of the CME have remained elusive. Although several approaches overcome the infinite dimensional nature of the CME through projections or other means, a common feature of proposed approaches is their susceptibility to the curse of dimensionality, i.e. the exponential growth in memory and computational requirements in the number of problem dimensions. We present a novel approach that has the potential to “lift” this curse of dimensionality. The approach is based on the use of the recently proposed Quantized Tensor Train (QTT) formatted numerical linear algebra for the low parametric, numerical representation of tensors. The QTT decomposition admits both, algorithms for basic tensor arithmetics with complexity scaling linearly in the dimension (number of species) and sub-linearly in the mode size (maximum copy number), and a numerical tensor rounding procedure which is stable and quasi-optimal. We show how the CME can be represented in QTT format, then use the exponentially-converging -discontinuous Galerkin discretization in time to reduce the CME evolution problem to a set of QTT-structured linear equations to be solved at each time step using an algorithm based on Density Matrix Renormalization Group (DMRG) methods from quantum chemistry. Our method automatically adapts the “basis” of the solution at every time step guaranteeing that it is large enough to capture the dynamics of interest but no larger than necessary, as this would increase the computational complexity. Our approach is demonstrated by applying it to three different examples from systems biology: independent birth-death process, an example of enzymatic futile cycle, and a stochastic switch model. The numerical results on these examples demonstrate that the proposed QTT method achieves dramatic speedups and several orders of magnitude

  18. Direct solution of the Chemical Master Equation using quantized tensor trains.

    PubMed

    Kazeev, Vladimir; Khammash, Mustafa; Nip, Michael; Schwab, Christoph

    2014-03-01

    The Chemical Master Equation (CME) is a cornerstone of stochastic analysis and simulation of models of biochemical reaction networks. Yet direct solutions of the CME have remained elusive. Although several approaches overcome the infinite dimensional nature of the CME through projections or other means, a common feature of proposed approaches is their susceptibility to the curse of dimensionality, i.e. the exponential growth in memory and computational requirements in the number of problem dimensions. We present a novel approach that has the potential to "lift" this curse of dimensionality. The approach is based on the use of the recently proposed Quantized Tensor Train (QTT) formatted numerical linear algebra for the low parametric, numerical representation of tensors. The QTT decomposition admits both, algorithms for basic tensor arithmetics with complexity scaling linearly in the dimension (number of species) and sub-linearly in the mode size (maximum copy number), and a numerical tensor rounding procedure which is stable and quasi-optimal. We show how the CME can be represented in QTT format, then use the exponentially-converging hp-discontinuous Galerkin discretization in time to reduce the CME evolution problem to a set of QTT-structured linear equations to be solved at each time step using an algorithm based on Density Matrix Renormalization Group (DMRG) methods from quantum chemistry. Our method automatically adapts the "basis" of the solution at every time step guaranteeing that it is large enough to capture the dynamics of interest but no larger than necessary, as this would increase the computational complexity. Our approach is demonstrated by applying it to three different examples from systems biology: independent birth-death process, an example of enzymatic futile cycle, and a stochastic switch model. The numerical results on these examples demonstrate that the proposed QTT method achieves dramatic speedups and several orders of magnitude storage

  19. Fabrication of a multifunctional carbon nanotube "cotton" yarn by the direct chemical vapor deposition spinning process.

    PubMed

    Zhong, Xiao-Hua; Li, Ya-Li; Feng, Jian-Min; Kang, Yan-Ru; Han, Shuai-Shuai

    2012-09-21

    A continuous cotton-like carbon nanotube fiber yarn, consisting of multiple threads of high purity double walled carbon nanotubes, was fabricated in a horizontal CVD gas flow reactor with water vapor densification by the direct chemical vapor deposition spinning process. The water vapor interaction leads to homogeneous shrinking of the CNT sock-like assembly in the gas flow. This allows well controlled continuous winding of the dense thread inside the reactor. The CNT yarn is quite thick (1-3 mm), has a highly porous structure (99%) while being mechanically strong and electrically conductive. The water vapor interaction leads to homogeneous oxidation of the CNTs, offering the yarn oxygen-functionalized surfaces. The unique structure and surface of the CNT yarn provide it multiple processing advantages and properties. It can be mechanically engineered into a dense yarn, infiltrated with polymers to form a composite and mixed with other yarns to form a blend, as demonstrated in this research. Therefore, this CNT yarn can be used as a "basic yarn" for various CNT based structural and functional applications. PMID:22864939

  20. The direct simulation of high-speed mixing-layers without and with chemical heat release

    NASA Technical Reports Server (NTRS)

    Sekar, B.; Mukunda, H. S.; Carpenter, M. H.

    1991-01-01

    A direct numerical simulation of high speed reacting and non-reacting flows for H2-air systems is presented. The calculations are made for a convective Mach number of 0.38 with hyperbolic tangent initial profile and finite rate chemical reactions. A higher-order numerical method is used in time accurate mode to time advance the solution to a statistical steady state. About 600 time slices of all the variables are then stored for statistical analysis. It is shown that most of the problems of high-speed combustion with air are characterized by relatively weak heat release. The present study shows that: (1) the convective speed is reduced by heat release by about 10 percent at this convective Mach number M(sub c) = 0.38; (2) the variation of the mean and rms fluctuation of temperature can be explained on the basis of temperature fluctuation between the flame temperature and the ambient; (3) the growth rate with heat release is reduced by 7 percent; and (4) the entrainment is reduced by 25 percent with heat release. These differences are small in comparison with incompressible flow dynamics, and are argued to be due to the reduced importance of heat release in comparison with the large enthalpy gradients resulting from the large-scale vortex dynamics. It is finally suggested that the problems of reduced mixing in high-speed flows are not severely complicated by heat release.

  1. Chemically related 4,5-linked aminoglycoside antibiotics drive subunit rotation in opposite directions

    NASA Astrophysics Data System (ADS)

    Wasserman, Michael R.; Pulk, Arto; Zhou, Zhou; Altman, Roger B.; Zinder, John C.; Green, Keith D.; Garneau-Tsodikova, Sylvie; Doudna Cate, Jamie H.; Blanchard, Scott C.

    2015-07-01

    Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. Here we show that aminoglycosides chemically related to neomycin--paromomycin, ribostamycin and neamine--each bind to sites within h44 and H69 to perturb bridge B2 and affect subunit rotation. Neomycin and paromomycin, which only differ by their ring-I 6'-polar group, drive subunit rotation in opposite directions. This suggests that their distinct actions hinge on the 6'-substituent and the drug's net positive charge. By solving the crystal structure of the paromomycin-ribosome complex, we observe specific contacts between the apical tip of H69 and the 6'-hydroxyl on paromomycin from within the drug's canonical h44-binding site. These results indicate that aminoglycoside actions must be framed in the context of bridge B2 and their regulation of subunit rotation.

  2. Direct formation of ZnO nanostructures by chemical solution deposition and EUV exposure.

    PubMed

    Auzelyte, V; Sigg, H; Schmitt, B; Solak, H H

    2010-05-28

    The development of effective methods for the fabrication of ZnO nanostructures is important for the use of this semiconductor material with interesting optical and electronic properties. Chemical solution deposition methods have been demonstrated for creating ZnO films and electron beam exposure of a precursor film, zinc naphthenate, has been shown to yield ZnO nanostructures. Here, we report on the fabrication of ZnO nanostructures with photon beam exposure of a precursor film in the extreme ultraviolet range followed by a high temperature anneal in air. Interference lithography at this wavelength (13.5 nm) led to the production of extremely smooth line/space and dot array type periodic nanostructures with sizes as small as 10 nm. ZnO films obtained through EUV exposure exhibit markedly improved PL spectra with a sharp emission line in the UV range and much suppressed green emission. Electron microscopy and x-ray diffraction measurements also show strong effects of the EUV exposure step in ZnO formation as a function of precursor film thickness, EUV exposure dose and anneal temperature. The use of energetic photons for direct formation of ZnO nanostructures is found to be a method that warrants more investigation for the fabrication of patterned ZnO films with controlled properties. PMID:20431188

  3. Direct Coherent Raman Temperature Imaging and Wideband Chemical Detection in a Hydrocarbon Flat Flame.

    PubMed

    Bohlin, Alexis; Kliewer, Christopher J

    2015-02-19

    A single-shot coherent Raman imaging technique has been developed for spatially correlated one-dimensional high-fidelity gas-phase thermometry and multiplex chemical detection in flames. The technique utilizes two-beam phase matching, operating a single ultrashort pump/Stokes excitation pulse (7 fs) and a narrowband picosecond probe pulse (70 ps), interrogating a Raman active window of ∼4200 cm(-1) with ∼0.3 cm(-1) spectral resolution. The measurement geometry is formed intersecting the two beams shaped as laser-sheets and the one-coordinate spatial information is retrieved with a linespread function of <40 μm. The advance provides the possibility for the multiplexed measurement of all combustion relevant major species simultaneously with gaseous temperature monitored over a several millimeter field of view. The current technique is optimized in a premixed hydrocarbon flat-flame. At the flame-front, it is shown that direct imaging renders the temperature profile within ∼1% inaccuracy, whereas typical point-wise raster scanning may have relative systematic deviations up to 15% due to spatial averaging effects. PMID:26262480

  4. Coal gasification: Direct applications and syntheses of chemicals and fuels: A research needs assessment

    SciTech Connect

    Penner, S.S.; Alpert, S.B.; Beer, J.M.; Denn, M.; Haag, W.; Magee, R.; Reichl, E.; Rubin, E.S.; Solomon, P.R.; Wender, I.

    1987-06-01

    The DOE Working Group for an Assessment of Coal-Gasification Research Needs (COGARN - coal gasification advanced research needs) has reviewed and evaluated US programs dealing with coal gasification for a variety of applications. Cost evaluations and environmental-impact assessments formed important components of the deliberations. We have examined in some depth each of the following technologies: coal gasification for electricity generation in combined-cycle systems, coal gasification for the production of synthetic natural gas, coal gasifiers for direct electricity generation in fuel cells, and coal gasification for the production of synthesis gas as a first step in the manufacture of a wide variety of chemicals and fuels. Both catalytic and non-catalytic conversion processes were considered. In addition, we have constructed an orderly, long-range research agenda on coal science, pyrolysis, and partial combustion in order to support applied research and development relating to coal gasification over the long term. The COGARN studies were performed in order to provide an independent assessment of research needs in fuel utilization that involves coal gasification as the dominant or an important component. The findings and research recommendations of COGARN are summarized in this publication.

  5. Plant Community Diversity Influences Allocation to Direct Chemical Defence in Plantago lanceolata

    PubMed Central

    Mraja, Anne; Unsicker, Sybille B.; Reichelt, Michael; Gershenzon, Jonathan; Roscher, Christiane

    2011-01-01

    Background Forecasting the consequences of accelerating rates of changes in biodiversity for ecosystem functioning requires a mechanistic understanding of the relationships between the structure of biological communities and variation in plant functional characteristics. So far, experimental data of how plant species diversity influences the investment of individual plants in direct chemical defences against herbivores and pathogens is lacking. Methodology/Principal Findings We used Plantago lanceolata as a model species in experimental grasslands differing in species richness and composition (Jena Experiment) to investigate foliar concentrations of the iridoid glycosides (IG), catalpol and its biosynthetic precursor aucubin. Total IG and aucubin concentrations decreased, while catalpol concentrations increased with increasing plant diversity in terms of species or functional group richness. Negative plant diversity effects on total IG and aucubin concentrations correlated with increasing specific leaf area of P. lanceolata, suggesting that greater allocation to light acquisition reduced the investment into these carbon-based defence components. In contrast, increasing leaf nitrogen concentrations best explained increasing concentrations of the biosynthetically more advanced IG, catalpol. Observed levels of leaf damage explained a significant proportion of variation in total IG and aucubin concentrations, but did not account for variance in catalpol concentrations. Conclusions/Significance Our results clearly show that plants growing in communities of varying species richness and composition differ in their defensive chemistry, which may modulate plant susceptibility to enemy attack and consequently their interactions with higher trophic level organisms. PMID:22174766

  6. Chemically related 4,5-linked aminoglycoside antibiotics drive subunit rotation in opposite directions.

    PubMed

    Wasserman, Michael R; Pulk, Arto; Zhou, Zhou; Altman, Roger B; Zinder, John C; Green, Keith D; Garneau-Tsodikova, Sylvie; Cate, Jamie H Doudna; Blanchard, Scott C

    2015-01-01

    Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. Here we show that aminoglycosides chemically related to neomycin-paromomycin, ribostamycin and neamine-each bind to sites within h44 and H69 to perturb bridge B2 and affect subunit rotation. Neomycin and paromomycin, which only differ by their ring-I 6'-polar group, drive subunit rotation in opposite directions. This suggests that their distinct actions hinge on the 6'-substituent and the drug's net positive charge. By solving the crystal structure of the paromomycin-ribosome complex, we observe specific contacts between the apical tip of H69 and the 6'-hydroxyl on paromomycin from within the drug's canonical h44-binding site. These results indicate that aminoglycoside actions must be framed in the context of bridge B2 and their regulation of subunit rotation. PMID:26224058

  7. Comparison: Direct thrust nuclear engine, nuclear electric engine, and a chemical engine for future space missions

    SciTech Connect

    Ramsthaler, J.H.; Sulmeisters, T.K.

    1988-01-01

    The need for an advanced direct thrust nuclear rocket propulsion engine has been identified in Project Forecast 2, Air Force Systems Command report which looks into future Air Force needs. The Air Force Astronautical Laboratory (AFAL) has been assigned responsibility for developing the nuclear engine, and they in turn have requested support from teams of contractors who have the full capability to assist in the development of the nuclear engine. The Idaho National Engineering Laboratory (INEL) has formed a team of experts with Martin Marietta for mission analysis. Science Applications International (SAIC) for flight safety analysis, Westinghouse for the nuclear subsystem, and Rocketdyne for the engine system. INEL is the overall program manager and manager for test facility design, construction and operation. The INEL team has produced plans for both the engine system and the ground test facility. AFAL has funded the INEL team to perform mission analyses to evaluate the cost, performance and operational advantages for a nuclear rocket engine in performing Air Force Space Missions. For those studies, the Advanced Nuclear Rocket Engine (ANRE), a scaled down NERVA derivative, was used as the baseline nuclear engine to compare against chemical engines and nuclear electric engines for performance of orbital transfer and maneuvering missions. 3 tabs.

  8. Chemically related 4,5-linked aminoglycoside antibiotics drive subunit rotation in opposite directions

    PubMed Central

    Wasserman, Michael R.; Pulk, Arto; Zhou, Zhou; Altman, Roger B.; Zinder, John C.; Green, Keith D.; Garneau-Tsodikova, Sylvie; Doudna Cate, Jamie H.; Blanchard, Scott C.

    2015-01-01

    Dynamic remodelling of intersubunit bridge B2, a conserved RNA domain of the bacterial ribosome connecting helices 44 (h44) and 69 (H69) of the small and large subunit, respectively, impacts translation by controlling intersubunit rotation. Here we show that aminoglycosides chemically related to neomycin—paromomycin, ribostamycin and neamine—each bind to sites within h44 and H69 to perturb bridge B2 and affect subunit rotation. Neomycin and paromomycin, which only differ by their ring-I 6′-polar group, drive subunit rotation in opposite directions. This suggests that their distinct actions hinge on the 6′-substituent and the drug's net positive charge. By solving the crystal structure of the paromomycin–ribosome complex, we observe specific contacts between the apical tip of H69 and the 6′-hydroxyl on paromomycin from within the drug's canonical h44-binding site. These results indicate that aminoglycoside actions must be framed in the context of bridge B2 and their regulation of subunit rotation. PMID:26224058

  9. Lactase nonpersistence is directed by DNA-variation-dependent epigenetic aging.

    PubMed

    Labrie, Viviane; Buske, Orion J; Oh, Edward; Jeremian, Richie; Ptak, Carolyn; Gasiūnas, Giedrius; Maleckas, Almantas; Petereit, Rūta; Žvirbliene, Aida; Adamonis, Kęstutis; Kriukienė, Edita; Koncevičius, Karolis; Gordevičius, Juozas; Nair, Akhil; Zhang, Aiping; Ebrahimi, Sasha; Oh, Gabriel; Šikšnys, Virginijus; Kupčinskas, Limas; Brudno, Michael; Petronis, Arturas

    2016-06-01

    The inability to digest lactose, due to lactase nonpersistence, is a common trait in adult mammals, except in certain human populations that exhibit lactase persistence. It is not known how the lactase gene is dramatically downregulated with age in most individuals but remains active in some individuals. We performed a comprehensive epigenetic study of human and mouse small intestines, by using chromosome-wide DNA-modification profiling and targeted bisulfite sequencing. Epigenetically controlled regulatory elements accounted for the differences in lactase mRNA levels among individuals, intestinal cell types and species. We confirmed the importance of these regulatory elements in modulating lactase mRNA levels by using CRISPR-Cas9-induced deletions. Genetic factors contribute to epigenetic changes occurring with age at the regulatory elements, because lactase-persistence and lactase-nonpersistence DNA haplotypes demonstrated markedly different epigenetic aging. Thus, genetic factors enable a gradual accumulation of epigenetic changes with age, thereby influencing phenotypic outcome. PMID:27159559

  10. The Significance of Multivalent Bonding Motifs and "Bond Order" in DNA-Directed Nanoparticle Crystallization.

    PubMed

    Thaner, Ryan V; Eryazici, Ibrahim; Macfarlane, Robert J; Brown, Keith A; Lee, Byeongdu; Nguyen, SonBinh T; Mirkin, Chad A

    2016-05-18

    Multivalent oligonucleotide-based bonding elements have been synthesized and studied for the assembly and crystallization of gold nanoparticles. Through the use of organic branching points, divalent and trivalent DNA linkers were readily incorporated into the oligonucleotide shells that define DNA-nanoparticles and compared to monovalent linker systems. These multivalent bonding motifs enable the change of "bond strength" between particles and therefore modulate the effective "bond order." In addition, the improved accessibility of strands between neighboring particles, either due to multivalency or modifications to increase strand flexibility, gives rise to superlattices with less strain in the crystallites compared to traditional designs. Furthermore, the increased availability and number of binding modes also provide a new variable that allows previously unobserved crystal structures to be synthesized, as evidenced by the formation of a thorium phosphide superlattice. PMID:27148838

  11. Development of highly fluorescent silica nanoparticles chemically doped with organic dye for sensitive DNA microarray detection.

    PubMed

    Liu, Aihua; Wu, Liyou; He, Zhili; Zhou, Jizhong

    2011-10-01

    Increasing the sensitivity in DNA microarray hybridization can significantly enhance the capability of microarray technology for a wide range of research and clinical diagnostic applications, especially for those with limited sample biomass. To address this issue, using reverse microemulsion method and surface chemistry, a novel class of homogenous, photostable, highly fluorescent streptavidin-functionalized silica nanoparticles was developed, in which Alexa Fluor 647 (AF647) molecules were covalently embedded. The coating of bovine serum albumin on the resultant fluorescent particles can greatly eliminate nonspecific background signal interference. The thus-synthesized fluorescent nanoparticles can specifically recognize biotin-labeled target DNA hybridized to the microarray via streptavidin-biotin interaction. The response of this DNA microarray technology exhibited a linear range within 0.2 to 10 pM complementary DNA and limit of detection of 0.1 pM, enhancing microarray hybridization sensitivity over tenfold. This promising technology may be potentially applied to other binding events such as specific interactions between proteins. PMID:21822973

  12. Effects of Tumor Suppressor Lysyl Oxidase Propeptide on Prostate Cancer Xenograft Growth and Its Direct Interactions with DNA Repair Pathways

    PubMed Central

    Bais, Manish V.; Ozdener, Gokhan Baris; Sonenshein, Gail E.; Trackman, Philip C.

    2014-01-01

    Lysyl oxidase (LOX) is a multifunctional protein required for normal collagen and elastin biosynthesis and maturation. In addition, LOX has complex roles in cancer in which the lysyl oxidase propeptide (LOX-PP) domain of secreted pro-LOX has tumor suppressor activity, while the active enzyme promotes metastasis. In prostate cancer cell lines, recombinant LOX-PP (rLOX-PP) inhibits the growth of PC3 cells in vitro by mechanisms which were not characterized, while in DU145 cells rLOX-PP targeted FGF signaling. Because rLOX-PP can enhance effects of a genotoxic chemotherapeutic on breast cancer cell apoptosis, we reasoned that rLOX-PP could target DNA repair pathways typically elevated in cancer. Here we demonstrate for the first time that rLOX-PP inhibits prostate xenograft growth in vivo and that activating phosphorylations of the key DNA repair molecules ATM and CHK2 are inhibited by rLOX-PP expression in vivo. In addition, in vitro studies showed that rLOX-PP inhibits radiation induced activating phosphorylations of ATM and CHK2, and that exogenously added rLOX-PP protein can localize to the nucleus in both DU145 and PC3 cells. rLOX-PP pull-down studies resulted in detection of a protein complex with the nuclear DNA repair regulator MRE11 in both cell lines, and rLOX-PP localized to radiation-induced nuclear DNA repair foci. Finally, rLOX-PP was shown to sensitize both DU145 and PC3 cells to radiation-induced cell death determined in colony formation assays. These data provide evidence that rLOX-PP has a nuclear mechanism of action in which it directly interacts with DNA repair proteins to sensitize prostate cancer cells to the effects of ionizing radiation. PMID:24882580

  13. Curriculum Assessment as a Direct Tool in ABET Outcomes Assessment in a Chemical Engineering Programme

    ERIC Educational Resources Information Center

    Abu-Jdayil, Basim; Al-Attar, Hazim

    2010-01-01

    The chemical engineering programme at the United Arab Emirates University is designed to fulfil the Accreditation Board for Engineering and Technology (ABET) (A-K) EC2000 criteria. The Department of Chemical & Petroleum Engineering has established a well-defined process for outcomes assessment for the chemical engineering programme in order to…

  14. Effects of counterion valency on the damping of phonons propagating along the axial direction of liquid-crystalline DNA

    NASA Astrophysics Data System (ADS)

    Liu, Yun; Chen, Sow-Hsin; Berti, Debora; Baglioni, Piero; Alatas, Ahmet; Sinn, Harald; Alp, Ercan; Said, Ayman

    2005-12-01

    The phonon propagation and damping along the axial direction of films of aligned 40wt% calf-thymus DNA rods are studied by inelastic x-ray scattering (IXS). The IXS spectra are analyzed with the generalized three effective eigenmode theory, from which we extract the dynamic structure factor S (Q,E) as a function of transferred energy E =ℏω, and the magnitude of the transferred wave vector Q. S (Q,E) of a DNA sample typically consists of three peaks, one central Rayleigh scattering peak, and two symmetric Stokes and anti-Stokes Brillouin side peaks. By analyzing the Brillouin peaks, the phonon excitation energy and damping can be extracted at different Q values from about 4 to 30nm-1. A high-frequency sound speed is obtained from the initial slope of the linear portion of the dispersion relation below Q =4nm-1. The high-frequency sound speed obtained in this Q range is 3100m /s, which is about twice faster than the ultrasound speed of 1800m/s, measured by Brillouin light scattering at Q ˜0.01nm-1 at the similar hydration level. Our observations provide further evidence of the strong coupling between the internal dynamics of a DNA molecule and the dynamics of the solvent. The effect on damping and propagation of phonons along the axial direction of DNA rods due to divalent and trivalent counterions has been studied. It is found that the added multivalent counterions introduce stronger phonon damping. The phonons at the range between ˜12.5 and ˜22.5nm-1 are overdamped by the added counterions according to our model analyses. The intermediate scattering function is extracted and it shows a clear two-step relaxation with the fast relaxation time ranging from 0.1 to 4ps.

  15. Direct analysis of native and chimeric GATA specific DNA binding proteins from Aspergillus nidulans.

    PubMed Central

    Peters, D G; Caddick, M X

    1994-01-01

    In Aspergillus nidulans the regulatory gene areA is responsible for mediating nitrogen metabolite repression. The areA product (AREA) represents an example of the GATA family of DNA binding proteins, which are characterised by the presence of a GATA domain consisting of a zinc finger within a highly conserved region of 52 amino acids. Among the other transcription factors included in this family is the principal erythroid transcription factor, GATA-1, which contains two GATA domains. In order to demonstrate high specificity binding of native AREA to DNA containing the sequence -GATA-, and investigate the presence in A.nidulans of other proteins with related specificities, we have used gel mobility shift assays. Both AREA-dependent and independent complexes have been identified. Two strains bearing chimeric genes were also characterised. In these, the region encoding the native GATA domain of AREA was replaced by sequences from murine GATA-1 cDNA encoding either the equivalent C-terminal domain or both the N and C-terminal domains. Strains bearing the areA::NC-GATA construct, which includes the sequence encoding both the N and C-terminal domains of GATA-1, leads to a pronounced increase in one of two AREA-dependent complexes and implicates the N-terminal domain of GATA-1 in mediating protein-protein interactions. Images PMID:7816601

  16. Direct visualization of both DNA and RNA quadruplexes in human cells via an uncommon spectroscopic method.

    PubMed

    Laguerre, Aurélien; Wong, Judy M Y; Monchaud, David

    2016-01-01

    Guanine-rich DNA or RNA sequences can fold into higher-order, four-stranded structures termed quadruplexes that are suspected to play pivotal roles in cellular mechanisms including the control of the genome integrity and gene expression. However, the biological relevance of quadruplexes is still a matter of debate owing to the paucity of unbiased evidences of their existence in cells. Recent reports on quadruplex-specific antibodies and small-molecule fluorescent probes help dispel reservations and accumulating evidences now pointing towards the cellular relevance of quadruplexes. To better assess and comprehend their biology, developing new versatile tools to detect both DNA and RNA quadruplexes in cells is essential. We report here a smart fluorescent probe that allows for the simple detection of quadruplexes thanks to an uncommon spectroscopic mechanism known as the red-edge effect (REE). We demonstrate that this effect could open avenues to greatly enhance the ability to visualize both DNA and RNA quadruplexes in human cells, using simple protocols and fluorescence detection facilities. PMID:27535322

  17. Direct visualization of both DNA and RNA quadruplexes in human cells via an uncommon spectroscopic method

    PubMed Central

    Laguerre, Aurélien; Wong, Judy M. Y.; Monchaud, David

    2016-01-01

    Guanine-rich DNA or RNA sequences can fold into higher-order, four-stranded structures termed quadruplexes that are suspected to play pivotal roles in cellular mechanisms including the control of the genome integrity and gene expression. However, the biological relevance of quadruplexes is still a matter of debate owing to the paucity of unbiased evidences of their existence in cells. Recent reports on quadruplex-specific antibodies and small-molecule fluorescent probes help dispel reservations and accumulating evidences now pointing towards the cellular relevance of quadruplexes. To better assess and comprehend their biology, developing new versatile tools to detect both DNA and RNA quadruplexes in cells is essential. We report here a smart fluorescent probe that allows for the simple detection of quadruplexes thanks to an uncommon spectroscopic mechanism known as the red-edge effect (REE). We demonstrate that this effect could open avenues to greatly enhance the ability to visualize both DNA and RNA quadruplexes in human cells, using simple protocols and fluorescence detection facilities. PMID:27535322

  18. Direct assay of radiation-induced DNA base lesions in mammalian cells

    SciTech Connect

    Not Available

    1992-01-01

    Adenine (Ade), 2'-deoxyadenosine (dAdo), 5'-deoxyadenosine monophosphate (dAUT), single stranded poly adenylic acid [poly (dA)], double stranded deoxyadenylic-thymidylic acid [ds poly (dA-T)] and salmon testis DNA were irradiated with 500 Gy under oxic and anoxic conditions. The major damage products were analyzed by BPLC with optical detection and quantitated in terms of the percentage of the adenosine in each model compound found as a specific damage product. Outside of the Ade free base, 8-OH-dAdo was the major oxic damage product from each model compound. The type and quantity of the major damage products depended on the sequence and conformation of the model compounds under anoxic conditions. When dAdo and dAMP were irradiated under anoxic conditions, the major damage product was either the R or S isomer of 8,5'cdAdo and little Ade or [alpha]-dAdo was observed. However, when poly(dA), poly(dA-dT), and salmon testis DNA were [gamma]-irradiated under nitrogen, the major deoxyadenosine damage product was identified as the [alpha]-anomer of deoxyadenosine. No [alpha]-deoxyadenosine was detected after irradiation under oxic conditions. The presence of nucleotides with the [alpha]-configuration at the anomeric carbon atom in the DNA chain may have a significant effect on its tertiary structure and possibly modify its biological activity.

  19. Factors that influence the mutagenic patterns of DNA adducts from chemical carcinogens.

    PubMed

    Seo, K Y; Jelinsky, S A; Loechler, E L

    2000-10-01

    Carcinogens are generally mutagens, which is understandable given that tumor cells grow uncontrollably because they have mutations in critical genes involved in growth control. Carcinogens often induce a complex pattern of mutations (e.g., GC-->TA, GC-->AT, etc.). These mutations are thought to be initiated when a DNA polymerase encounters a carcinogen-DNA adduct during replication. In principle, mutational complexity could be due to either a collection of different adducts each inducing a single kind of mutation (Hypothesis 1a), or a single adduct inducing different kinds of mutations (Hypothesis 1b). Examples of each are discussed. Regarding Hypothesis 1b, structural factors (e.g., DNA sequence context) and biological factors (e.g., differing DNA polymerases) that can affect the pattern of adduct mutagenesis are discussed. This raises the question: how do structural and biological factors influence the pattern of adduct mutagenesis. For structural factors, three possibilities are considered: (Hypothesis 2a) a single conformation of an adduct giving rise to multiple mutations -- dNTP insertion by DNA polymerase being influenced by (e.g.) the surrounding DNA sequence context; (Hypothesis 2b) a variation on this ("dislocation mutagenesis"); or (Hypothesis 2c) a single adduct adopting multiple conformations, each capable of giving a different pattern of mutations. Hypotheses 2a, 2b and 2c can each in principle rationalize many mutational results, including how the pattern of adduct mutagenesis might be influenced by factors, such as DNA sequence context. Five lines of evidence are discussed suggesting that Hypothesis 2c can be correct for base substitution mutagenesis. For example, previous work from our laboratory was interpreted to indicate that [+ta]-B[a]P-N(2)-dG in a 5'-CGG sequence context (G115) could be trapped in a conformation giving predominantly G-->T mutations, but heating caused the adduct to equilibrate to its thermodynamic mixture of conformations

  20. Synthesis and Functionalization of Gold Nanoparticles Using Chemically Modified ssDNA

    NASA Astrophysics Data System (ADS)

    Calabrese, P. G.

    In the first part of this thesis, methods for functionalizing spherical gold nanoparticles with nucleic acid binding ligands (aptamers) that target the VEGF receptor complex were developed. In order to provide a multiplexed labeling strategy for imaging the VEGF receptor complex in electron microscopy, gold nanoparticles of distinct sizes were conjugated to modified ssDNA aptamers that target the VEGF-A cytokine, the VEGFR-2 RTK receptor and a membrane associated co-receptor, Nrp-1. The modified ssDNA gold nanoparticle conjugates were applied to a human lung carcinoma cell line (A549) which has been shown to express each of these proteins and used as a model system for VEGF signaling. Binding constants for the modified aptamers were also determined using a fluorescence polarization anisotropy assay to determine KD and KOFF for the aptamers with their respective proteins. In the latter part of this thesis, a modied ssDNA SELEX protocol was also developed in order to evolve imidazole modied ssDNA sequences that assemble gold nanoparticles from Au3+ precursor ions in aqueous solution. Active sequences bound to nanoparticles were partitioned from inactive sequences based on density via ultracentrifugation through a discontinuous sucrose gradient. Colloidal gold solutions produced by the evolved pool had a distinct absorbance spectra and produced nanoparticles with a narrower distribution of sizes compared to colloidal gold solutions produced by the starting randomized pool of imidazole modified ssDNA. Sequencing data from the evolved pool shows that conserved 5 and 6 nt motifs were shared amongst many of the isolates, which indicates that these motifs could serve as chelation sites for gold atoms or help stabilize colloidal gold solutions in a base specific manner.

  1. Polymers as directing agents for motions of chemical and biological species

    NASA Astrophysics Data System (ADS)

    Tanyeri, Nihan Yonet

    This thesis involves descriptions of solid surface modifications with various polymeric materials which were used as a guiding agent for motion of chemical and biological species. Quasi-two dimensional poly(oligoethylene glycol) acrylate polymer brush based molecular conduits have been designed with the goal of regulating and controlling the diffusive transport of molecular, e.g. organic dyes, and ionic species, e.g. AuCl4-, and Cu2+ ions, along predefined 2-D pathways. The transport of these chemical species has been examined by both fluorescence and dark field microscopy. The polymer brushes were formed through microcontact printing of an initiator, followed by surface-initiated Atom Transfer Radical Polymerization (SI-ATRP). SI-ATRP enables both 2-D patterning with a resolution of about 1 micrometer, and control over the resultant polymer brush thickness (which was varied from 10-100 nm). A hydrophilic poly(oligoethylene glycol) acrylate brushe was selected because of its potential to dissolve a wide range of hydrophilic species. The transport of fluorescent species can be directly followed. A non-lithographic fabrication method was developed for mufluidic devices used in the diffusion studies. Singular channel mufluidic device was utilized to study the directed organic dye diffusion. The AuCl4-, and Cu 2+ ion transport was studied by designing molecular devices with two mufluidic channels. We have demonstrated that the various species of interest diffuse much more rapidly along the predefined pathway than along the bare (polymer brush free) regions of the substrate, demonstrating that diffusive conduits for molecular transport can indeed be formed. The protein resistance of poly(N-isopropylacrylamide) (PNIPAM) brushes grafted from silicon wafers was investigated as a function of the chain molecular weight, grafting density, and temperature. Above the lower critical solution temperature (LCST) of 32°C, the collapse of the water swollen chains, determined by

  2. Chemical synthesis of human papillomavirus type 16 E7 oncoprotein: autonomous protein domains for induction of cellular DNA synthesis and for trans activation.

    PubMed

    Rawls, J A; Pusztai, R; Green, M

    1990-12-01

    The human papillomavirus type 16 E7 protein belongs to a family of nuclear oncoproteins that share amino acid sequences and functional homology. To localize biochemical activities associated with E7, we chemically synthesized the full-length 98-amino-acid polypeptide and several deletion mutant peptides. We show that the E7 polypeptide is biologically active and possesses at least two functional domains; the first induces cellular DNA synthesis in quiescent rodent cells, and the second trans activates the adenovirus E1A-inducible early E2 promoter and binds zinc. Further, each domain is autonomous and can function on separate peptides. DNA synthesis induction activity maps within the N-terminal portion of the molecule, which contains sequences related to adenovirus E1A conserved domains 1 and 2 required for cell transformation and binding of the retinoblastoma gene product. trans-Activation and Zn-binding activities map within the C-terminal portion of the molecule, a region which contains Cys-X-X-Cys motifs. trans Activation does not require protein synthesis, implying a mechanism that involves interaction with a preexisting cellular factor(s). E7 trans activates the adenovirus E2 promoter but not other E1A-inducible viral promoters, suggesting the possibility that E7 trans activation involves interaction, directly or indirectly, with cellular transcription factor E2F. PMID:2173783

  3. The DNA Methyltransferase Dnmt1 Directly Interacts with the SET and RING Finger-associated (SRA) Domain of the Multifunctional Protein Uhrf1 to Facilitate Accession of the Catalytic Center to Hemi-methylated DNA*

    PubMed Central

    Berkyurek, Ahmet Can; Suetake, Isao; Arita, Kyohei; Takeshita, Kohei; Nakagawa, Atsushi; Shirakawa, Masahiro; Tajima, Shoji

    2014-01-01

    Dnmt1 is responsible for the maintenance DNA methylation during replication to propagate methylation patterns to the next generation. The replication foci targeting sequence (RFTS), which plugs the catalytic pocket, is necessary for recruitment of Dnmt1 to the replication site. In the present study we found that the DNA methylation activity of Dnmt1 was DNA length-dependent and scarcely methylated 12-bp short hemi-methylated DNA. Contrarily, the RFTS-deleted Dnmt1 and Dnmt1 mutants that destroyed the hydrogen bonds between the RFTS and catalytic domain showed significant DNA methylation activity even toward 12-bp hemi-methylated DNA. The DNA methylation activity of the RFTS-deleted Dnmt1 toward 12-bp hemi-methylated DNA was strongly inhibited on the addition of RFTS, but to a lesser extent by Dnmt1 harboring the mutations that impair the hydrogen bond formation. The SRA domain of Uhrf1, which is a prerequisite factor for maintenance methylation and selectively binds to hemi-methylated DNA, stimulated the DNA methylation activity of Dnmt1. The SRA to Dnmt1 concentration ratio was the determinant for the maximum stimulation. In addition, a mutant SRA, which had lost the DNA binding activity but was able to bind to Dnmt1, stimulated the DNA methylation activity of Dnmt1. The results indicate that the DNA methylation activity of Dnmt1 was stimulated on the direct interaction of the SRA and Dnmt1. The SRA facilitated acceptance of the 12-bp fluorocytosine-containing DNA by the catalytic center. We propose that the SRA removes the RFTS plug from the catalytic pocket to facilitate DNA acceptance by the catalytic center. PMID:24253042

  4. Accessible DNA and Relative Depletion of H3K9me2 at Maize Loci Undergoing RNA-Directed DNA Methylation[W][OPEN

    PubMed Central

    Gent, Jonathan I.; Madzima, Thelma F.; Bader, Rechien; Kent, Matthew R.; Zhang, Xiaoyu; Stam, Maike; McGinnis, Karen M.; Dawe, R. Kelly

    2014-01-01

    RNA-directed DNA methylation (RdDM) in plants is a well-characterized example of RNA interference-related transcriptional gene silencing. To determine the relationships between RdDM and heterochromatin in the repeat-rich maize (Zea mays) genome, we performed whole-genome analyses of several heterochromatic features: dimethylation of lysine 9 and lysine 27 (H3K9me2 and H3K27me2), chromatin accessibility, DNA methylation, and small RNAs; we also analyzed two mutants that affect these processes, mediator of paramutation1 and zea methyltransferase2. The data revealed that the majority of the genome exists in a heterochromatic state defined by inaccessible chromatin that is marked by H3K9me2 and H3K27me2 but that lacks RdDM. The minority of the genome marked by RdDM was predominantly near genes, and its overall chromatin structure appeared more similar to euchromatin than to heterochromatin. These and other data indicate that the densely staining chromatin defined as heterochromatin differs fundamentally from RdDM-targeted chromatin. We propose that small interfering RNAs perform a specialized role in repressing transposons in accessible chromatin environments and that the bulk of heterochromatin is incompatible with small RNA production. PMID:25465407

  5. Characterization of a T-DNA promoter trap line of Arabidopsis thaliana uncovers a cryptic bi-directional promoter.

    PubMed

    Pratibha, Pritu; Singh, Sunil Kumar; Sharma, Isha; Kumar, Ravi; Srinivasan, Ramamurthy; Bhat, Shripad Ramachandra; Ahuja, Paramvir Singh; Sreenivasulu, Yelam

    2013-07-15

    Investigation of the transgenic Arabidopsis promoter trap line GFP-868 that showed GFP expression only in anthers revealed the T-DNA insertion at 461bp upstream to the hypothetical gene At4g10596 with the GFP reporter gene in head-to-head orientation to the At4g10596 gene. The expression of the At4g10596 gene in wild type and in GFP-868 plant homozygous for T-DNA insertion was comparable and found in all tissues tested, while the GFP expression was restricted to anthers of the GFP-868 plants suggesting that the 461bp fragment separating the two genes in the GFP-868 line is functioning as bi-directional promoter. This 461bp fragment was cloned upstream to the GUS gene in two orientations to test for bi-directional promoter activity. Transgenic Arabidopsis plants carrying either of these constructs showed GUS activity in anthers indicating that this fragment behaves as bi-directional promoter specific to anthers. These results were also supported by the presence of cis-acting motifs such as TATA box and POLLEN1LELAT52 (AGAAA) within the 461bp sequence in both orientations. However, transcripts corresponding to the upstream sequences beyond -461 nucleotides were not detected in the wild type suggesting that this 461bp fragment is a cryptic promoter. The significance of the promoter trap approach and the usefulness of this type of promoter are discussed. PMID:23612249

  6. SHAMS: Combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids

    PubMed Central

    Turner, Kevin B.; Yi-Brunozzi, Hye Young; Brinson, Robert G.; Marino, John P.; Fabris, Daniele; Le Grice, Stuart F.J.

    2009-01-01

    Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) has gained popularity as a facile method of examining RNA structure both in vitro and in vivo, exploiting accessibility of the ribose 2′-OH to acylation by N-methylisatoic anhydride (NMIA) in unpaired or flexible configurations. Subsequent primer extension terminates at the site of chemical modification, and these products are fractionated by high-resolution gel electrophoresis. When applying SHAPE to investigate structural features associated with the wild-type and analog-substituted polypurine tract (PPT)–containing RNA/DNA hybrids, their size (20–25 base pairs) rendered primer extension impractical. As an alternative method of detection, we reasoned that chemical modification could be combined with tandem mass spectrometry, relying on the mass increment of RNA fragments containing the NMIA adduct (Mr = 133 Da). Using this approach, we demonstrate both specific modification of the HIV-1 PPT RNA primer and variations in its acylation pattern induced by replacing template nucleotides with a non-hydrogen-bonding thymine isostere. Our selective 2′-hydroxyl acylation analyzed by mass spectrometry strategy (SHAMS) should find utility when examining the structure of small RNA fragments or RNA/DNA hybrids where primer extension cannot be performed. PMID:19535461

  7. Single-cell, locus-specific bisulfite sequencing (SLBS) for direct detection of epimutations in DNA methylation patterns

    PubMed Central

    Gravina, Silvia; Ganapathi, Shireen; Vijg, Jan

    2015-01-01

    Stochastic epigenetic changes drive biological processes, such as development, aging and disease. Yet, epigenetic information is typically collected from millions of cells, thereby precluding a more precise understanding of cell-to-cell variability and the pathogenic history of epimutations. Here we present a novel procedure for directly detecting epimutations in DNA methylation patterns using single-cell, locus-specific bisulfite sequencing (SLBS). We show that within gene promoter regions of mouse hepatocytes the epimutation rate is two orders of magnitude higher than the mutation rate. PMID:25897117

  8. Repair of DNA treated with lambda-irradiation and chemical carcinogens: Progress report, 1983-1987

    SciTech Connect

    Goldthwait, D.A.

    1987-08-01

    Studies on the in vitro enzymatic mechanisms of DNA repair of chromatin structures are described. In addition new studies on signal transduction in cancer and in carcinogenesis are emphasized. We are using molecular biological approaches to three model systems, which we are developing. The first involves signal transduction controlling sis gene (platelet derived growth factor-..beta..) mRNA levels in human glioblastoma cells. The second involves signal transduction in the activation of a long terminal repeat. The third involves an experiment designed to detect a tranposition event in a human cell. This progress report will summarize work on DNA repair of chromatin and then detail our progress in developing the three model systems. 59 refs., 14 figs., 7 tabs.

  9. Kinetics of cyclobutane thymine dimer splitting by DNA photolyase directly monitored in the UV

    PubMed Central

    Thiagarajan, Viruthachalam; Byrdin, Martin; Eker, André P. M.; Müller, Pavel; Brettel, Klaus

    2011-01-01

    CPD photolyase uses light to repair cyclobutane pyrimidine dimers (CPDs) formed between adjacent pyrimidines in UV-irradiated DNA. The enzyme harbors an FAD cofactor in fully reduced state (FADH-). The CPD repair mechanism involves electron transfer from photoexcited FADH- to the CPD, splitting of its intradimer bonds, and electron return to restore catalytically active FADH-. The two electron transfer processes occur on time scales of 10-10 and 10-9 s, respectively. Until now, CPD splitting itself has only been poorly characterized by experiments. Using a previously unreported transient absorption setup, we succeeded in monitoring cyclobutane thymine dimer repair in the main UV absorption band of intact thymine at 266 nm. Flavin transitions that overlay DNA-based absorption changes at 266 nm were monitored independently in the visible and subtracted to obtain the true repair kinetics. Restoration of intact thymine showed a short lag and a biexponential rise with time constants of 0.2 and 1.5 ns. We assign these two time constants to splitting of the intradimer bonds (creating one intact thymine and one thymine anion radical T∘-) and electron return from T∘- to the FAD cofactor with recovery of the second thymine, respectively. Previous model studies and computer simulations yielded various CPD splitting times between < 1 ps and < 100 ns. Our experimental results should serve as a benchmark for future efforts to model enzymatic photorepair. The technique and methods developed here may be applied to monitor other photoreactions involving DNA. PMID:21606324

  10. Assessment of direct versus indirect magnetic bead-based T-cell isolation procedures followed by magnetic bead-based DNA isolation

    PubMed Central

    Rosenbaum, Anna; Bleck, Ellen; Schneider, Matthias; Pongratz, Georg; Vordenbäumen, Stefan

    2016-01-01

    Objective To compare direct and indirect bead-based T-cell isolation followed by magnetic bead-based DNA isolation. Methods T-cells were isolated by direct or indirect selection with magnetic bead coated antbiodies followed by magnetic bead-based automated DNA isolation in 10 healthy subjects. Purity of T-cells, purity of DNA (by A260/A280 ratio measurement) and DNA concentration were assessed. Results Direct and indirect labelling resulted in comparable T-cell purity (93.11±1.47% vs. 94.99±1.54%, p= 0.125) and DNA concentration per cell (50.97±14.15 ng/(mlxcell) vs. 49.53±13.62 ng/(mlxcell), p=0.492), while DNA purity was significantly higher after direct labelling (1.82±0.05 vs. 1.78±0.03, p=0.0488). Conclusions Both direct and indirect magnetic bead-based T-cell selection may be used prior to magnetic bead-based DNA isolation procedures. PMID:27547441

  11. Atypical hereditary neuropathy with liability to pressure palsies (HNPP): the value of direct DNA diagnosis.

    PubMed Central

    Sessa, M; Nemni, R; Quattrini, A; Del Carro, U; Wrabetz, L; Canal, N

    1997-01-01

    We report two patients with suspected hereditary liability to pressure palsies. Neurophysiological studies showed a mixed axonal-demyelinating sensory-motor polyneuropathy with focal slowing of conduction velocities at the common sites of entrapment. Morphological studies on sural nerve biopsy from the proband showed active axonal regeneration without typical tomacula. Molecular analysis confirmed the presence of a deletion of chromosome 17p11.2 in both patients. Our observation confirms the heterogeneity of hereditary liability to pressure palsies and the relevance of DNA testing for the diagnosis of this hereditary neuropathy. Images PMID:9391880

  12. DNA origami-directed, discrete three-dimensional plasmonic tetrahedron nanoarchitectures with tailored optical chirality.

    PubMed

    Dai, Gaole; Lu, Xuxing; Chen, Zhong; Meng, Chun; Ni, Weihai; Wang, Qiangbin

    2014-04-23

    Discrete, three-dimensional (3D) gold nanoparticle (AuNP) tetrahedron nanoarchitectures are successfully self-assembled with DNA origami as template with high purity (>85%). A distinct plasmonic chiral response is experimentally observed from the AuNP tetrahedron nanoarchitectures and appears in a configuration-dependent manner. The chiral optical properties are then rationally engineered by modifying the structural parameters including the AuNP size and interparticle distance. Theoretical study of the AuNP tetrahedron nanoarchitectures shows the dependence of the chiral optical property on the AuNP size and interparticle distance, consistent with the ensemble averaged measurements. PMID:24716524

  13. Responses of genes involved in cell cycle control to diverse DNA damaging chemicals in human lung adenocarcinoma A549 cells

    PubMed Central

    Zhu, Huijun; Smith, Catherine; Ansah, Charles; Gooderham, Nigel J

    2005-01-01

    Background Many anticancer agents and carcinogens are DNA damaging chemicals and exposure to such chemicals results in the deregulation of cell cycle progression. The molecular mechanisms of DNA damage-induced cell cycle alteration are not well understood. We have studied the effects of etoposide (an anticancer agent), cryptolepine (CLP, a cytotoxic alkaloid), benzo [a]pyrene (BaP, a carcinogenic polycyclic aromatic hydrocarbon) and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP, a cooked-meat derived carcinogen) on the expression of cell cycle regulatory genes to understand the molecular mechanisms of the cell cycle disturbance. Results A549 cells were treated with DMSO or chemicals for up to 72 h and periodically sampled for cell cycle analysis, mRNA and protein expression. DMSO treated cells showed a dominant G1 peak in cell cycle at all times examined. Etoposide and CLP both induced G2/M phase arrest yet the former altered the expression of genes functioning at multiple phases, whilst the latter was more effective in inhibiting the expression of genes in G2-M transition. Both etoposide and CLP induced an accumulation of p53 protein and upregulation of p53 transcriptional target genes. Neither BaP nor PhIP had substantial phase-specific cell cycle effect, however, they induced distinctive changes in gene expression. BaP upregulated the expression of CYP1B1 at 6–24 h and downregulated many cell cycle regulatory genes at 48–72 h. By contrast, PhIP increased the expression of many cell cycle regulatory genes. Changes in the expression of key mRNAs were confirmed at protein level. Conclusion Our experiments show that DNA damaging agents with different mechanisms of action induced distinctive changes in the expression pattern of a panel of cell cycle regulatory genes. We suggest that examining the genomic response to chemical exposure provides an exceptional opportunity to understand the molecular mechanism involved in cellular response to toxicants. PMID

  14. Native chemical ligation for conversion of sequence-defined oligomers into targeted pDNA and siRNA carriers.

    PubMed

    Zhang, Can Yang; Kos, Petra; Müller, Katharina; Schrimpf, Waldemar; Troiber, Christina; Lächelt, Ulrich; Scholz, Claudia; Lamb, Don C; Wagner, Ernst

    2014-04-28

    Native chemical ligation (NCL) was established for the conversion of sequence-defined oligomers of different topologies into targeted and PEG shielded pDNA and siRNA carriers. From an existing library of non-targeted oligoethanamino amides, six oligomers containing N-terminal cysteines were selected as cationic cores, to which monodisperse polyethylene glycol (PEG) containing terminal folic acid as targeting ligand (or terminal alanine as targeting negative control ligand) were attached by NCL. Ligated conjugates plus controls (in sum 18 oligomers) were evaluated for pDNA or siRNA gene delivery. Biophysical characteristics including nucleic acid binding in the absence or presence of serum, as well as biological activities in cellular uptake and gene transfer (or gene silencing, respectively) were determined. In most cases, the folic acid-PEG-ligated oligomers displayed a strongly improved cellular binding, uptake and gene transfer into receptor-positive KB cells as compared to the alanine-PEG controls. Changing the topological structures by increasing the number of cationic arms, adding tyrosine trimers as polyplex stabilizing domains, or histidines facilitating endosomal escape resulted in beneficial gene transfer characteristics. The screen revealed different requirements for pDNA and siRNA delivery. A folate-PEG ligated histidinylated four-arm oligomer was most effective for pDNA delivery but inactive for siRNA, whereas a folate-PEG-ligated three-arm oligomer with tyrosine trimer modifications was most effective in siRNA mediated gene silencing. The results demonstrate the site-selective NCL reaction as powerful method to modify existing oligomers. Thus multifunctional targeted carriers can be obtained with ease and used to identify lead structures for subsequent in vivo delivery. PMID:24566255

  15. Peroxone mineralization of chemical oxygen demand for direct potable water reuse: Kinetics and process control.

    PubMed

    Wu, Tingting; Englehardt, James D

    2015-04-15

    Mineralization of organics in secondary effluent by the peroxone process was studied at a direct potable water reuse research treatment system serving an occupied four-bedroom, four bath university residence hall apartment. Organic concentrations were measured as chemical oxygen demand (COD) and kinetic runs were monitored at varying O3/H2O2 dosages and ratios. COD degradation could be accurately described as the parallel pseudo-1st order decay of rapidly and slowly-oxidizable fractions, and effluent COD was reduced to below the detection limit (<0.7 mg/L). At dosages ≥4.6 mg L(-1) h(-1), an O3/H2O2 mass ratio of 3.4-3.8, and initial COD <20 mg/L, a simple first order decay was indicated for both single-passed treated wastewater and recycled mineral water, and a relationship is proposed and demonstrated to estimate the pseudo-first order rate constant for design purposes. At this O3/H2O2 mass ratio, ORP and dissolved ozone were found to be useful process control indicators for monitoring COD mineralization in secondary effluent. Moreover, an average second order rate constant for OH oxidation of secondary effluent organics (measured as MCOD) was found to be 1.24 × 10(7) ± 0.64 × 10(7) M(-1) S(-1). The electric energy demand of the peroxone process is estimated at 1.73-2.49 kW h electric energy for removal of one log COD in 1 m(3) secondary effluent, comparable to the energy required for desalination of medium strength seawater. Advantages/disadvantages of the two processes for municipal wastewater reuse are discussed. PMID:25704155

  16. Bioassay-directed chemical analysis and detection of mutagenicity in ambient air of the coke oven.

    PubMed

    Dobiás, L; Kůsová, J; Gajdos, O; Vidová, P; Gajdosová, D; Havránková, J; Fried, M; Binková, B; Topinka, J

    1999-09-30

    In the present study, we summarize the results of studies on the mutagenic potential of the main fractions and subfractions of extractable organic material (EOM) in the ambient air at the workplaces of the coke oven. The objective of our experiments was to apply the Bioassay-Directed Chemical Analysis (with the use of the Ames test) for the identification of the differences in the mutagenicity of these fractions, in relationship to the complex mixture of EOM in occupational air. From the evaluation of results, it is possible to deduce the following conclusions: (1) The comparison of the mutagenicity in the main fractions (basic, acidic, neutral) demonstrates the existence of differences in mutagenic potential. Of the total mutagenicity, 20.4% is in the basic fraction, 25.4% in the acidic fraction and 54.2% in the neutral fraction. (2) In general, 90.1% of the mutagenicity found in the basic, acidic and neutral fractions together was associated with the requirement of metabolic activation in vitro (+S9). In the case of the neutral fraction, it was 51.8%. (3) These results also suggest that frameshift mutations are the major component (53.8%) of the total mutagenicity of the main fractions. (4) With regards to the mutagenicity of organic compounds in the neutral fraction it appeared that genotoxicants of its subfractions (slightly and moderately polar and aromatic) play the main role. Carcinogenic aromatic hydrocarbons (PAH) and genotoxic nitrocompounds play an important role as determinants of the mutagenic potential of complex mixtures of harmful compounds in ambient air. This is confirmed first by the results of short-term bacterial tests. PMID:10575437

  17. Directed evolution strategies for enantiocomplementary haloalkane dehalogenases: from chemical waste to enantiopure building blocks.

    PubMed

    van Leeuwen, Jan G E; Wijma, Hein J; Floor, Robert J; van der Laan, Jan-Metske; Janssen, Dick B

    2012-01-01

    We used directed evolution to obtain enantiocomplementary haloalkane dehalogenase variants that convert the toxic waste compound 1,2,3-trichloropropane (TCP) into highly enantioenriched (R)- or (S)-2,3-dichloropropan-1-ol, which can easily be converted into optically active epichlorohydrins-attractive intermediates for the synthesis of enantiopure fine chemicals. A dehalogenase with improved catalytic activity but very low enantioselectivity was used as the starting point. A strategy that made optimal use of the limited capacity of the screening assay, which was based on chiral gas chromatography, was developed. We used pair-wise site-saturation mutagenesis (SSM) of all 16 noncatalytic active-site residues during the initial two rounds of evolution. The resulting best R- and S-enantioselective variants were further improved in two rounds of site-restricted mutagenesis (SRM), with incorporation of carefully selected sets of amino acids at a larger number of positions, including sites that are more distant from the active site. Finally, the most promising mutations and positions were promoted to a combinatorial library by using a multi-site mutagenesis protocol with restricted codon sets. To guide the design of partly undefined (ambiguous) codon sets for these restricted libraries we employed structural information, the results of multiple sequence alignments, and knowledge from earlier rounds. After five rounds of evolution with screening of only 5500 clones, we obtained two strongly diverged haloalkane dehalogenase variants that give access to (R)-epichlorohydrin with 90 % ee and to (S)-epichlorohydrin with 97 % ee, containing 13 and 17 mutations, respectively, around their active sites. PMID:22109980

  18. Long-Range Vibrational Dynamics Are Directed by Watson-Crick Base Pairing in Duplex DNA.

    PubMed

    Hithell, Gordon; Shaw, Daniel J; Donaldson, Paul M; Greetham, Gregory M; Towrie, Michael; Burley, Glenn A; Parker, Anthony W; Hunt, Neil T

    2016-05-01

    Ultrafast two-dimensional infrared (2D-IR) spectroscopy of a 15-mer A-T DNA duplex in solution has revealed structure-dependent vibrational coupling and energy transfer processes linking bases with the sugar-phosphate backbone. Duplex melting induces significant changes in the positions of off-diagonal peaks linking carbonyl and ring-stretching vibrational modes of the adenine and thymine bases with vibrations of the phosphate group and phosphodiester linkage. These indicate that Watson-Crick hydrogen bonding and helix formation lead to a unique vibrational coupling arrangement of base vibrational modes with those of the phosphate unit. On the basis of observations from time-resolved 2D-IR data, we conclude that rapid energy transfer processes occur between base and backbone, mediated by additional modes located on the deoxyribose moiety within the same nucleotide. These relaxation dynamics are insensitive to duplex melting, showing that efficient intramolecular energy relaxation to the solvent via the phosphate groups is the key to excess energy dissipation in both single- and double-stranded DNA. PMID:27079484

  19. Next-generation DNA sequencing of HEXA: a step in the right direction for carrier screening.

    PubMed

    Hoffman, Jodi D; Greger, Valerie; Strovel, Erin T; Blitzer, Miriam G; Umbarger, Mark A; Kennedy, Caleb; Bishop, Brian; Saunders, Patrick; Porreca, Gregory J; Schienda, Jaclyn; Davie, Jocelyn; Hallam, Stephanie; Towne, Charles

    2013-11-01

    Tay-Sachs disease (TSD) is the prototype for ethnic-based carrier screening, with a carrier rate of ∼1/27 in Ashkenazi Jews and French Canadians. HexA enzyme analysis is the current gold standard for TSD carrier screening (detection rate ∼98%), but has technical limitations. We compared DNA analysis by next-generation DNA sequencing (NGS) plus an assay for the 7.6 kb deletion to enzyme analysis for TSD carrier screening using 74 samples collected from participants at a TSD family conference. Fifty-one of 74 participants had positive enzyme results (46 carriers, five late-onset Tay-Sachs [LOTS]), 16 had negative, and seven had inconclusive results. NGS + 7.6 kb del screening of HEXA found a pathogenic mutation, pseudoallele, or variant of unknown significance (VUS) in 100% of the enzyme-positive or obligate carrier/enzyme-inconclusive samples. NGS detected the B1 allele in two enzyme-negative obligate carriers. Our data indicate that NGS can be used as a TSD clinical carrier screening tool. We demonstrate that NGS can be superior in detecting TSD carriers compared to traditional enzyme and genotyping methodologies, which are limited by false-positive and false-negative results and ethnically focused, limited mutation panels, respectively, but is not ready for sole use due to lack of information regarding some VUS. PMID:24498621

  20. Direct Involvement of Retinoblastoma Family Proteins in DNA Repair by Non-homologous End-Joining

    PubMed Central

    Cook, Rebecca; Zoumpoulidou, Georgia; Luczynski, Maciej T.; Rieger, Simone; Moquet, Jayne; Spanswick, Victoria J.; Hartley, John A.; Rothkamm, Kai; Huang, Paul H.; Mittnacht, Sibylle

    2015-01-01

    Summary Deficiencies in DNA double-strand break (DSB) repair lead to genetic instability, a recognized cause of cancer initiation and evolution. We report that the retinoblastoma tumor suppressor protein (RB1) is required for DNA DSB repair by canonical non-homologous end-joining (cNHEJ). Support of cNHEJ involves a mechanism independent of RB1’s cell-cycle function and depends on its amino terminal domain with which it binds to NHEJ components XRCC5 and XRCC6. Cells with engineered loss of RB family function as well as cancer-derived cells with mutational RB1 loss show substantially reduced levels of cNHEJ. RB1 variants disabled for the interaction with XRCC5 and XRCC6, including a cancer-associated variant, are unable to support cNHEJ despite being able to confer cell-cycle control. Our data identify RB1 loss as a candidate driver of structural genomic instability and a causative factor for cancer somatic heterogeneity and evolution. PMID:25818292

  1. Possible mechanism of psoralen phototoxicity not involving direct interaction with DNA

    SciTech Connect

    Laskin, J.D.; Lee, E.; Yurkow, E.J.; Laskin, D.L.; Gallo, M.A.

    1985-09-01

    Psoralens in combination with ultraviolet light (UVA; 320-400 nm) are used in the photochemical treatment of a variety of skin diseases including vitiligo, a skin depigmentational disorder, and psoriasis, a disease of accelerated epidermal cell proliferation. Although it is generally assumed that the major site of action of the psoralens is DNA, the authors have obtained evidence that another site may be the primary target for these compounds. They have identified specific, saturable, high-affinity binding sites for 8-methoxypsoralen on HeLa cells and have detected specific binding of 8-methoxypsoralen to four other human cell lines and five mouse cell lines. In HeLa cells, specific binding is reversible and independent of the ability of the compound to intercalate into DNA. In addition, binding sites become covalently modified by the psoralen after UVA exposure. Specific binding of 8-(methyoxy-/sup 3/H)methoxypsoralen constitutes 79% of the label bound to the cells. Scatchard analysis indicated two classes of psoralen binding sites. Based on these findings, the authors hypothesize that specific binding sites for psoralens on mammalian cells mediate, at least in part, psoralen-induced phototoxicity.

  2. Chemical repair of base lesions, AP-sites, and strand breaks on plasmid DNA in dilute aqueous solution by ascorbic acid

    SciTech Connect

    Hata, Kuniki; Urushibara, Ayumi; Yamashita, Shinichi; Shikazono, Naoya; Yokoya, Akinari; Katsumura, Yosuke

    2013-05-03

    Highlights: •We report a novel mechanism of radiation protection of DNA by chemical activity of ascorbic acid. •The “chemical repair” of DNA damage was revealed using biochemical assay and chemical kinetics analysis. •We found that ascorbic acid significantly repairs precursors of nucleobase lesions and abasic sites. •However, ascorbic acid seldom repairs precursors of DNA-strand breaks. -- Abstract: We quantified the damage yields produced in plasmid DNA by γ-irradiation in the presence of low concentrations (10–100 μM) of ascorbic acid, which is a major antioxidant in living systems, to clarify whether it chemically repairs radiation damage in DNA. The yield of DNA single strand breaks induced by irradiation was analyzed with agarose gel electrophoresis as conformational changes in closed circular plasmids. Base lesions and abasic sites were also observed as additional conformational changes by treating irradiated samples with glycosylase proteins. By comparing the suppression efficiencies to the induction of each DNA lesion, in addition to scavenging of the OH radicals derived from water radiolysis, it was found that ascorbic acid promotes the chemical repair of precursors of AP-sites and base lesions more effectively than those of single strand breaks. We estimated the efficiency of the chemical repair of each lesion using a kinetic model. Approximately 50–60% of base lesions and AP-sites were repaired by 10 μM ascorbic acid, although strand breaks were largely unrepaired by ascorbic acid at low concentrations. The methods in this study will provide a route to understanding the mechanistic aspects of antioxidant activity in living systems.

  3. USE OF BIOASSAY-DIRECTED CHEMICAL ANALYSIS FOR IDENTIFYING MUTAGENIC COMPOUNDS IN URBAN AIR AND COMBUSTION EMISSIONS

    EPA Science Inventory

    Bioassay-directed chemical analysis fractionation has been used for 30 years to identify mutagenic classes of compounds in complex mixtures. Most studies have used the Salmonella (Ames) mutagenicity assay, and we have recently applied this methodology to two standard reference sa...

  4. Microintaglio Printing of In situ Synthesized Proteins Enables Rapid Printing of High-Density Protein Microarrays Directly from DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Biyani, Manish; Moriyasu, Junpei; Tanaka, Yoko; Sato, Shusuke; Ueno, Shingo; Ichiki, Takanori

    2013-08-01

    A simple and versatile approach to the simultaneous on-chip synthesis and printing of proteins has been studied for high-density protein microarray applications. The method used is based on the principle of intaglio printing using microengraved plates. Unlike conventional approaches that require multistep reactions for synthesizing proteins off the chip followed by printing using a robotic spotter, our approach demonstrates the following: (i) parallel and spotter-free printing of high-density protein microarrays directly from a type of DNA microarray and (ii) microcompartmentalization of cell-free coupled transcription/translation reaction and direct transferring of picoliter protein solution per spot to pattern microarrays of 25-100 µm features.

  5. 76 FR 12556 - Airworthiness Directives; Various Transport Category Airplanes Equipped With Chemical Oxygen...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-08

    ... 12866, (2) Is not a ``significant rule'' under DOT Regulatory Policies and Procedures (44 FR 11034... Category Airplanes Equipped With Chemical Oxygen Generators Installed in a Lavatory AGENCY: Federal... affected airplanes identified above. This AD requires modifying the chemical oxygen generators in...

  6. Direct measurement of the intermolecular forces between counterion-condensed DNA double helices. Evidence for long range attractive hydration forces.

    PubMed Central

    Rau, D C; Parsegian, V A

    1992-01-01

    Rather than acting by modifying van der Waals or electrostatic double layer interactions or by directly bridging neighboring molecules, polyvalent ligands bound to DNA double helices appear to act by reconfiguring the water between macromolecular surfaces to create attractive long range hydration forces. We have reached this conclusion by directly measuring the repulsive forces between parallel B-form DNA double helices pushed together from the separations at which they have self organized into hexagonal arrays of parallel rods. For all of the wide variety of "condensing agents" from divalent Mn to polymeric protamines, the resulting intermolecular force varies exponentially with a decay rate of 1.4-1.5 A, exactly one-half that seen previously for hydration repulsion. Such behavior qualitatively contradicts the predictions of all electrostatic double layer and van der Waals force potentials previously suggested. It fits remarkably well with the idea, developed and tested here, that multivalent counterion adsorption reorganizes the water at discrete sites complementary to unadsorbed sites on the apposing surface. The measured strength and range of these attractive forces together with their apparent specificity suggest the presence of a previously unexpected force in molecular organization. Images FIGURE 1 PMID:1540693

  7. Direct measurement of formation of loops in DNA by a human tumor suppressor protein

    NASA Astrophysics Data System (ADS)

    Migliori, Amy; Kung, Samuel; Wang, Danielle; Smith, Douglas E.

    2013-09-01

    In previous work we developed methods using optical tweezers to measure protein-mediated formation of loops in DNA structures that can play an important role in regulating gene expression. We previously applied this method to study two-site restriction endonucleases, which were convenient model systems for studying this phenomenon. Here we report preliminary work in which we have applied this method to study p53, a human tumor suppressor protein, and show that we can measure formation of loops. Previous biophysical evidence for loops comes from relatively limited qualitative studies of fixed complexes by electron microscopy4. Our results provide independent corroboration and future opportunities for more quantitative studies investigating structure and mechanics.

  8. A FLUORESCENCE-BASED SCREENING ASSAY FOR DNA DAMAGE INDUCED BY GENOTOXIC INDUSTRIAL CHEMICALS

    EPA Science Inventory

    The possibility of deliberate or accidental release of toxic chemicals in industrial, commercial or residential settings has indicated a need for rapid, cost-effective and versatile monitoring methods to prevent exposures to humans and ecosystems. Because many toxic industrial c...

  9. DNA-Mutation Inventory to Refine and Enhance Cancer Treatment (DIRECT): A Catalog of Clinically Relevant Cancer Mutations to Enable Genome-Directed Anticancer Therapy

    PubMed Central

    Yeh, Paul; Chen, Heidi; Andrews, Jenny; Naser, Riyad; Pao, William; Horn, Leora

    2014-01-01

    Purpose Tumor gene mutation status is becoming increasingly important in the treatment of patients with cancer. A comprehensive catalog of tumor gene–response outcomes from individual patients is needed, especially for actionable mutations and rare variants. We created a proof-of-principle database [DNA-mutation Inventory to Refine and Enhance Cancer Treatment (DIRECT)], starting with lung cancer-associated EGF receptor (EGFR) mutations, to provide a resource for clinicians to prioritize treatment decisions based on a patient’s tumor mutations at the point of care. Methods A systematic search of literature published between June 2005 and May 2011 was conducted through PubMed to identify patient-level, mutation–drug response in patients with non–small cell lung cancer (NSCLC) with EGFR mutant tumors. Minimum inclusion criteria included patient’s EGFR mutation, corresponding treatment, and an associated radiographic outcome. Results A total of 1,021 patients with 1,070 separate EGFR tyrosine kinase inhibitor therapy responses from 116 different publications were included. About 188 unique EGFR mutations occurring in 207 different combinations were identified: 149 different mutation combinations were associated with disease control and 42 were associated with disease progression. Four secondary mutations, in 16 different combinations, were associated with acquired resistance. Conclusions As tumor sequencing becomes more common in oncology, this comprehensive electronic catalog can enable genome-directed anticancer therapy. DIRECT will eventually encompass all tumor mutations associated with clinical outcomes on targeted therapies. Users can make specific queries at http:// www.mycancergenome.org/about/direct to obtain clinically relevant data associated with various mutations. PMID:23344264

  10. Standard role for a conserved aspartate or more direct involvement in deglycosylation? An ONIOM and MD investigation of adenine-DNA glycosylase.

    PubMed

    Kellie, Jennifer L; Wilson, Katie A; Wetmore, Stacey D

    2013-12-01

    8-Oxoguanine (OG) is one of the most frequently occurring forms of DNA damage and is particularly deleterious since it forms a stable Hoogsteen base pair with adenine (A). The repair of an OG:A mispair is initiated by adenine-DNA glycosylase (MutY), which hydrolyzes the sugar-nucleobase bond of the adenine residue before the lesion is processed by other proteins. MutY has been proposed to use a two-part chemical step involving protonation of the adenine nucleobase, followed by SN1 hydrolysis of the glycosidic bond. However, differences between a recent (fluorine recognition complex, denoted as the FLRC) crystal structure and the structure on which most mechanistic conclusions have been based to date (namely, the lesion recognition complex or LRC) raise questions regarding the mechanism used by MutY and the discrete role of various active-site residues. The present work uses both molecular dynamics (MD) and quantum mechanical (ONIOM) models to compare the active-site conformational dynamics in the two crystal structures, which suggests that only the understudied FLRC leads to a catalytically competent reactant. Indeed, all previous computational studies on MutY have been initiated from the LRC structure. Subsequently, for the first time, various mechanisms are examined with detailed ONIOM(M06-2X:PM6) reaction potential energy surfaces (PES) based on the FLRC structure, which significantly extends the mechanistic picture. Specifically, our work reveals that the reaction proceeds through a different route than the commonly accepted mechanism and the catalytic function of various active-site residues (Geobacillus stearothermophilus numbering). Specifically, contrary to proposals based on the LRC, E43 is determined to solely be involved in the initial adenine protonation step and not the deglycosylation reaction as the general base. Additionally, a novel catalytic role is proposed for Y126, whereby this residue plays a significant role in stabilizing the highly charged

  11. Improved solubility of DNA in recyclable and reusable bio-based deep eutectic solvents with long-term structural and chemical stability.

    PubMed

    Mondal, Dibyendu; Sharma, Mukesh; Mukesh, Chandrakant; Gupta, Vishal; Prasad, Kamalesh

    2013-10-25

    The solubility of DNA in bio-based deep eutectic solvents (DESs) consisting of mixtures of choline chloride with levulinic acid, glycerol, ethylene glycol, sorbitol and resorcinol was investigated. The macromolecule was found to be soluble and chemically and structurally stable in DESs consisting of mixtures containing glycerol and ethylene glycol. Furthermore recyclability of the DESs was demonstrated over three consecutive reuses in DNA dissolution. PMID:24022824

  12. Final report : LDRD project 79824 carbon nanotube sorting via DNA-directed self-assembly.

    SciTech Connect

    Robinson, David B; Leung, Kevin; Rempe, Susan B.; Dossa, Paul D.; Frischknecht, Amalie Lucile; Martin, Marcus Gary

    2007-10-01

    Single-wall carbon nanotubes (SWNTs) have shown great promise in novel applications in molecular electronics, biohazard detection, and composite materials. Commercially synthesized nanotubes exhibit a wide dispersion of geometries and conductivities, and tend to aggregate. Hence the key to using these materials is the ability to solubilize and sort carbon nanotubes according to their geometric/electronic properties. One of the most effective dispersants is single-stranded DNA (ssDNA), but there are many outstanding questions regarding the interaction between nucleic acids and SWNTs. In this work we focus on the interactions of SWNTs with single monomers of nucleic acids, as a first step to answering these outstanding questions. We use atomistic molecular dynamics simulations to calculate the binding energy of six different nucleotide monophosphates (NMPs) to a (6,0) single-wall carbon nanotube in aqueous solution. We find that the binding energies are generally favorable, of the order of a few kcal/mol. The binding energies of the different NMPs were very similar in salt solution, whereas we found a range of binding energies for NMPs in pure water. The binding energies are sensitive to the details of the association of the sodium ions with the phosphate groups and also to the average conformations of the nucleotides. We use electronic structure (Density Functional Theory (DFT) and Moller-Plesset second order perturbation to uncorrelated Hartree Fock theory (MP2)) methods to complement the classical force field study. With judicious choices of DFT exchange correlation functionals, we find that DFT, MP2, and classical force field predictions are in qualitative and even quantitative agreement; all three methods should give reliable and valid predictions. However, in one important case, the interactions between ions and metallic carbon nanotubes--the SWNT polarization-induced affinity for ions, neglected in most classical force field studies, is found to be extremely

  13. Evidence of L10 chemical order in CoPt nanoclusters: Direct observation and magnetic signature

    NASA Astrophysics Data System (ADS)

    Tournus, Florent; Tamion, Alexandre; Blanc, Nils; Hannour, Abdelkrim; Bardotti, Laurent; Prével, Brigitte; Ohresser, Philippe; Bonet, Edgar; Epicier, Thierry; Dupuis, Véronique

    2008-04-01

    We report the synthesis and characterization of well-defined CoPt clusters with a mean diameter of 3 nm, produced in ultrahigh vacuum conditions following a physical route. Samples made of diluted layers of CoPt clusters embedded in amorphous carbon have been studied by transmission electron microscopy. High-resolution observations have revealed the appearance of L10 chemical order upon annealing, even for clusters with a 2 nm diameter, without cluster coalescence. The magnetic properties of both chemically disordered and ordered CoPt clusters embedded in amorphous carbon have then been measured by x-ray magnetic circular dichroism and superconducting quantum interference device magnetometry. Despite a striking change of the Co magnetic moment, the magnetic anisotropy of chemically ordered nanoparticles increases, with respect to the chemically disordered A1 phase, in much lower proportions than what is observed for the bulk.

  14. Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3'-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease.

    PubMed

    Yamamoto, Junpei; Takahata, Chiaki; Kuraoka, Isao; Hirota, Kouji; Iwai, Shigenori

    2016-01-01

    Nucleoside/nucleotide analogs that lack the 3'-hydroxy group are widely utilized for HIV therapy. These chain-terminating nucleoside analogs (CTNAs) block DNA synthesis after their incorporation into growing DNA, leading to the antiviral effects. However, they are also considered to be DNA damaging agents, and tyrosyl-DNA phosphodiesterase 1, a DNA repair enzyme, is reportedly able to remove such CTNA-modifications of DNA. Here, we have synthesized phosphoramidite building blocks of representative CTNAs, such as acyclovir, abacavir, carbovir, and lamivudine, and oligonucleotides with the 3'-CTNAs were successfully synthesized on solid supports. Using the chemically synthesized oligonucleotides, we investigated the excision of the 3'-CTNAs in DNA by the human excision repair cross complementing protein 1-xeroderma pigmentosum group F (ERCC1-XPF) endonuclease, which is one of the main components of the nucleotide excision repair pathway. A biochemical analysis demonst