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Sample records for fen1-like nuclease required

  1. Dual Nuclease and Helicase Activities of Helicobacter pylori AddAB Are Required for DNA Repair, Recombination, and Mouse Infectivity*

    OpenAIRE

    Amundsen, Susan K.; Fero, Jutta; Salama, Nina R.; Smith, Gerald R.

    2009-01-01

    Helicobacter pylori infection of the human stomach is associated with disease-causing inflammation that elicits DNA damage in both bacterial and host cells. Bacteria must repair their DNA to persist. The H. pylori AddAB helicase-exonuclease is required for DNA repair and efficient stomach colonization. To dissect the role of each activity in DNA repair and infectivity, we altered the AddA and AddB nuclease (NUC) domains and the AddA helicase (HEL) domain by site-directed mutagenesis. Extracts...

  2. Mre11 nuclease and C-terminal tail-mediated DDR functions are required for initiating yeast telomere healing.

    Science.gov (United States)

    Bhattacharyya, M K; Matthews, K M; Lustig, A J

    2008-08-01

    Mre11 is a central factor in creating an optimal substrate for telomerase loading and elongation. We have used a G2/M synchronized telomere-healing assay as a tool to separate different functions of Mre11 that are not apparent in null alleles. An analysis of healing efficiencies of several mre11 alleles revealed that both nuclease and C-terminal mutations led to a loss of healing. Interestingly, trans-complementation of the 49 amino acid C-terminal deletion (DeltaC49) and the D16A mutant, deficient in nuclease activity and partially defective in MRX complex formation, restores healing. DeltaC49 provokes Rad53 phosphorylation after treatment with the radiomimetic agent MMS exclusively through the Tel1 pathway, suggesting that a Tel1-mediated function is initiated through the C-terminal tail.

  3. Coincident resection at both ends of random, γ-induced double-strand breaks requires MRX (MRN, Sae2 (Ctp1, and Mre11-nuclease.

    Directory of Open Access Journals (Sweden)

    James W Westmoreland

    2013-03-01

    Full Text Available Resection is an early step in homology-directed recombinational repair (HDRR of DNA double-strand breaks (DSBs. Resection enables strand invasion as well as reannealing following DNA synthesis across a DSB to assure efficient HDRR. While resection of only one end could result in genome instability, it has not been feasible to address events at both ends of a DSB, or to distinguish 1- versus 2-end resections at random, radiation-induced "dirty" DSBs or even enzyme-induced "clean" DSBs. Previously, we quantitatively addressed resection and the role of Mre11/Rad50/Xrs2 complex (MRX at random DSBs in circular chromosomes within budding yeast based on reduced pulsed-field gel electrophoretic mobility ("PFGE-shift". Here, we extend PFGE analysis to a second dimension and demonstrate unique patterns associated with 0-, 1-, and 2-end resections at DSBs, providing opportunities to examine coincidence of resection. In G2-arrested WT, Δrad51 and Δrad52 cells deficient in late stages of HDRR, resection occurs at both ends of γ-DSBs. However, for radiation-induced and I-SceI-induced DSBs, 1-end resections predominate in MRX (MRN null mutants with or without Ku70. Surprisingly, Sae2 (Ctp1/CtIP and Mre11 nuclease-deficient mutants have similar responses, although there is less impact on repair. Thus, we provide direct molecular characterization of coincident resection at random, radiation-induced DSBs and show that rapid and coincident initiation of resection at γ-DSBs requires MRX, Sae2 protein, and Mre11 nuclease. Structural features of MRX complex are consistent with coincident resection being due to an ability to interact with both DSB ends to directly coordinate resection. Interestingly, coincident resection at clean I-SceI-induced breaks is much less dependent on Mre11 nuclease or Sae2, contrary to a strong dependence on MRX complex, suggesting different roles for these functions at "dirty" and clean DSB ends. These approaches apply to resection at

  4. The yeast Pan2 protein is required for poly(A)-binding protein-stimulated poly(A)-nuclease activity.

    Science.gov (United States)

    Boeck, R; Tarun, S; Rieger, M; Deardorff, J A; Müller-Auer, S; Sachs, A B

    1996-01-05

    The removal of the mRNA poly(A) tail in the yeast Saccharomyces cerevisiae is stimulated by the poly(A)-binding protein (Pab1p). A large scale purification of the Pab1p-stimulated poly(A) ribonuclease (PAN) identifies a 76-kDa and two 135-Da polypeptides as candidate enzyme subunits. Antibodies against the Pan1p protein, which is the minor 135-kDa protein in the preparation, can immunodeplete Pan1p but not PAN activity. The protein sequence of the major 135-kDa protein, Pan2p, reveals a novel protein that was also found in the previously reported PAN purification (Sachs, A. B., and Deardorff, J. A. (1992) Cell 70, 961-973). Deletion of the non-essential PAN2 gene results in an increase of the average length of mRNA poly(A) tails in vivo, and a loss of Pab1p-stimulated PAN activity in crude extracts. These data confirm that Pan2p and not Pan1p is required for PAN activity, and they suggest that ribonucleases other than the Pab1p-stimulated PAN are capable of shortening poly(A) tails in vivo.

  5. Cloning of a gene from Thermus filiformis and characterization of the thermostable nuclease.

    Science.gov (United States)

    Fomenkov, A; Xu, S Y

    1995-09-22

    A gene coding for a thermostable nuclease was cloned from the thermophilic microorganism, Thermus filiformis (Tf), using an indicator strain containing a dinD::lacZ fusion. The gene, designated nuc17, has been mapped within a 2300-bp fragment. The 55-kDa Tf nuclease was purified to over 95% homogeneity. Single-stranded (ss) DNA is the preferred substrate for the Tf nuclease, although double-stranded (ds) DNA can also be digested. Nuclease activity increases with increasing temperature up to 80 degrees C and requires the metal ions Ca++ or Mg++ for catalysis. Tf nuclease is primarily an endonuclease that leaves 5' phosphates in the digested products. The ssDNA extensions remaining after exonuclease III digestion of dsDNA can be removed by the Tf nuclease, making it a useful reagent to generate unidirectional deletions.

  6. Zinc-finger nucleases: a panoramic view.

    Science.gov (United States)

    Carroll, Dana

    2011-02-01

    Zinc-finger nucleases (ZFNs) are emerging as very powerful tools for directed genome modifications. Their key features are: a DNA-binding domain comprised of zinc fingers that can be designed to favor very specific targets; a nonspecific cleavage domain that must dimerize to cut DNA--this requirement enhances specificity and minimizes random cleavage. ZFNs have been shown to be effective in a wide range of organisms and cell types. This article reviews discoveries that led to the development of ZFNs, cites examples of successes in genome engineering, and projects how ZFNs may be used in the future, particularly in applications to humans.

  7. Copper complexes as chemical nucleases

    Indian Academy of Sciences (India)

    Unknown

    Chemical nucleases are redox active coordination complexes that cleave DNA by an oxidative pathway. ... with a reductant like ascorbate, reduced glutathione or NADH in DNA strand breaking giving the order T > G > C > A 16,17. .... The emission intensity of CT DNA-bound ethidium bromide (12⋅5 µM) at different complex ...

  8. Nucleases from Prevotella intermedia can degrade neutrophil extracellular traps.

    Science.gov (United States)

    Doke, M; Fukamachi, H; Morisaki, H; Arimoto, T; Kataoka, H; Kuwata, H

    2017-08-01

    Periodontitis is an inflammatory disease caused by periodontal bacteria in subgingival plaque. These bacteria are able to colonize the periodontal region by evading the host immune response. Neutrophils, the host's first line of defense against infection, use various strategies to kill invading pathogens, including neutrophil extracellular traps (NETs). These are extracellular net-like fibers comprising DNA and antimicrobial components such as histones, LL-37, defensins, myeloperoxidase, and neutrophil elastase from neutrophils that disarm and kill bacteria extracellularly. Bacterial nuclease degrades the NETs to escape NET killing. It has now been shown that extracellular nucleases enable bacteria to evade this host antimicrobial mechanism, leading to increased pathogenicity. Here, we compared the DNA degradation activity of major Gram-negative periodontopathogenic bacteria, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. We found that Pr. intermedia showed the highest DNA degradation activity. A genome search of Pr. intermedia revealed the presence of two genes, nucA and nucD, putatively encoding secreted nucleases, although their enzymatic and biological activities are unknown. We cloned nucA- and nucD-encoding nucleases from Pr. intermedia ATCC 25611 and characterized their gene products. Recombinant NucA and NucD digested DNA and RNA, which required both Mg 2+ and Ca 2+ for optimal activity. In addition, NucA and NucD were able to degrade the DNA matrix comprising NETs. © 2016 The Authors Molecular Oral Microbiology Published by John Wiley & Sons Ltd.

  9. Designed nucleases for targeted genome editing.

    Science.gov (United States)

    Lee, Junwon; Chung, Jae-Hee; Kim, Ho Min; Kim, Dong-Wook; Kim, Hyongbum

    2016-02-01

    Targeted genome-editing technology using designed nucleases has been evolving rapidly, and its applications are widely expanding in research, medicine and biotechnology. Using this genome-modifying technology, researchers can precisely and efficiently insert, remove or change specific sequences in various cultured cells, micro-organisms, animals and plants. This genome editing is based on the generation of double-strand breaks (DSBs), repair of which modifies the genome through nonhomologous end-joining (NHEJ) or homology-directed repair (HDR). In addition, designed nickase-induced generation of single-strand breaks can also lead to precise genome editing through HDR, albeit at relatively lower efficiencies than that induced by nucleases. Three kinds of designed nucleases have been used for targeted DSB formation: zinc-finger nucleases, transcription activator-like effector nucleases, and RNA-guided engineered nucleases derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) system. A growing number of researchers are using genome-editing technologies, which have become more accessible and affordable since the discovery and adaptation of CRISPR-Cas9. Here, the repair mechanism and outcomes of DSBs are reviewed and the three types of designed nucleases are discussed with the hope that such understanding will facilitate applications to genome editing. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  10. Elimination of double strand nuclease activity from S1 nuclease prepared from crude alpha amylase.

    Science.gov (United States)

    Hahn, W E; Van Ness, J

    1976-01-01

    Single strand-specific s1 nuclease prepared as previously described from crude alpha amylase by DEAE-cellulose chromatography also contains nuclease which degrades double strand nucleic acid. The double strand activity can be removed by repeating the DEAE-cellulose chromatography procedure at least two additional times. S1 nuclease prepared by this procedure does not degrade double strand sheared DNA as measured by Sephadex chromatography. Under the same conditions single strand DNA is completely degraded. Thus, S1 nuclease prepared by this procedure is suitable for use in removing single strand regions in DNA/DNA duplexes and DNA/RNA hybrids. PMID:940774

  11. Tudor staphylococcal nuclease: biochemistry and functions

    OpenAIRE

    Gutierrez-Beltran, Emilio; Denisenko, Tatiana V; Zhivotovsky, Boris; Bozhkov, Peter V

    2016-01-01

    Tudor staphylococcal nuclease (TSN, also known as Tudor-SN, SND1 or p100) is an evolutionarily conserved protein with invariant domain composition, represented by tandem repeat of staphylococcal nuclease domains and a tudor domain. Conservation along significant evolutionary distance, from protozoa to plants and animals, suggests important physiological functions for TSN. It is known that TSN is critically involved in virtually all pathways of gene expression, ranging from transcription to RN...

  12. Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases

    Directory of Open Access Journals (Sweden)

    Kentaro Ishida

    2015-10-01

    Full Text Available Programmable nucleases, such as zinc finger nucleases (ZFNs, transcription activator like effector nucleases (TALENs, and clustered regularly interspersed short palindromic repeats associated protein-9 (CRISPR-Cas9, hold tremendous potential for applications in the clinical setting to treat genetic diseases or prevent infectious diseases. However, because the accuracy of DNA recognition by these nucleases is not always perfect, off-target mutagenesis may result in undesirable adverse events in treated patients such as cellular toxicity or tumorigenesis. Therefore, designing nucleases and analyzing their activity must be carefully evaluated to minimize off-target mutagenesis. Furthermore, rigorous genomic testing will be important to ensure the integrity of nuclease modified cells. In this review, we provide an overview of available nuclease designing platforms, nuclease engineering approaches to minimize off-target activity, and methods to evaluate both on- and off-target cleavage of CRISPR-Cas9.

  13. Synthesis, characterisation, nuclease and cytotoxic activity of ...

    Indian Academy of Sciences (India)

    Complexes 1 and 2 were evaluated for their nuclease and in vitro anti-tumor activities against human breast and colorectal cancer cell lines. The DNA cleavage and cytotoxic assays revealed that both 1 and 2 are effective in cleaving DNA, while the cytotoxic activity of 1 is better than 2 in both human colon and breast cancer ...

  14. Synthesis, characterisation, nuclease and cytotoxic activity of ...

    Indian Academy of Sciences (India)

    GULZAR A BHAT

    2018-02-07

    Feb 7, 2018 ... Synthesis, characterisation, nuclease and cytotoxic activity of phosphate-free and phosphate-containing copper. 4 -(N-methylpyridinium)-2,2 :6 ,2 terpyridine complexes. GULZAR A BHATa, RAIHANA MAQBOOLb and RAMASWAMY MURUGAVELa,∗. aDepartment of Chemistry, Indian Institute of ...

  15. The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

    Science.gov (United States)

    Hilbert, Brendan J.; Hayes, Janelle A.; Stone, Nicholas P.; Xu, Rui-Gang

    2017-01-01

    Abstract Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA. PMID:28082398

  16. Eukaryotic zinc-dependent multifunctional nuclease I

    Czech Academy of Sciences Publication Activity Database

    Koval, Tomáš; Lipovová, P.; Podzimek, T.; Matoušek, Jaroslav; Stránský, Jan; Dušková, Jarmila; Fejfarová, Karla; Skálová, Tereza; Hašek, Jindřich; Dohnálek, Jan

    2014-01-01

    Roč. 70, Supplement /August/ (2014), C211 ISSN 0108-7673. [Congress and General Assembly of the International Union of Crystallography /23./ - IUCr 2014. 05.08.2014-12.08.2014, Montreal] R&D Projects: GA MŠk(CZ) EE2.3.30.0029; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61389013 ; RVO:86652036 ; RVO:60077344 Keywords : nuclease * tomato * crystal structure Subject RIV: CE - Biochemistry

  17. Genome editing with engineered nucleases in plants.

    Science.gov (United States)

    Osakabe, Yuriko; Osakabe, Keishi

    2015-03-01

    Numerous examples of successful 'genome editing' now exist. Genome editing uses engineered nucleases as powerful tools to target specific DNA sequences to edit genes precisely in the genomes of both model and crop plants, as well as a variety of other organisms. The DNA-binding domains of zinc finger (ZF) proteins were the first to be used as genome editing tools, in the form of designed ZF nucleases (ZFNs). More recently, transcription activator-like effector nucleases (TALENs), as well as the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system, which utilizes RNA-DNA interactions, have proved useful. A key step in genome editing is the generation of a double-stranded DNA break that is specific to the target gene. This is achieved by custom-designed endonucleases, which enable site-directed mutagenesis via a non-homologous end-joining (NHEJ) repair pathway and/or gene targeting via homologous recombination (HR) to occur efficiently at specific sites in the genome. This review provides an overview of recent advances in genome editing technologies in plants, and discusses how these can provide insights into current plant molecular biology research and molecular breeding technology. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  18. Origins of Programmable Nucleases for Genome Engineering.

    Science.gov (United States)

    Chandrasegaran, Srinivasan; Carroll, Dana

    2016-02-27

    Genome engineering with programmable nucleases depends on cellular responses to a targeted double-strand break (DSB). The first truly targetable reagents were the zinc finger nucleases (ZFNs) showing that arbitrary DNA sequences could be addressed for cleavage by protein engineering, ushering in the breakthrough in genome manipulation. ZFNs resulted from basic research on zinc finger proteins and the FokI restriction enzyme (which revealed a bipartite structure with a separable DNA-binding domain and a non-specific cleavage domain). Studies on the mechanism of cleavage by 3-finger ZFNs established that the preferred substrates were paired binding sites, which doubled the size of the target sequence recognition from 9 to 18bp, long enough to specify a unique genomic locus in plant and mammalian cells. Soon afterwards, a ZFN-induced DSB was shown to stimulate homologous recombination in cells. Transcription activator-like effector nucleases (TALENs) that are based on bacterial TALEs fused to the FokI cleavage domain expanded this capability. The fact that ZFNs and TALENs have been used for genome modification of more than 40 different organisms and cell types attests to the success of protein engineering. The most recent technology platform for delivering a targeted DSB to cellular genomes is that of the RNA-guided nucleases, which are based on the naturally occurring Type II prokaryotic CRISPR-Cas9 system. Unlike ZFNs and TALENs that use protein motifs for DNA sequence recognition, CRISPR-Cas9 depends on RNA-DNA recognition. The advantages of the CRISPR-Cas9 system-the ease of RNA design for new targets and the dependence on a single, constant Cas9 protein-have led to its wide adoption by research laboratories around the world. These technology platforms have equipped scientists with an unprecedented ability to modify cells and organisms almost at will, with wide-ranging implications across biology and medicine. However, these nucleases have also been shown to cut

  19. Interaction of nuclease colicins with membranes: insertion depth correlates with bilayer perturbation.

    Directory of Open Access Journals (Sweden)

    Mireille Vankemmelbeke

    Full Text Available Protein transport across cellular membranes is an important aspect of toxin biology. Escherichia coli cell killing by nuclease colicins occurs through DNA (DNases or RNA (RNases hydrolysis and to this end their cytotoxic domains require transportation across two sets of membranes. In order to begin to unravel the molecular mechanisms underlying the membrane translocation of colicin nuclease domains, we have analysed the membrane association of four DNase domains (E9, a charge reduction E9 mutant, E8, and E7 and one ribosomal RNase domain (E3 using a biomembrane model system.We demonstrate, through the use of large unilamellar vesicles composed of synthetic and E. coli lipids and a membrane surface potential sensor, that the colicin nuclease domains bind anionic membranes only, with micromolar affinity and via a cooperative binding mechanism. The evaluation of the nuclease bilayer insertion depth, through a fluorescence quenching analysis using brominated lipids, indicates that the nucleases locate to differential regions in the bilayer. Colicin DNases target the interfacial region of the lipid bilayer, with the DNase E7 showing the deepest insertion, whereas the ribosomal RNase E3 penetrates into the hydrophobic core region of the bilayer. Furthermore, the membrane association of the DNase E7 and the ribosomal RNase E3 induces vesicle aggregation, lipid mixing and content leakage to a much larger extent than that of the other DNases analysed.Our results show, for the first time, that after the initial electrostatically driven membrane association, the pleiotropic membrane effects induced by colicin nuclease domains relate to their bilayer insertion depth and may be linked to their in vivo membrane translocation.

  20. A guide to genome engineering with programmable nucleases.

    Science.gov (United States)

    Kim, Hyongbum; Kim, Jin-Soo

    2014-05-01

    Programmable nucleases - including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases (RGENs) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) system - enable targeted genetic modifications in cultured cells, as well as in whole animals and plants. The value of these enzymes in research, medicine and biotechnology arises from their ability to induce site-specific DNA cleavage in the genome, the repair (through endogenous mechanisms) of which allows high-precision genome editing. However, these nucleases differ in several respects, including their composition, targetable sites, specificities and mutation signatures, among other characteristics. Knowledge of nuclease-specific features, as well as of their pros and cons, is essential for researchers to choose the most appropriate tool for a range of applications.

  1. Genome editing with engineered zinc finger nucleases.

    Science.gov (United States)

    Urnov, Fyodor D; Rebar, Edward J; Holmes, Michael C; Zhang, H Steve; Gregory, Philip D

    2010-09-01

    Reverse genetics in model organisms such as Drosophila melanogaster, Arabidopsis thaliana, zebrafish and rats, efficient genome engineering in human embryonic stem and induced pluripotent stem cells, targeted integration in crop plants, and HIV resistance in immune cells - this broad range of outcomes has resulted from the application of the same core technology: targeted genome cleavage by engineered, sequence-specific zinc finger nucleases followed by gene modification during subsequent repair. Such 'genome editing' is now established in human cells and a number of model organisms, thus opening the door to a range of new experimental and therapeutic possibilities.

  2. Structure analysis of group I plant nucleases

    Czech Academy of Sciences Publication Activity Database

    Dohnálek, Jan; Kovaľ, Tomáš; Lipovová, P.; Podzimek, Tomáš; Matoušek, Jaroslav

    2011-01-01

    Roč. 18, Part 1 (2011), s. 29-30 ISSN 0909-0495. [International Symposium on Diffraction Structural Biology /3./. Paris - Orsay, 25.05.2010-28.05.2010] R&D Projects: GA ČR GA310/09/1407; GA ČR GA521/09/1214 Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z10100521; CEZ:AV0Z50510513 Keywords : nuclease * protein structure * crystal structure Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.726, year: 2011

  3. TALE nucleases and next generation GM crops.

    KAUST Repository

    Mahfouz, Magdy M.

    2011-04-01

    Site-specific and adaptable DNA binding domains are essential modules to develop genome engineering technologies for crop improvement. Transcription activator-like effectors (TALEs) proteins are used to provide a highly specific and adaptable DNA binding modules. TALE chimeric nucleases (TALENs) were used to generate site-specific double strand breaks (DSBs) in vitro and in yeast, Caenorhabditis elegans, mammalian and plant cells. The genomic DSBs can be generated at predefined and user-selected loci and repaired by either the non-homologous end joining (NHEJ) or homology dependent repair (HDR). Thus, TALENs can be used to achieve site-specific gene addition, stacking, deletion or inactivation. TALE-based genome engineering tools should be powerful to develop new agricultural biotechnology approaches for crop improvement. Here, we discuss the recent research and the potential applications of TALENs to accelerate the generation of genomic variants through targeted mutagenesis and to produce a non-transgenic GM crops with the desired phenotype.

  4. Role of recBC nuclease in Escherichia coli transformation.

    OpenAIRE

    Hoekstra, W P; Bergmans, J E; Zuidweg, E M

    1980-01-01

    In Escherichia coli transformation with linear donor deoxyribonucleic acid, the recBC pathway is functional, but genetic analysis shows that the recBC nuclease is deleterious to linear deoxyribonucleic acid.

  5. Catalytic activity of nuclease P1: Experiment and theory

    International Nuclear Information System (INIS)

    Miller, J.H.; Falcone, J.M.; Shibata, M.; Box, H.C.

    1994-10-01

    Nuclease P1 from Penicillium citrinum is a zinc dependent glyco-enzyme that recognizes single stranded DNA and RNA as substrates and hydrolyzes the phosphate ester bond. Nuclease Pl seems to recognize particular conformations of the phosphodiester backbone and shows significant variation in the rate of hydrolytic activity depending upon which nucleosides are coupled by the phosphodiester bond. The efficiency of nuclease Pl in hydrolyzing the phosphodiester bonds of a substrate can be altered by modifications to one of the substrate bases induced by ionizing radiation or oxidative stress. Measurements have been made of the effect of several radiation induced lesions on the catalytic rate of nuclease Pl. A model of the structure of the enzyme has been constructed in order to better understand the binding and activity of this enzyme on various ssDNA substrates

  6. Solving RNA's structural secrets: interaction with antibodies and crystal structure of a nuclease resistant RNA

    International Nuclear Information System (INIS)

    Wallace, S.T.

    1998-10-01

    This Ph.D. thesis concerns the structural characterization of RNA. The work is split into two sections: 1) in vitro selection and characterization of RNAs which bind antibiotics and 2) crystal structure of a nuclease resistant RNA molecule used in antisense applications. Understanding antibiotic-RNA interactions is crucial in aiding rational drug design. We were interested in studying antibiotic interactions with RNAs small enough to characterize at the molecular and possibly at the atomic level. In order to do so, we previously performed in vitro selection to find small RNAs which bind to the peptide antibiotic viomycin and the aminoglycoside antibiotic streptomycin. The characterization of the viomycin-binding RNAs revealed the necessity of a pseudoknot-structure in order to interact with the antibiotic. The RNAs which were selected to interact with streptomycin require the presence of magnesium to bind the antibiotic. One of the RNAs, upon interacting with streptomycin undergoes a significant conformational change spanning the entire RNA sequence needed to bind the antibiotic. In a quest to design oligodeoxynucleotides (ODNs) which are able to specifically bid and inactivate the mRNA of a gene, it is necessary to fulfill two criteria: 1) increase binding affinity between the ODN and the target RNA and 2) increase the ODN's resistance to nuclease degradation. An ODN with an aminopropyl modification at the 2' position of its ribose has emerged as the most successful candidate at fulfilling both criteria. It is the most nuclease resistant modification known to date. We were interested in explaining how this modification is able to circumvent degradation by nucleases. A dodecamer containing a single 2'-O-aminopropyl modified nucleotide was crystallized and the structure was solved to a resolution of 1.6 A. In an attempt to explain the nuclease resistance, the crystal coordinates were modeled into the active exonuclease site of DNA polymerase I. We propose the

  7. Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

    Science.gov (United States)

    Nerys-Junior, Arildo; Costa, Lendel C.; Braga-Dias, Luciene P.; Oliveira, Márcia; Rossi, Átila D.; da Cunha, Rodrigo Delvecchio; Gonçalves, Gabriel S.; Tanuri, Amilcar

    2014-01-01

    Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity. PMID:24688299

  8. Genetic correction using engineered nucleases for gene therapy applications.

    Science.gov (United States)

    Li, Hongmei Lisa; Nakano, Takao; Hotta, Akitsu

    2014-01-01

    Genetic mutations in humans are associated with congenital disorders and phenotypic traits. Gene therapy holds the promise to cure such genetic disorders, although it has suffered from several technical limitations for decades. Recent progress in gene editing technology using tailor-made nucleases, such as meganucleases (MNs), zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs) and, more recently, CRISPR/Cas9, has significantly broadened our ability to precisely modify target sites in the human genome. In this review, we summarize recent progress in gene correction approaches of the human genome, with a particular emphasis on the clinical applications of gene therapy. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

  9. Crystallization and preliminary X-ray analysis of a RecB-family nuclease from the archaeon Pyrococcus abyssi

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Bin, E-mail: ren@csb.ki.se [Center for Structural Biochemistry, Karolinska Institute, NOVUM, S-141 57 Huddinge (Sweden); Kuhn, Joëlle; Meslet-Cladiere, Laurence; Myllykallio, Hannu [Université Paris-Sud, Institut de Génétique et Microbiologie, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8621, F-91405 Orsay CEDEX (France); Ladenstein, Rudolf [Center for Structural Biochemistry, Karolinska Institute, NOVUM, S-141 57 Huddinge (Sweden)

    2007-05-01

    A RecB-like nuclease from the archaeon Pyrococcus abyssi was expressed, purified and crystallized. The crystals belong to the orthorhombic space group C222{sub 1} with a = 81.5, b = 159.8, c = 100.8 Å, and a native data set was collected to 2.65 Å resolution. Nucleases are required to process and repair DNA damage in living cells. One of the best studied nucleases is the RecB protein, which functions in Escherichia coli as a component of the RecBCD enzyme complex that amends double-strand breaks in DNA. Although archaea do not contain the RecBCD complex, a RecB-like nuclease from Pyrococcus abyssi has been cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant. The crystals belong to the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 81.5, b = 159.8, c = 100.8 Å. Self-rotation function and native Patterson map calculations revealed that there is a dimer in the asymmetric unit with its local twofold axis running parallel to the crystallographic twofold screw axis. The crystals diffracted to about 2 Å and a complete native data set was collected to 2.65 Å resolution.

  10. Isolation, characterization and molecular cloning of Duplex-Specific Nuclease from the hepatopancreas of the Kamchatka crab

    Directory of Open Access Journals (Sweden)

    Vagner Laura L

    2008-05-01

    Full Text Available Abstract Background Nucleases, which are key components of biologically diverse processes such as DNA replication, repair and recombination, antiviral defense, apoptosis and digestion, have revolutionized the field of molecular biology. Indeed many standard molecular strategies, including molecular cloning, studies of DNA-protein interactions, and analysis of nucleic acid structures, would be virtually impossible without these versatile enzymes. The discovery of nucleases with unique properties has often served as the basis for the development of modern molecular biology methods. Thus, the search for novel nucleases with potentially exploitable functions remains an important scientific undertaking. Results Using degenerative primers and the rapid amplification of cDNA ends (RACE procedure, we cloned the Duplex-Specific Nuclease (DSN gene from the hepatopancreas of the Kamchatka crab and determined its full primary structure. We also developed an effective method for purifying functional DSN from the crab hepatopancreas. The isolated enzyme was highly thermostable, exhibited a broad pH optimum (5.5 – 7.5 and required divalent cations for activity, with manganese and cobalt being especially effective. The enzyme was highly specific, cleaving double-stranded DNA or DNA in DNA-RNA hybrids, but not single-stranded DNA or single- or double-stranded RNA. Moreover, only DNA duplexes containing at least 9 base pairs were effectively cleaved by DSN; shorter DNA duplexes were left intact. Conclusion We describe a new DSN from Kamchatka crab hepatopancreas, determining its primary structure and developing a preparative method for its purification. We found that DSN had unique substrate specificity, cleaving only DNA duplexes longer than 8 base pairs, or DNA in DNA-RNA hybrids. Interestingly, the DSN primary structure is homologous to well-known Serratia-like non-specific nucleases structures, but the properties of DSN are distinct. The unique substrate

  11. Antitumor activity od apoptotic nuclease TBN1 from L. esculentum

    Czech Academy of Sciences Publication Activity Database

    Matoušek, Jaroslav; Podzimek, Tomáš; Poučková, P.; Stehlík, Jan; Škvor, J.; Lipovová, P.; Matoušek, Josef

    2010-01-01

    Roč. 57, č. 4 (2010), s. 339-348 ISSN 0028-2685 R&D Projects: GA ČR GA521/06/1149; GA ČR GA521/09/1214 Institutional research plan: CEZ:AV0Z50510513; CEZ:AV0Z50450515 Keywords : anticancerogenic and antiproliferative nuclease * dsRNase * human solid malignant tumors Subject RIV: FD - Oncology ; Hematology Impact factor: 1.449, year: 2010

  12. Antitumor Effects and Cytotoxicity of Recombinant Plant Nucleases

    Czech Academy of Sciences Publication Activity Database

    Matoušek, Jaroslav; Podzimek, Tomáš; Pouckova, P.; Stehlík, Jan; Škvor, J.; Souček, J.; Matoušek, Josef

    2009-01-01

    Roč. 18, č. 4 (2009), s. 163-171 ISSN 0965-0407 R&D Projects: GA ČR GA521/09/1214 Institutional research plan: CEZ:AV0Z50510513; CEZ:AV0Z50450515 Keywords : Anticarcinogenic and antiproliferative nucleases * Human melanoma * Tumor xenografts * Nicotiana benthamina Subject RIV: FD - Oncology ; Hematology Impact factor: 1.478, year: 2009

  13. Nanoplasmonic molecular ruler for nuclease activity and DNAfootprinting

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Fanqing Frank; Liu, Gang L.; Yin, Yadong; Gerion, Daniele; Kunchakarra, Siri; Mukherjee, Bipasha; Jett, Stephen D.; Bear, David G.; Alivisatos, Paul; Lee, Luke P.

    2006-08-15

    We have constructed a nanoplasmonic molecular ruler, which can perform label-free and real-time monitoring of DNA length changes and perform DNA footprinting. The ruler was created by tethering double-stranded DNA to single Au nanoparticles. The scattering spectra of Au-DNA nanoconjugates showed red-shifted peak plasmon resonance wavelength dependent on DNA length, which can be measured with sub-nanometer axial resolution, averaging {approx}1.24 nm peak wavelength shift per DNA base pair. The spectra of individual Au-DNA nanoconjugates in the presence of nuclease showed a time-resolved dependence on the reaction dynamics, allowing quantitative, kinetic and real-time measurement of nuclease activity. The ruler was further developed into a new DNA footprinting platform. We showed the specific binding of a protein to DNA and the accurate mapping of its footprint. This work promises a very fast and convenient platform for mapping DNA-protein interactions, for nuclease activity monitoring, and for other DNA size-based methods.

  14. Phylogenomic analysis of the GIY-YIG nuclease superfamily

    Directory of Open Access Journals (Sweden)

    Bujnicki Janusz M

    2006-04-01

    Full Text Available Abstract Background The GIY-YIG domain was initially identified in homing endonucleases and later in other selfish mobile genetic elements (including restriction enzymes and non-LTR retrotransposons and in enzymes involved in DNA repair and recombination. However, to date no systematic search for novel members of the GIY-YIG superfamily or comparative analysis of these enzymes has been reported. Results We carried out database searches to identify all members of known GIY-YIG nuclease families. Multiple sequence alignments together with predicted secondary structures of identified families were represented as Hidden Markov Models (HMM and compared by the HHsearch method to the uncharacterized protein families gathered in the COG, KOG, and PFAM databases. This analysis allowed for extending the GIY-YIG superfamily to include members of COG3680 and a number of proteins not classified in COGs and to predict that these proteins may function as nucleases, potentially involved in DNA recombination and/or repair. Finally, all old and new members of the GIY-YIG superfamily were compared and analyzed to infer the phylogenetic tree. Conclusion An evolutionary classification of the GIY-YIG superfamily is presented for the very first time, along with the structural annotation of all (subfamilies. It provides a comprehensive picture of sequence-structure-function relationships in this superfamily of nucleases, which will help to design experiments to study the mechanism of action of known members (especially the uncharacterized ones and will facilitate the prediction of function for the newly discovered ones.

  15. The involvement of nuclear nucleases in rat thymocyte DNA degradation after γ-irradiation

    International Nuclear Information System (INIS)

    Nikonova, L.V.; Nelipovich, P.A.; Umansky, S.R.

    1982-01-01

    Possible mechanisms of internucleosomal DNA fragmentation in thymocytes of irradiated rats were studied. It was shown that thymocyte nuclei contain at least two nucleases that cleave DNA between nucleosomes - a Ca 2+ /Mg 2+ -dependent nuclease and an acidic one which does not depend on bivalent ions. 2 and 3 h after irradiation at a dose of 10 Gy the initial rate of DNA cleavage by Ca 2+ /Mg 2+ -dependent nuclease in isolated nuclei increased three and seven times, respectively, but the kinetics of DNA digestion by acidic nuclease did not change. The experiments with cycloheximide indicated that Ca 2+ /Mg 2+ -dependent endonuclease turns over at a high rate. The activity of the cytoplasmic acidic and Mg 2+ -dependent nucleases was shown to increase (by 40 and 50%, respectively) 3h after irradiation. The effect is caused by the de novo synthesis of the nucleases. At the same time the activity of nuclear nucleases did not essentially change. The chromatin isolated from rat thymocytes 3 h after irradiation did not differ in its sensitivity to some exogenic nucleases (DNAase I, micrococcal nuclease and nuclease from Serratia marcescens) from the control. Thus, Ca 2+ /Mg 2+ -dependent endonuclease seems to be responsible for the postirradiation internucleosomal DNA fragmentation in dying thymocytes. (Auth.)

  16. Applications of Alternative Nucleases in the Age of CRISPR/Cas9

    Directory of Open Access Journals (Sweden)

    Tuhin K. Guha

    2017-11-01

    Full Text Available Breakthroughs in the development of programmable site-specific nucleases, including zinc-finger nucleases (ZFNs, transcription activator-like effector nucleases (TALENs, meganucleases (MNs, and most recently, the clustered regularly interspaced short palindromic repeats (CRISPR associated proteins (including Cas9 have greatly enabled and accelerated genome editing. By targeting double-strand breaks to user-defined locations, the rates of DNA repair events are greatly enhanced relative to un-catalyzed events at the same sites. However, the underlying biology of each genome-editing nuclease influences the targeting potential, the spectrum of off-target cleavages, the ease-of-use, and the types of recombination events at targeted double-strand breaks. No single genome-editing nuclease is optimized for all possible applications. Here, we focus on the diversity of nuclease domains available for genome editing, highlighting biochemical properties and the potential applications that are best suited to each domain.

  17. Current and future delivery systems for engineered nucleases: ZFN, TALEN and RGEN.

    Science.gov (United States)

    Ul Ain, Qurrat; Chung, Jee Young; Kim, Yong-Hee

    2015-05-10

    Gene therapy by engineered nucleases is a genetic intervention being investigated for curing the hereditary disorders by targeting selected genes with specific nucleotides for establishment, suppression, abolishment of a function or correction of mutation. Here, we review the fast developing technology of targeted genome engineering using site specific programmable nucleases zinc finger nucleases (ZFNs), transcription activator like nucleases (TALENs) and cluster regulatory interspaced short palindromic repeat/CRISPR associated proteins (CRISPR/Cas) based RNA-guided DNA endonucleases (RGENs) and their different characteristics including pros and cons of genome modifications by these nucleases. We have further discussed different types of delivery methods to induce gene editing, novel development in genetic engineering other than nucleases and future prospects. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Crystallization and preliminary X-ray analysis of a RecB-family nuclease from the archaeon Pyrococcus abyssi.

    Science.gov (United States)

    Ren, Bin; Kuhn, Joëlle; Meslet-Cladiere, Laurence; Myllykallio, Hannu; Ladenstein, Rudolf

    2007-05-01

    Nucleases are required to process and repair DNA damage in living cells. One of the best studied nucleases is the RecB protein, which functions in Escherichia coli as a component of the RecBCD enzyme complex that amends double-strand breaks in DNA. Although archaea do not contain the RecBCD complex, a RecB-like nuclease from Pyrococcus abyssi has been cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method using polyethylene glycol 8000 as the precipitant. The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 81.5, b = 159.8, c = 100.8 A. Self-rotation function and native Patterson map calculations revealed that there is a dimer in the asymmetric unit with its local twofold axis running parallel to the crystallographic twofold screw axis. The crystals diffracted to about 2 A and a complete native data set was collected to 2.65 A resolution.

  19. Intra and extracellular nuclease production by Aspergillus niger and Aspergillus nidulans

    Directory of Open Access Journals (Sweden)

    Ferreira Adlane V. B.

    1998-01-01

    Full Text Available Intra and extracellular nuclease production by strains of Aspergillus niger and Aspergillus nidulans was estimated using a modified DNAse test agar and cell-free extract assays. Differences in the production of nucleases by A. niger and A. nidulans were observed. These observations suggest that the DNAse test agar can be helpful for a quick screening for some types of nucleases in filamentous fungi. The assays using cell-free extracts can also be useful for initial characterization of other types of nucleases.

  20. Chemical Approach to Biological Safety: Molecular-Level Control of an Integrated Zinc Finger Nuclease

    DEFF Research Database (Denmark)

    Németh, Eszter; Asaka, Masamitsu N; Kato, Kohsuke

    2018-01-01

    Application of artificial nucleases (ANs) in genome editing is still hindered by their cytotoxicity related to off-target cleavages. This problem can be targeted by regulation of the nuclease domain. Here, we provide an experimental survey of computationally designed integrated zinc finger...... nucleases, constructed by linking the inactivated catalytic centre and the allosteric activator sequence of the colicin E7 nuclease domain to the two opposite termini of a zinc finger array. DNA specificity and metal binding were confirmed by electrophoretic mobility shift assays, synchrotron radiation...

  1. Crystal structure of a Fanconi anemia-associated nuclease homolog bound to 5' flap DNA: basis of interstrand cross-link repair by FAN1

    Energy Technology Data Exchange (ETDEWEB)

    Gwon, Gwang Hyeon; Kim, Youngran; Liu, Yaqi; Watson, Adam T.; Jo, Aera; Etheridge, Thomas J.; Yuan, Fenghua; Zhang, Yanbin; Kim, YoungChang; Carr, Anthony M.; Cho, Yunje

    2014-10-15

    Fanconi anemia (FA) is an autosomal recessive genetic disorder caused by defects in any of 15 FA genes responsible for processing DNA interstrand cross-links (ICLs). The ultimate outcome of the FA pathway is resolution of cross-links, which requires structure-selective nucleases. FA-associated nuclease 1 (FAN1) is believed to be recruited to lesions by a monoubiquitinated FANCI–FANCD2 (ID) complex and participates in ICL repair. Here, we determined the crystal structure of Pseudomonas aeruginosa FAN1 (PaFAN1) lacking the UBZ (ubiquitin-binding zinc) domain in complex with 5' flap DNA. All four domains of the right-hand-shaped PaFAN1 are involved in DNA recognition, with each domain playing a specific role in bending DNA at the nick. The six-helix bundle that binds the junction connects to the catalytic viral replication and repair (VRR) nuclease (VRR nuc) domain, enabling FAN1 to incise the scissile phosphate a few bases distant from the junction. The six-helix bundle also inhibits the cleavage of intact Holliday junctions. PaFAN1 shares several conserved features with other flap structure-selective nucleases despite structural differences. A clamping motion of the domains around the wedge helix, which acts as a pivot, facilitates nucleolytic cleavage. The PaFAN1 structure provides insights into how archaeal Holliday junction resolvases evolved to incise 5' flap substrates and how FAN1 integrates with the FA complex to participate in ICL repair.

  2. Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1

    Energy Technology Data Exchange (ETDEWEB)

    Gruenig, Marielle C.; Lu, Duo; Won, Sang Joon; Dulberger, Charles L.; Manlick, Angela J.; Keck, James L.; Cox, Michael M. (UW)

    2012-03-16

    The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosen DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 {angstrom} resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded {beta}-hairpin that is sandwiched between several {alpha}-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.

  3. Transcription Activator-Like Effectors (TALEs) Hybrid Nucleases for Genome Engineering Application

    KAUST Repository

    Wibowo, Anjar

    2011-06-06

    Gene targeting is a powerful genome engineering tool that can be used for a variety of biotechnological applications. Genomic double-strand DNA breaks generated by engineered site-specific nucleases can stimulate gene targeting. Hybrid nucleases are composed of DNA binding module and DNA cleavage module. Zinc Finger Nucleases were used to generate double-strand DNA breaks but it suffers from failures and lack of reproducibility. The transcription activator–like effectors (TALEs) from plant pathogenic Xanthomonas contain a unique type of DNA-binding domain that bind specific DNA targets. The purpose of this study is to generate novel sequence specific nucleases by fusing a de novo engineered Hax3 TALE-based DNA binding domain to a FokI cleavage domain. Our data show that the de novo engineered TALE nuclease can bind to its target sequence and create double-strand DNA breaks in vitro. We also show that the de novo engineered TALE nuclease is capable of generating double-strand DNA breaks in its target sequence in vivo, when transiently expressed in Nicotiana benthamiana leaves. In conclusion, our data demonstrate that TALE-based hybrid nucleases can be tailored to bind a user-selected DNA sequence and generate site-specific genomic double-strand DNA breaks. TALE-based hybrid nucleases hold much promise as powerful molecular tools for gene targeting applications.

  4. Diversity and distribution of nuclease bacteriocins in bacterial genomes revealed using Hidden Markov Models.

    Directory of Open Access Journals (Sweden)

    Connor Sharp

    2017-07-01

    Full Text Available Bacteria exploit an arsenal of antimicrobial peptides and proteins to compete with each other. Three main competition systems have been described: type six secretion systems (T6SS; contact dependent inhibition (CDI; and bacteriocins. Unlike T6SS and CDI systems, bacteriocins do not require contact between bacteria but are diffusible toxins released into the environment. Identified almost a century ago, our understanding of bacteriocin distribution and prevalence in bacterial populations remains poor. In the case of protein bacteriocins, this is because of high levels of sequence diversity and difficulties in distinguishing their killing domains from those of other competition systems. Here, we develop a robust bioinformatics pipeline exploiting Hidden Markov Models for the identification of nuclease bacteriocins (NBs in bacteria of which, to-date, only a handful are known. NBs are large (>60 kDa toxins that target nucleic acids (DNA, tRNA or rRNA in the cytoplasm of susceptible bacteria, usually closely related to the producing organism. We identified >3000 NB genes located on plasmids or on the chromosome from 53 bacterial species distributed across different ecological niches, including human, animals, plants, and the environment. A newly identified NB predicted to be specific for Pseudomonas aeruginosa (pyocin Sn was produced and shown to kill P. aeruginosa thereby validating our pipeline. Intriguingly, while the genes encoding the machinery needed for NB translocation across the cell envelope are widespread in Gram-negative bacteria, NBs are found exclusively in γ-proteobacteria. Similarity network analysis demonstrated that NBs fall into eight groups each with a distinct arrangement of protein domains involved in import. The only structural feature conserved across all groups was a sequence motif critical for cell-killing that is generally not found in bacteriocins targeting the periplasm, implying a specific role in translocating the

  5. Extracellular Nucleases of Streptococcus equi subsp. zooepidemicus Degrade Neutrophil Extracellular Traps and Impair Macrophage Activity of the Host.

    Science.gov (United States)

    Ma, Fang; Guo, Xiao; Fan, Hongjie

    2017-01-15

    bacteremia in its hosts. However, little is known about how S. equi subsp. zooepidemicus interacts with the host innate immune system, particularly innate cells found in the blood. S. equi subsp. zooepidemicus is capable of evading NET-mediated killing via the actions of its potent extracellular nucleases, ENuc and 5Nuc, which directly degrade the NET DNA backbone to deoxyadenosine. In previous studies, other pathogens have required the synergism of nuclease and 5'-nucleotidase to engage in this self-protective process; however, ENuc and 5Nuc both possess nuclease activity and 5'-nucleotidase activity, highlighting the novelty of this discovery. Furthermore, deoxyadenosine impairs phagocytosis but not the intracellular bactericidal activity of macrophages. Here we describe a novel mechanism for S. equi subsp. zooepidemicus extracellular nucleases in NET degradation, which may provide new insights into the pathogen immune evasion mechanism and the prevention and treatment of bacterial disease. Copyright © 2016 American Society for Microbiology.

  6. Automated 5 ' nuclease PCR assay for identification of Salmonella enterica

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Ahrens, Peter; Rådström, P.

    2000-01-01

    A simple and ready-to-go test based on a 5' nuclease (TaqMan) PCR technique was developed for identification of presumptive Salmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica...... isolates, including 100 problematic "rough" isolates. An internal positive control was designed to use the same Salmonella primers for amplification of a spiked nonrelevant template (116 bp) in the sample tube. The PCR test correctly identified all the Salmonella strains by resulting in positive end......-point fluorescence (FAM) signals for the samples and positive control (TET) signals (relative sensitivity [Delta Rn], >0.6). The diagnostic specificity of the method was assessed using 120 non-Salmonella strains, which all resulted in negative FAM signals (Delta Rn, less than or equal to 0.5). All 100 rough...

  7. Genome editing in plant cells by zinc finger nucleases.

    Science.gov (United States)

    Weinthal, Dan; Tovkach, Andriy; Zeevi, Vardit; Tzfira, Tzvi

    2010-06-01

    Gene targeting is a powerful tool for functional gene studies. However, only a handful of reports have been published describing the successful targeting of genome sequences in model and crop plants. Gene targeting can be stimulated by induction of double-strand breaks at specific genomic sites. The expression of zinc finger nucleases (ZFNs) can induce genomic double-strand breaks. Indeed, ZFNs have been used to drive the replacement of native DNA sequences with foreign DNA molecules, to mediate the integration of the targeted transgene into native genome sequences, to stimulate the repair of defective transgenes, and as site-specific mutagens in model and crop plant species. This review introduces the principles underlying the use of ZFNs for genome editing, with an emphasis on their recent use for plant research and biotechnology.

  8. Critical parameters for genome editing using zinc finger nucleases.

    Science.gov (United States)

    Camenisch, Todd D; Brilliant, Murray H; Segal, David J

    2008-06-01

    The possibility to make precise modifications to the genome at high frequency holds tremendous potential for biotechnology, conventional drug development and gene therapy. Homologous recombination is a powerful method for introducing such modifications in organisms such as mice. However, in mammals and plants, the frequency of gene modification by homologous recombination is quite low, precluding the therapeutic use of this methodology. In the past few years, tremendous progress has been made in overcoming one of primary barriers to efficient recombination, namely the introduction of a targeted double-strand break near the intended recombination site. This review will discuss the advances in engineering custom zinc-finger nucleases and their application in stimulating homologous recombination in higher eukaryotic cells at efficiencies approaching 1 in 2 cells.

  9. A comprehensive overview of computational resources to aid in precision genome editing with engineered nucleases.

    Science.gov (United States)

    Periwal, Vinita

    2017-07-01

    Genome editing with engineered nucleases (zinc finger nucleases, TAL effector nucleases s and Clustered regularly inter-spaced short palindromic repeats/CRISPR-associated) has recently been shown to have great promise in a variety of therapeutic and biotechnological applications. However, their exploitation in genetic analysis and clinical settings largely depends on their specificity for the intended genomic target. Large and complex genomes often contain highly homologous/repetitive sequences, which limits the specificity of genome editing tools and could result in off-target activity. Over the past few years, various computational approaches have been developed to assist the design process and predict/reduce the off-target activity of these nucleases. These tools could be efficiently used to guide the design of constructs for engineered nucleases and evaluate results after genome editing. This review provides a comprehensive overview of various databases, tools, web servers and resources for genome editing and compares their features and functionalities. Additionally, it also describes tools that have been developed to analyse post-genome editing results. The article also discusses important design parameters that could be considered while designing these nucleases. This review is intended to be a quick reference guide for experimentalists as well as computational biologists working in the field of genome editing with engineered nucleases. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  10. Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases.

    Science.gov (United States)

    Cai, Yujia; Bak, Rasmus O; Mikkelsen, Jacob Giehm

    2014-04-24

    Future therapeutic use of engineered site-directed nucleases, like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), relies on safe and effective means of delivering nucleases to cells. In this study, we adapt lentiviral vectors as carriers of designer nuclease proteins, providing efficient targeted gene disruption in vector-treated cell lines and primary cells. By co-packaging pairs of ZFN proteins with donor RNA in 'all-in-one' lentiviral particles, we co-deliver ZFN proteins and the donor template for homology-directed repair leading to targeted DNA insertion and gene correction. Comparative studies of ZFN activity in a predetermined target locus and a known nearby off-target locus demonstrate reduced off-target activity after ZFN protein transduction relative to conventional delivery approaches. Additionally, TALEN proteins are added to the repertoire of custom-designed nucleases that can be delivered by protein transduction. Altogether, our findings generate a new platform for genome engineering based on efficient and potentially safer delivery of programmable nucleases.DOI: http://dx.doi.org/10.7554/eLife.01911.001. Copyright © 2014, Cai et al.

  11. Plant plasma membrane-bound staphylococcal-like DNases as a novel class of eukaryotic nucleases

    Directory of Open Access Journals (Sweden)

    Leśniewicz Krzysztof

    2012-10-01

    Full Text Available Abstract Background The activity of degradative nucleases responsible for genomic DNA digestion has been observed in all kingdoms of life. It is believed that the main function of DNA degradation occurring during plant programmed cell death is redistribution of nucleic acid derived products such as nitrogen, phosphorus and nucleotide bases. Plant degradative nucleases that have been studied so far belong mainly to the S1-type family and were identified in cellular compartments containing nucleic acids or in the organelles where they are stored before final application. However, the explanation of how degraded DNA components are exported from the dying cells for further reutilization remains open. Results Bioinformatic and experimental data presented in this paper indicate that two Arabidopsis staphylococcal-like nucleases, named CAN1 and CAN2, are anchored to the cell membrane via N-terminal myristoylation and palmitoylation modifications. Both proteins possess a unique hybrid structure in their catalytic domain consisting of staphylococcal nuclease-like and tRNA synthetase anticodon binding-like motifs. They are neutral, Ca2+-dependent nucleaces showing a different specificity toward the ssDNA, dsDNA and RNA substrates. A study of microarray experiments and endogenous nuclease activity revealed that expression of CAN1 gene correlates with different forms of programmed cell death, while the CAN2 gene is constitutively expressed. Conclusions In this paper we present evidence showing that two plant staphylococcal-like nucleases belong to a new, as yet unidentified class of eukaryotic nucleases, characterized by unique plasma membrane localization. The identification of this class of nucleases indicates that plant cells possess additional, so far uncharacterized, mechanisms responsible for DNA and RNA degradation. The potential functions of these nucleases in relation to their unique intracellular location are discussed.

  12. Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis

    DEFF Research Database (Denmark)

    Lonowski, Lindsey A; Narimatsu, Yoshiki; Riaz, Anjum

    2017-01-01

    ). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator. Second...... the testing of new nuclease reagents and the generation of edited cell pools or clonal cell lines, reducing the number of clones that need to be generated and increasing the ease with which they are screened. The pipeline shortens the time line, but it most prominently reduces the workload of cell...

  13. Is BAC transgenesis obsolete? State of the art in the era of designer nucleases.

    Science.gov (United States)

    Beil, J; Fairbairn, L; Pelczar, P; Buch, T

    2012-01-01

    DNA constructs based on bacterial artificial chromosomes (BACs) are frequently used to generate transgenic animals as they reduce the influence of position effects and allow predictable expression patterns for genes whose regulatory sequences are not fully identified. Despite these advantages BAC transgenics suffer from drawbacks such as complicated vector construction, low efficiency of transgenesis, and some remaining expression variegation. The recent development of transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs) has resulted in new transgenic techniques which do not have the drawbacks associated with BAC transgenesis. Initial reports indicate that such designer nucleases (DNs) allow the targeted insertion of transgenes into endogenous loci by direct injection of the targeting vector and mRNA/DNA encoding the predesigned nucleases into oocytes. This results in the transgene being inserted at a specific locus in the mouse genome, thus circumventing the drawbacks associated with BAC transgenesis.

  14. Synthesis, X-ray crystal structure, DNA binding and Nuclease activity ...

    Indian Academy of Sciences (India)

    s12039-016-1125-x. Synthesis, X-ray crystal structure, DNA binding and Nuclease activity of lanthanide(III) complexes of 2-benzoylpyridine acetylhydrazone. KARREDDULA RAJA, AKKILI SUSEELAMMA and KATREDDI HUSSAIN REDDY. ∗.

  15. Delivery methods for site-specific nucleases: Achieving the full potential of therapeutic gene editing.

    Science.gov (United States)

    Liu, Jia; Shui, Sai-Lan

    2016-12-28

    The advent of site-specific nucleases, particularly CRISPR/Cas9, provides researchers with the unprecedented ability to manipulate genomic sequences. These nucleases are used to create model cell lines, engineer metabolic pathways, produce transgenic animals and plants, perform genome-wide functional screen and, most importantly, treat human diseases that are difficult to tackle by traditional medications. Considerable efforts have been devoted to improving the efficiency and specificity of nucleases for clinical applications. However, safe and efficient delivery methods remain the major obstacle for therapeutic gene editing. In this review, we summarize the recent progress on nuclease delivery methods, highlight their impact on the outcomes of gene editing and discuss the potential of different delivery approaches for therapeutic gene editing. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Generation of knockout rabbits using transcription activator-like effector nucleases

    Directory of Open Access Journals (Sweden)

    Yu Wang

    2014-01-01

    Full Text Available Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

  17. Potential relationships between morphological differentiation and mutants with high nuclease P1 production of Penicillium citrinum

    International Nuclear Information System (INIS)

    Liang Xinle; Shou Qian; Zhang Hong; Chen Min; Liu Xuan

    2009-01-01

    Diversification of colony characteristics of mutants derived from Penicillium citrinum CICC 4011 treated with 60 Co γ-irradiation and protoplast fusion were analyzed. There were distinct differences among mutants with different nuclease P1 activity, especially in pigment productivity. Color of colony was changed from the original green to white, grey-green, or yellow-green etc., while the nuclease P1 activity would be fluctuated with the color change. The hypothesis was suggested that there would be a relationship between pigments and nuclease P1 production. Mutants with grey-green colony would give out high nuclease P1 outputs in a high probability such as mutant J1Y6( nuclease P1 activity, 167.3U/ml) and fusant F-13 (nuclease P1 activity, 568.7U/ml), while others with deep-green colony observed low nuclease outputs. Four variation strains didn't show any significant difference in growth rate. Broom branches of conidiophore stem in J1Y6 and F-13 were obviously reduced, conidiophores productivity reduced, but hyphae growth haled. These suggested that nuclease P1 production was associated with growth phase, but pigment synthesis course wasn't. RAPD from 6 randomly selected primers was used to analyze the polymorphic rich of the four strains, the results showed that there were 70 percent polymorphism detection rate among those. UPGMA cluster analysis and genetic map constructed by NTSYS-PC software, which showed that J1Y6 and F-14 were clustered as one group at similar coefficient 0.9, where there was an appear distance from the group of 4011 and F-R-33 strains (similar coefficient 0.8). (authors)

  18. Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases

    Czech Academy of Sciences Publication Activity Database

    Kašpárek, Petr; Krausová, Michaela; Hanečková, Radka; Kříž, Vítězslav; Žbodáková, Olga; Kořínek, Vladimír; Sedláček, Radislav

    2014-01-01

    Roč. 588, č. 21 (2014), 3982-3988 ISSN 0014-5793 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109; GA ČR(CZ) GA14-33952S; GA MŠk(CZ) LM2011032 Institutional support: RVO:68378050 Keywords : Gene targeting * Homologous recombination * Rosa26 locus * Transgenic * Transcription activator-like effector nucleases * Zinc-finger nucleases Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.169, year: 2014

  19. Molecular Cloning and Characterization of P4 Nuclease from Leishmania infantum

    Directory of Open Access Journals (Sweden)

    Safar Farajnia

    2011-01-01

    Full Text Available Parasite of the genus Leishmania is reliant on the salvage pathway for recycling of ribonucleotides. A class I nuclease enzyme also known as P4 nuclease is involved in salvage of purines in cutaneous Leishmania species but the relevant enzymes have not been characterized in Leishmania infantum (L. infantum. The aim of this study was to clone and characterize the gene encoding class I nuclease in L. infantum. DNA extracted from L. infantum was used for amplification of P4 nuclease gene (Li-P4 by PCR. The product was cloned, sequenced, and expressed in E. coli for further characterization. Analysis of the sequence of Li-P4 revealed that the gene consists of an ORF of 951 bp. Sequence similarity analysis indicated that Li-P4 has a high homology to relevant enzymes of other kintoplastids with the highest homology (88% to p1/s1 class I nuclease from L. donovani. Western blotting of antirecombinant Li-P4 with promastigote and amastigote stages of L. infantum showed that this nuclease is present in both stages of parasite with higher expression in amastigote stage. The highly conserved nature of this essential enzyme in Leishmania parasites suggests it as a promising drug target for leishmaniasis.

  20. A baculovirus alkaline nuclease knockout construct produces fragmented DNA and aberrant capsids

    International Nuclear Information System (INIS)

    Okano, Kazuhiro; Vanarsdall, Adam L.; Rohrmann, George F.

    2007-01-01

    DNA replication of bacmid-derived constructs of the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) was analyzed by field inversion gel electrophoresis (FIGE) in combination with digestion at a unique Eco81I restriction enzyme site. Three constructs were characterized: a parental bacmid, a bacmid deleted for the alkaline nuclease gene, and a bacmid from which the gp64 gene had been deleted. The latter was employed as a control for comparison with the alkaline nuclease knockout because neither yields infectious virus and their replication is limited to the initially transfected cells. The major difference between DNA replicated by the different constructs was the presence in the alkaline nuclease knockout of high concentrations of relatively small, subgenome length DNA in preparations not treated with Eco81I. Furthermore, upon Eco81I digestion, the alkaline nuclease knockout bacmid also yielded substantially more subgenome size DNA than the other constructs. Electron microscopic examination of cells transfected with the alkaline nuclease knockout indicated that, in addition to a limited number of normal-appearing electron-dense nucleocapsids, numerous aberrant capsid-like structures were observed indicating a defect in nucleocapsid maturation or in a DNA processing step that is necessary for encapsidation. Because of the documented role of the baculovirus alkaline nuclease and its homologs from other viruses in homologous recombination, these data suggest that DNA recombination may play a major role in the production of baculovirus genomes

  1. RNA-protein analysis using a conditional CRISPR nuclease.

    Science.gov (United States)

    Lee, Ho Young; Haurwitz, Rachel E; Apffel, Alex; Zhou, Kaihong; Smart, Brian; Wenger, Craig D; Laderman, Stephen; Bruhn, Laurakay; Doudna, Jennifer A

    2013-04-02

    RNA-binding proteins control the fate and function of the transcriptome in all cells. Here we present technology for isolating RNA-protein partners efficiently and accurately using an engineered clustered regularly interspaced short palindromic repeats (CRISPR) endoribonuclease. An inactive version of the Csy4 nuclease binds irreversibly to transcripts engineered with a 16-nt hairpin sequence at their 5' ends. Once immobilized by Csy4 on a solid support, contaminating proteins and other molecules can be removed by extensive washing. Upon addition of imidazole, Csy4 is activated to cleave the RNA, removing the hairpin tag and releasing the native transcript along with its specifically bound protein partners. This conditional Csy4 enzyme enables recovery of specific RNA-binding partners with minimal false-positive contamination. We use this method, coupled with quantitative MS, to identify cell type-specific human pre-microRNA-binding proteins. We also show that this technology is suitable for analyzing diverse size transcripts, and that it is suitable for adaptation to a high-throughput discovery format.

  2. Mung Bean nuclease mapping of RNAs 3' end

    Directory of Open Access Journals (Sweden)

    Barbieri Rainer

    2009-05-01

    Full Text Available Abstract A method is described that allows an accurate mapping of 3' ends of RNAs. In this method a labeled DNA probe, containing the presumed 3' end of the RNA under analysis is allowed to anneals to the RNA itself. Mung-bean nuclease is then used to digest single strands of both RNA and DNA. Electrophoretic fractionation of "protected" undigested, labeled DNA is than performed using a sequence reaction of a known DNA as length marker. This procedure was applied to the analysis of both a polyA RNA (Interleukin 10 mRNA and non polyA RNAs (sea urchin 18S and 26S rRNAs. This method might be potentially relevant for the evaluation of the role of posttrascriptional control of IL-10 in the pathogenesis of the immune and inflammatory mediated diseases associated to ageing. This might allow to develop new strategies to approach to the diagnosis and therapy of age related diseases.

  3. Knockout of targeted gene in porcine somatic cells using zinc-finger nuclease.

    Science.gov (United States)

    Hisamatsu, Shin; Sakaue, Motoharu; Takizawa, Akiko; Kato, Tsubasa; Kamoshita, Maki; Ito, Junya; Kashiwazaki, Naomi

    2015-02-01

    Targeted genome editing is a widely applicable approach for efficiently modifying any sequence of interest in animals. It is very difficult to generate knock-out and knock-in animals except for mice up to now. Very recently, a method of genome editing using zinc-finger nucleases (ZFNs) has been developed to produce knockout rats. Since only injection of ZFNs into the pronuclear (PN) embryo is required, it seems to be useful for generating gene-targeted animals, including domestic species. However, no one has reported the successful production of knockout pigs by direct injection of ZFNs into PN embryos. We examined whether ZFN works on editing the genome of porcine growth hormone receptor in two kinds of cell lines (ST and PT-K75) derived from the pig as a preliminary study. Our data showed that pZFN1/2 vectors were efficiently transfected into both ST and PT-K75 cells. In both cell lines, results from Cel-I assay showed that modification of the targeted gene was confirmed. We injected ZFN1/2 mRNAs into the nucleus of PN stage embryos and then they were transferred to the recipients. However, pups were not delivered. Taken together, ZFN can be an available technology of genome editing even in the pig but further improvement will be required for generating genome-modified pigs. © 2014 Japanese Society of Animal Science.

  4. Caffeine impairs resection during DNA break repair by reducing the levels of nucleases Sae2 and Dna2

    Science.gov (United States)

    Tsabar, Michael; Eapen, Vinay V.; Mason, Jennifer M.; Memisoglu, Gonen; Waterman, David P.; Long, Marcus J.; Bishop, Douglas K.; Haber, James E.

    2015-01-01

    In response to chromosomal double-strand breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint, which is orchestrated by the PI3 kinase-like protein kinases ATR and ATM (Mec1 and Tel1 in budding yeast). Following DSB formation, Mec1 and Tel1 phosphorylate histone H2A on serine 129 (known as γ-H2AX). We used caffeine to inhibit the checkpoint kinases after DSB induction. We show that prolonged phosphorylation of H2A-S129 does not require continuous Mec1 and Tel1 activity. Unexpectedly, caffeine treatment impaired homologous recombination by inhibiting 5′ to 3′ end resection, independent of Mec1 and Tel1 inhibition. Caffeine treatment led to the rapid loss, by proteasomal degradation, of both Sae2, a nuclease that plays a role in early steps of resection, and Dna2, a nuclease that facilitates one of two extensive resection pathways. Sae2's instability is evident in the absence of DNA damage. A similar loss is seen when protein synthesis is inhibited by cycloheximide. Caffeine treatment had similar effects on irradiated HeLa cells, blocking the formation of RPA and Rad51 foci that depend on 5′ to 3′ resection of broken chromosome ends. Our findings provide insight toward the use of caffeine as a DNA damage-sensitizing agent in cancer cells. PMID:26019182

  5. Functional Interplay of the Mre11 Nuclease and Ku in the Response to Replication-Associated DNA Damage ▿

    Science.gov (United States)

    Foster, Steven S.; Balestrini, Alessia; Petrini, John H. J.

    2011-01-01

    The Mre11 complex is a central component of the DNA damage response, with roles in damage sensing, molecular bridging, and end resection. We have previously shown that in Saccharomyces cerevisiae, Ku70 (yKu70) deficiency reduces the ionizing radiation sensitivity of mre11Δ mutants. In this study, we show that yKu70 deficiency suppressed the camptothecin (CPT) and methyl methanesulfonate (MMS) sensitivity of nuclease-deficient mre11-3 and sae2Δ mutants in an Exo1-dependent manner. CPT-induced G2/M arrest, γ-H2AX persistence, and chromosome breaks were elevated in mre11-3 mutants. These outcomes were reduced by yKu70 deficiency. Given that the genotoxic effects of CPT are manifest during DNA replication, these data suggest that Ku limits Exo1-dependent double-strand break (DSB) resection during DNA replication, inhibiting the initial processing steps required for homology-directed repair. We propose that Mre11 nuclease- and Sae2-dependent DNA end processing, which initiates DSB resection prevents Ku from engaging DSBs, thus promoting Exo1-dependent resection. In agreement with this idea, we show that Ku affinity for binding to short single-stranded overhangs is much lower than for blunt DNA ends. Collectively, the data define a nonhomologous end joining (NHEJ)-independent, S-phase-specific function of the Ku heterodimer. PMID:21876003

  6. Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases.

    Directory of Open Access Journals (Sweden)

    Craig B Wilen

    2011-04-01

    Full Text Available HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5 virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4 in place of or in addition to CCR5 (R5X4 remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5Δ32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals.

  7. Developing a puncture-free in ovo chicken transfection strategy based on bypassing albumen nucleases.

    Science.gov (United States)

    Amini, Hamid-Reza; Pakdel, Abbas; Shahr-Babak, Hossein Moradi; Eghbalsaied, Shahin

    2017-03-15

    Chicken is a dual-purpose animal important from both agricultural and medical aspects. Even though significant improvements have been made in chicken transgenesis technologies, chicken genome manipulation has not been widely used in developmental biology. This study was aimed to evaluate chicken egg white nuclease properties and thereof plausibility of devising an in vivo transfection technology without causing physical damage to the embryo. First, the nuclease activity of egg albumen was assessed. The egg white nucleases were strongly active in degrading DNA and RNA. The egg white DNase activity was comparable to commercially available DNase-I. Nuclease activities were also assessed after heating, proteinase K, or EDTA treatment. Unlike proteinase K, both heating and EDTA were noticeably effective for the nuclease inactivation. Simultaneous application of lipoplex form of DNA (1 μg pDB2: 3 μl Lipofectamine2000) and EDTA showed a synergistic effect in protection against egg white nucleases. Finally, we injected the lipoplexes with or without EDTA close to the embryo at day0, but outside the embryonic epiblast. Implementation of a scrutinized PCR assay indicated that transfection took place only when EDTA was complemented to the lipoplexes. The transfection rate of day4 embryos and the hatched chicks were 54.5 and 30.0%, respectively. EGFP expression was detected in two out of three transgenic chicks. In conclusion, this study provided a detail analysis of chicken egg albumen nuclease properties and suggested the feasibility of developing a puncture-free handmade technology for transfection of the chicken embryo. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. DNA Oxidation Profiles of Copper Phenanthrene Chemical Nucleases

    Directory of Open Access Journals (Sweden)

    Zara eMolphy

    2015-04-01

    Full Text Available 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(phen2}2(μ-terph](terph (Cu-Terph, with results being compared directly to Sigman’s reagent [Cu(phen2]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(NH36]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.

  9. DNA Oxidation Profiles of Copper Phenanthrene Chemical Nucleases

    Science.gov (United States)

    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.

  10. Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

    Science.gov (United States)

    Kleinstiver, Benjamin P; Prew, Michelle S; Tsai, Shengdar Q; Topkar, Ved V; Nguyen, Nhu T; Zheng, Zongli; Gonzales, Andrew P W; Li, Zhuyun; Peterson, Randall T; Yeh, Jing-Ruey Joanna; Aryee, Martin J; Joung, J Keith

    2015-07-23

    Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

  11. [Tale nucleases--new tool for genome editing].

    Science.gov (United States)

    Glazkova, D V; Shipulin, G A

    2014-01-01

    The ability to introduce targeted changes in the genome of living cells or entire organisms enables researchers to meet the challenges of basic life sciences, biotechnology and medicine. Knockdown of target genes in the zygotes gives the opportunity to investigate the functions of these genes in different organisms. Replacement of single nucleotide in the DNA sequence allows to correct mutations in genes and thus to cure hereditary diseases. Adding transgene to specific genomic.loci can be used in biotechnology for generation of organisms with certain properties or cell lines for biopharmaceutical production. Such manipulations of gene sequences in their natural chromosomal context became possible after the emergence of the technology called "genome editing". This technology is based on the induction of a double-strand break in a specific genomic target DNA using endonucleases that recognize the unique sequences in the genome and on subsequent recovery of DNA integrity through the use of cellular repair mechanisms. A necessary tool for the genome editing is a custom-designed endonuclease which is able to recognize selected sequences. The emergence of a new type of programmable endonucleases, which were constructed on the basis of bacterial proteins--TAL-effectors (Transcription activators like effector), has become an important stage in the development of technology and promoted wide spread of the genome editing. This article reviews the history of the discovery of TAL effectors and creation of TALE nucleases, and describes their advantages over zinc finger endonucleases that appeared earlier. A large section is devoted to description of genetic modifications that can be performed using the genome editing.

  12. Suppression of HBV replication by the expression of nickase- and nuclease dead-Cas9.

    Science.gov (United States)

    Kurihara, Takeshi; Fukuhara, Takasuke; Ono, Chikako; Yamamoto, Satomi; Uemura, Kentaro; Okamoto, Toru; Sugiyama, Masaya; Motooka, Daisuke; Nakamura, Shota; Ikawa, Masato; Mizokami, Masashi; Maehara, Yoshihiko; Matsuura, Yoshiharu

    2017-07-21

    Complete removal of hepatitis B virus (HBV) DNA from nuclei is difficult by the current therapies. Recent reports have shown that a novel genome-editing tool using Cas9 with a single-guide RNA (sgRNA) system can cleave the HBV genome in vitro and in vivo. However, induction of a double-strand break (DSB) on the targeted genome by Cas9 risks undesirable off-target cleavage on the host genome. Nickase-Cas9 cleaves a single strand of DNA, and thereby two sgRNAs are required for inducing DSBs. To avoid Cas9-induced off-target mutagenesis, we examined the effects of the expressions of nickase-Cas9 and nuclease dead Cas9 (d-Cas9) with sgRNAs on HBV replication. The expression of nickase-Cas9 with a pair of sgRNAs cleaved the target HBV genome and suppressed the viral-protein expression and HBV replication in vitro. Moreover, nickase-Cas9 with the sgRNA pair cleaved the targeted HBV genome in mouse liver. Interestingly, d-Cas9 expression with the sgRNAs also suppressed HBV replication in vitro without cleaving the HBV genome. These results suggest the possible use of nickase-Cas9 and d-Cas9 with a pair of sgRNAs for eliminating HBV DNA from the livers of chronic hepatitis B patients with low risk of undesirable off-target mutation on the host genome.

  13. LEM-3 is a midbody-tethered DNA nuclease that resolves chromatin bridges during late mitosis.

    Science.gov (United States)

    Hong, Ye; Sonneville, Remi; Wang, Bin; Scheidt, Viktor; Meier, Bettina; Woglar, Alexander; Demetriou, Sarah; Labib, Karim; Jantsch, Verena; Gartner, Anton

    2018-02-20

    Faithful chromosome segregation and genome maintenance requires the removal of all DNA bridges that physically link chromosomes before cells divide. Using C. elegans embryos we show that the LEM-3/Ankle1 nuclease defines a previously undescribed genome integrity mechanism by processing DNA bridges right before cells divide. LEM-3 acts at the midbody, the structure where abscission occurs at the end of cytokinesis. LEM-3 localization depends on factors needed for midbody assembly, and LEM-3 accumulation is increased and prolonged when chromatin bridges are trapped at the cleavage plane. LEM-3 locally processes chromatin bridges that arise from incomplete DNA replication, unresolved recombination intermediates, or the perturbance of chromosome structure. Proper LEM-3 midbody localization and function is regulated by AIR-2/Aurora B kinase. Strikingly, LEM-3 acts cooperatively with the BRC-1/BRCA1 homologous recombination factor to promote genome integrity. These findings provide a molecular basis for the suspected role of the LEM-3 orthologue Ankle1 in human breast cancer.

  14. Tudor Staphylococcal Nuclease plays two antagonistic roles in RNA metabolism under stress.

    Science.gov (United States)

    Gutiérrez-Beltran, Emilio; Bozhkov, Peter V; Moschou, Panagiotis N

    2015-01-01

    Adaptation to stress entails a repertoire of molecular pathways that remodel the proteome, thereby promoting selective translation of pro-survival proteins. Yet, translation of other proteins, especially those which are harmful for stress adaptation is, on the contrary, transiently suppressed through mRNA decay or storage. Proteome remodeling under stress is intimately associated with the cytoplasmic ribonucleoprotein (RNP) complexes called stress granules (SGs) and processing bodies (PBs). The molecular composition and regulation of SGs and PBs in plants remain largely unknown. Recently, we identified the Arabidopsis Tudor Staphylococcal Nuclease (TSN, Tudor-SN or SND1) as a SG- and PB-associated protein required for mRNA decapping under stress conditions. Here we show that SGs localize in close proximity to PBs within plant cells that enable the exchange of molecular components. Furthermore, we provide a meta-analysis of mRNA degradome of TSN-deficient plants suggesting that TSN might inhibit the degradation of mRNAs which are involved in stress adaptation. Our results establish TSN as a versatile mRNA regulator during stress.

  15. Plant genome editing using engineered nucleases and success of CRISPR/Cas9 system

    Directory of Open Access Journals (Sweden)

    Moon Sajid

    2017-08-01

    Full Text Available Development of new plant breeding techniques have facilitated easy manipulation of plants at genetic level. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/CRISPR associated protein9 (Cas9 system is a valuable addition in programmable nucleases. The CRISPR/Cas9 system uses an RNA component to recognize a target DNA sequences and it has shown promising results with respect to simultaneous editing of multigenic plant traits. In this review, components of CRISPR/Cas9, their construction and its methods of delivery to plant cells are analyzed. Variation in nucleotide sequence of the protospacer adjacent motif, codon optimization and progress in web-based bioinformatic tools, will make CRISPR/Cas9 systems more efficient for plants. Development and optimization of protocols to efficiently target all plant species is still under development. Along with this, methods to inspect induced mutation and efficiency of the system have also been reviewed. Auxiliary improvements and understanding are still required to expand the CRISPR/Cas9 systems to target complex genome architectures and epigenetic elements.

  16. Removing residual DNA from Vero-cell culture-derived human rabies vaccine by using nuclease.

    Science.gov (United States)

    Li, Si-Ming; Bai, Fu-Liang; Xu, Wen-Juan; Yang, Yong-Bi; An, Ying; Li, Tian-He; Yu, Yin-Hang; Li, De-Shan; Wang, Wen-Fei

    2014-09-01

    The clearance of host cell DNA is a critical indicator for Vero-cell culture-derived rabies vaccine. In this study, we evaluated the clearance of DNA in Vero-cell culture-derived rabies vaccine by purification process utilizing ultrafiltration, nuclease digestion, and gel filtration chromatography. The results showed that the bioprocess of using nuclease decreased residual DNA. Dot-blot hybridization analysis showed that the residual host cell DNA was rabies vaccine was less than 0.1 ng/ml protein. The residual nuclease could not paly the biologically active role of digestion of DNA. Experiments of stability showed that the freeze-drying rabies virus vaccine was stable and titers were >5.0 IU/ml. Immunogenicity test and protection experiments indicated mice were greatly induced generation of neutralizing antibodies and invoked protective effects immunized with intraperitoneal injections of the rabies vaccine. These results demonstrated that the residual DNA was removed from virus particles and nuclease was removed by gel filtration chromatography. The date indicated that technology was an efficient method to produce rabies vaccine for human use by using nuclease. Copyright © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  17. Analysis of pyrimidine dimer content of isolated DNA by nuclease digestion

    International Nuclear Information System (INIS)

    Farland, W.H.; Sutherland, B.M.

    1980-01-01

    Isolated DNA is highly susceptible to degradation by exogenous nucleases. Complete digestion is possible with a number of well-characterized enzymes from a variety of sources. Treatment of DNA with a battery of enzymes including both phosphodiesterase and phosphatase activities yields a mixture of nucleosides and inorganic phosphate (P/sub i/) as a final product. Unlike native DNA, ultraviolet-irradiated DNA is resistant to complete digestion. Setlow et al. demonstrated that the structural changes in the DNA responsible for the nuclease resistance were the formation of cyclobutyl pyrimidine dimers, the major photoproduct in UV-irradiated DNA. Using venom phosphodiesterase, they demonstrated that UV irradiation of DNA affected both the rate and extent of enzymatic hydrolysis. In addition, it was demonstrated that the major nuclease-resistant product of this hydrolysis was an oligonucleotide containing dimerized pyrimidines. Treatment of the DNA to split the dimers, either photochemically or photoenzymatically, rendered the polymer more susceptible to hydrolysis by the phosphodiesterase. The specificity of photoreactivating enzyme for pyrimidine dimers lends support to the role of these structures in conferring nuclease resistance to UV-irradiated DNA. The nuclease resistance of DNA containing dimers has been the basis of several assays for the measurement of these photoproducts. Sutherland and Chamberlin reported the development of a rapid and sensitive assay for dimers in 32 P-labeled DNA

  18. Use of S1 nuclease in deep sequencing for detection of double-stranded RNA viruses.

    Science.gov (United States)

    Shimada, Saya; Nagai, Makoto; Moriyama, Hiromitsu; Fukuhara, Toshiyuki; Koyama, Satoshi; Omatsu, Tsutomu; Furuya, Tetsuya; Shirai, Junsuke; Mizutani, Tetsuya

    2015-09-01

    Metagenomic approach using next-generation DNA sequencing has facilitated the detection of many pathogenic viruses from fecal samples. However, in many cases, majority of the detected sequences originate from the host genome and bacterial flora in the gut. Here, to improve efficiency of the detection of double-stranded (ds) RNA viruses from samples, we evaluated the applicability of S1 nuclease on deep sequencing. Treating total RNA with S1 nuclease resulted in 1.5-28.4- and 10.1-208.9-fold increases in sequence reads of group A rotavirus in fecal and viral culture samples, respectively. Moreover, increasing coverage of mapping to reference sequences allowed for sufficient genotyping using analytical software. These results suggest that library construction using S1 nuclease is useful for deep sequencing in the detection of dsRNA viruses.

  19. GUIDEseq: a bioconductor package to analyze GUIDE-Seq datasets for CRISPR-Cas nucleases.

    Science.gov (United States)

    Zhu, Lihua Julie; Lawrence, Michael; Gupta, Ankit; Pagès, Hervé; Kucukural, Alper; Garber, Manuel; Wolfe, Scot A

    2017-05-15

    Genome editing technologies developed around the CRISPR-Cas9 nuclease system have facilitated the investigation of a broad range of biological questions. These nucleases also hold tremendous promise for treating a variety of genetic disorders. In the context of their therapeutic application, it is important to identify the spectrum of genomic sequences that are cleaved by a candidate nuclease when programmed with a particular guide RNA, as well as the cleavage efficiency of these sites. Powerful new experimental approaches, such as GUIDE-seq, facilitate the sensitive, unbiased genome-wide detection of nuclease cleavage sites within the genome. Flexible bioinformatics analysis tools for processing GUIDE-seq data are needed. Here, we describe an open source, open development software suite, GUIDEseq, for GUIDE-seq data analysis and annotation as a Bioconductor package in R. The GUIDEseq package provides a flexible platform with more than 60 adjustable parameters for the analysis of datasets associated with custom nuclease applications. These parameters allow data analysis to be tailored to different nuclease platforms with different length and complexity in their guide and PAM recognition sequences or their DNA cleavage position. They also enable users to customize sequence aggregation criteria, and vary peak calling thresholds that can influence the number of potential off-target sites recovered. GUIDEseq also annotates potential off-target sites that overlap with genes based on genome annotation information, as these may be the most important off-target sites for further characterization. In addition, GUIDEseq enables the comparison and visualization of off-target site overlap between different datasets for a rapid comparison of different nuclease configurations or experimental conditions. For each identified off-target, the GUIDEseq package outputs mapped GUIDE-Seq read count as well as cleavage score from a user specified off-target cleavage score prediction

  20. Processing of 3'-Phosphoglycolate-Terminated DNA Double-StrandBreaks by Artemis Nuclease

    Energy Technology Data Exchange (ETDEWEB)

    Povrik, Lawrence F.; Zhou, Tong; Zhou, Ruizhe; Cowan, Morton J.; Yannone, Steven M.

    2005-10-01

    The Artemis nuclease is required for V(D)J recombination and for repair of an as yet undefined subset of radiation-induced DNA double-strand breaks. To assess the possibility that Artemis functions on oxidatively modified double-strand break termini, its activity toward model DNA substrates, bearing either 3{prime}-hydroxyl or 3{prime}-phosphoglycolate moieties, was examined. A 3{prime}-phosphoglycolate had little effect on Artemis-mediated trimming of long 3{prime} overhangs (>9 nucleotides), which were efficiently trimmed to 4-5 nucleotides. However, 3{prime}-phosphoglycolates on overhangs of 4-5 bases promoted selective Artemis-mediated trimming of a single 3{prime}-terminal nucleotide, while at least 2 nucleotides were trimmed from identical hydroxyl-terminated substrates. Artemis also efficiently removed a single nucleotide from a phosphoglycolate-terminated 3-base 3{prime} overhang, while leaving an analogous hydroxyl-terminated overhang largely intact. Such removal was dependent upon Ku, DNA-dependent protein kinase, and ATP. Together, these data suggest that Artemis-mediated cleavage of 3{prime} overhangs requires a minimum of 2 nucleotides, or a nucleotide plus a phosphoglycolate, 3{prime} to the cleavage site. Shorter 3{prime}-phosphoglycolate-terminated overhangs and blunt ends were also processed by Artemis, but much less efficiently. Consistent with the in vitro substrate specificity of Artemis, human cells lacking Artemis exhibited hypersensitivity to X-rays, bleomycin and neocarzinostatin, which all induce 3{prime}-phosphoglycolate-terminated double-strand breaks. Collectively, these results suggest that 3{prime}-phosphoglycolate termini and/or specific classes of DNA ends that arise from such blocked termini are relevant Artemis substrates in vivo.

  1. From classical mutagenesis to nuclease-based breeding - directing natural DNA repair for a natural end-product.

    Science.gov (United States)

    Pacher, Michael; Puchta, Holger

    2017-05-01

    Production of mutants of crop plants by the use of chemical or physical genotoxins has a long tradition. These factors induce the natural DNA repair machinery to repair damage in an error-prone way. In the case of radiation, multiple double-strand breaks (DSBs) are induced randomly in the genome, leading in very rare cases to a desirable phenotype. In recent years the use of synthetic, site-directed nucleases (SDNs) - also referred to as sequence-specific nucleases - like the CRISPR/Cas system has enabled scientists to use exactly the same naturally occurring DNA repair mechanisms for the controlled induction of genomic changes at pre-defined sites in plant genomes. As these changes are not necessarily associated with the permanent integration of foreign DNA, the obtained organisms per se cannot be regarded as genetically modified as there is no way to distinguish them from natural variants. This applies to changes induced by DSBs as well as single-strand breaks, and involves repair by non-homologous end-joining and homologous recombination. The recent development of SDN-based 'DNA-free' approaches makes mutagenesis strategies in classical breeding indistinguishable from SDN-derived targeted genome modifications, even in regard to current regulatory rules. With the advent of new SDN technologies, much faster and more precise genome editing becomes available at reasonable cost, and potentially without requiring time-consuming deregulation of newly created phenotypes. This review will focus on classical mutagenesis breeding and the application of newly developed SDNs in order to emphasize similarities in the context of the regulatory situation for genetically modified crop plants. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  2. Enthalpy-driven nuclease-like activity and mechanism of peptide-chlorambucil conjugates.

    Science.gov (United States)

    Yang, Robin C K; Huang, Jonathan T B; Chen, Yu-Ling; Hung, Chia-Chun; Liao, Mokai; Yao, Wen-Chen; Chen, Chiu-Heng; Liou, Chien-Chung; Waring, Michael J; Sheh, Leung

    2014-07-21

    We report the results of attaching the anticancer drug chlorambucil (CLB) to two high-affinity DNA binding peptides: Met-Hyp-Arg-Lys-(Py)4-Lys-Arg-NH2 (HyM-10) and Gln-Hyp-Arg-Lys-(Py)4-Lys-Arg-NH2 (HyQ-10). These CLB-peptide conjugates cleave DNA very effectively and sequence-selectively without the use of chemicals, heat, or UV irradiation. Polyacrylamide gel electrophoresis identifies the sites where CLB-HyM-10 and CLB-HyQ-10 attack a complementary pair of 5'-(32)P-labeled duplexes derived from pBR322 in the absence of piperidine or other chemical additives. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has confirmed the preferential cleavage sites as well as a novel stepwise cleavage mechanism of sequence-selective DNA cleavage. Resembling restriction endonucleases, the CLB-peptide conjugates appear to be capable of producing double strand DNA breaks. Circular dichroism studies show that CLB-HyM-10 and CLB-HyQ-10 induce significant local conformational changes in DNA via the minor groove, possibly with dimeric binding stoichiometry. The energetic basis of DNA binding by these conjugates has been investigated by isothermal titration calorimetry, revealing that the binding of both the peptides and their CLB conjugates is overwhelmingly enthalpy-driven. The maintenance of a conserved negative binding free energy in DNA-conjugate interactions is a crucial feature of the universal enthalpy-entropy compensation phenomenon. The strongly enthalpy-driven binding of CLB-peptide conjugates to preferred loci in DNA furnishes the required proximity effect to generate the observed nuclease-like sequence-selective cleavage.

  3. Antitumor and biological effects of black pine (Pinus nigra) pollen nuclease

    Czech Academy of Sciences Publication Activity Database

    Lipovová, P.; Podzimek, T.; Orctová, Lidmila; Matoušek, Jaroslav; Poučková, P.; Souček, J.; Matoušek, Josef

    2008-01-01

    Roč. 55, - (2008), s. 158-164 ISSN 0028-2685 Institutional research plan: CEZ:AV0Z50510513; CEZ:AV0Z50450515 Keywords : pollen nuclease * Antitumor effect Subject RIV: FD - Oncology ; Hematology Impact factor: 1.179, year: 2008

  4. Plant multifunctional nuclease TBN1 with unexpected phospholipase activity: structural study and reaction-mechanism analysis

    Czech Academy of Sciences Publication Activity Database

    Kovaľ, Tomáš; Lipovová, P.; Podzimek, T.; Matoušek, J.; Dušková, J.; Skálová, T.; Štěpánková, A.; Hašek, J.; Dohnálek, Jan

    2013-01-01

    Roč. 69, č. 2 (2013), s. 213-226 ISSN 0907-4449 Grant - others:AVČR(CZ) Praemium Academiae Institutional support: RVO:68378271 Keywords : TBN1 * nuclease * protein * x-ray structure Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 7.232, year: 2013

  5. Characterization, crystallization and preliminary X-ray diffraction studies of tomato nuclease I

    Czech Academy of Sciences Publication Activity Database

    Koval, Tomáš; Dohnálek, Jan; Lipovová, P.; Podzimek, T.; Matoušek, Jaroslav

    2009-01-01

    Roč. 16, 1a (2009), b33 ISSN 1211-5894. [Discussions in Structural Molecular Biology /7./. 12.03.2009-14.03.2009, Nové Hrady] Institutional research plan: CEZ:AV0Z40500505 Keywords : X-ray diffraction * tomato nuclease Subject RIV: CD - Macromolecular Chemistry

  6. The nuclease specificity of the bacteriophage phi X174 A* protein

    NARCIS (Netherlands)

    Langeveld, S. A.; van Mansfeld, A. D.; van der Ende, A.; van de Pol, J. H.; van Arkel, G. A.; Weisbeek, P. J.

    1981-01-01

    The A* protein of bacteriophage phi X174 is a single-stranded DNA specific nuclease. It can cleave phi X viral ss DNA in many different places. The position of these sites have been determined within the known phi X174 nucleotide sequence (1). From the sequences at these sites it is clear that the

  7. Mung bean sprout (Phaseolus aureus) nuclease and its biological and antitumor effects

    Czech Academy of Sciences Publication Activity Database

    Souček, J.; Škvor, J.; Poučková, P.; Matoušek, Jaroslav; Slavík, Tomáš; Matoušek, Josef

    2006-01-01

    Roč. 53, - (2006), s. 402-409 ISSN 0028-2685 R&D Projects: GA ČR GA521/06/1149; GA ČR GA523/04/0755 Keywords : mung bean * nuclease Subject RIV: FD - Oncology ; Hematology Impact factor: 1.247, year: 2006

  8. Nucleases activities during French bean leaf aging and dark-induced senescence.

    Science.gov (United States)

    Lambert, Rocío; Quiles, Francisco Antonio; Gálvez-Valdivieso, Gregorio; Piedras, Pedro

    2017-11-01

    During leaf senescence resources are managed, with nutrients mobilized from older leaves to new sink tissues. The latter implies a dilemma in terms of resource utilization, the leaf senescence should increase seed quality whereas delay in senescence should improve the seed yield. Increased knowledge about nutrient recycling during leaf senescence could lead to advances in agriculture and improved seed quality. Macromolecules mobilized during leaf senescence include proteins and nucleic acids. Although nucleic acids have been less well studied than protein degradation, they are possible reservoirs of nitrogen and phosphorous. The present study investigated nuclease activities and gene expression patterns of five members of the S1/P1 family in French bean (Phaseolus vulgaris L. cv.)Page: 2 during leaf senescence. An in-gel assay was used to detect nuclease activity during natural and dark-induced senescence, with single-stranded DNA (ssDNA) used as a substrate. The results revealed two nucleases (glycoproteins), with molecular masses of 34 and 39kDa in the senescent leaves. The nuclease activities were higher at a neutral than at an acidic pH. EDTA treatment inhibited the activities of the nucleases, and the addition of zinc resulted in the recovery of these activities. Both the 34 and 39kDa nucleases were able to use RNA and double-stranded DNA (dsDNA) as substrates, although their activities were low when dsDNA was used as a substrate. In addition, two ribonucleases with molecular masses of 14 and 16kDa, both of which could only utilize RNA as a substrate, were detected in the senescent leaves. Two members of the S1/P1 family, PVN2 and PVN5, were expressed under the experimental conditions, suggesting that these two genes were involved in senescence. The nuclease activity of the glycoproteins and gene expression were similar under both natural senescence and dark-induced senescence conditions. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights

  9. Identification of Plasmodium falciparum DNA Repair Protein Mre11 with an Evolutionarily Conserved Nuclease Function.

    Directory of Open Access Journals (Sweden)

    Sugith Babu Badugu

    Full Text Available The eukaryotic Meiotic Recombination protein 11 (Mre11 plays pivotal roles in the DNA damage response (DDR. Specifically, Mre11 senses and signals DNA double strand breaks (DSB and facilitates their repair through effector proteins belonging to either homologous recombination (HR or non-homologous end joining (NHEJ repair mechanisms. In the human malaria parasite Plasmodium falciparum, HR and alternative-NHEJ have been identified; however, little is known about the upstream factors involved in the DDR of this organism. In this report, we identify a putative ortholog of Mre11 in P. falciparum (PfalMre11 that shares 22% sequence similarity to human Mre11. Homology modeling reveals striking structural resemblance of the predicted PfalMre11 nuclease domain to the nuclease domain of Saccharomyces cerevisiae Mre11 (ScMre11. Complementation analyses reveal functional conservation of PfalMre11 nuclease activity as demonstrated by the ability of the PfalMre11 nuclease domain, in conjunction with the C-terminal domain of ScMre11, to functionally complement an mre11 deficient yeast strain. Functional complementation was virtually abrogated by an amino acid substitution in the PfalMre11 nuclease domain (D398N. PfalMre11 is abundant in the mitotically active trophozoite and schizont stages of P. falciparum and is up-regulated in response to DNA damage, suggesting a role in the DDR. PfalMre11 exhibits physical interaction with PfalRad50. In addition, yeast 2-hybrid studies show that PfalMre11 interacts with ScRad50 and ScXrs2, two important components of the well characterized Mre11-Rad50-Xrs2 complex which is involved in DDR signaling and repair in S. cerevisiae, further supporting a role for PfalMre11 in the DDR. Taken together, these findings provide evidence that PfalMre11 is an evolutionarily conserved component of the DDR in Plasmodium.

  10. An inactivated nuclease-like domain in RecC with novel function: implications for evolution

    Directory of Open Access Journals (Sweden)

    Rigden Daniel

    2005-06-01

    Full Text Available Abstract Background The PD-(D/ExK superfamily, containing a wide variety of other exo- and endonucleases, is a notable example of general function conservation in the face of extreme sequence and structural variation. Almost all members employ a small number of shared conserved residues to bind catalytically essential metal ions and thereby effect DNA cleavage. The crystal structure of the RecBCD prokaryotic DNA repair machinery shows that RecB contains such a nuclease domain at its C-terminus. The RecC C-terminal region was reported as having a novel fold. Results The RecC C-terminal region can be divided into an alpha/beta domain and a smaller alpha-helical bundle domain. Here we show that the alpha/beta domain is homologous to the RecB nuclease domain but lacks the features necessary for catalysis. Instead, the domain has a novel function within the nuclease superfamily – providing a hoop through which single-stranded DNA passes. Comparison with other structures of nuclease domains bound to DNA reveals strikingly different modes of ligand binding. The alpha-helical bundle domain contributes the pin which splits the DNA duplex. Conclusion The demonstrated homology of RecB and RecC shows how evolution acted to produce the present RecBCD complex through aggregation of new domains as well as functional divergence and structural redeployment of existing domains. Distantly homologous nuclease(-like domains bind DNA in highly diverse manners.

  11. Efficient methods for targeted mutagenesis in zebrafish using zinc-finger nucleases: data from targeting of nine genes using CompoZr or CoDA ZFNs.

    Directory of Open Access Journals (Sweden)

    Raman Sood

    Full Text Available Recently, it has been shown that targeted mutagenesis using zinc-finger nucleases (ZFNs and transcription activator-like effector nucleases (TALENs can be used to generate knockout zebrafish lines for analysis of their function and/or developing disease models. A number of different methods have been developed for the design and assembly of gene-specific ZFNs and TALENs, making them easily available to most zebrafish researchers. Regardless of the choice of targeting nuclease, the process of generating mutant fish is similar. It is a time-consuming and multi-step process that can benefit significantly from development of efficient high throughput methods. In this study, we used ZFNs assembled through either the CompoZr (Sigma-Aldrich or the CoDA (context-dependent assembly platforms to generate mutant zebrafish for nine genes. We report our improved high throughput methods for 1 evaluation of ZFNs activity by somatic lesion analysis using colony PCR, eliminating the need for plasmid DNA extractions from a large number of clones, and 2 a sensitive founder screening strategy using fluorescent PCR with PIG-tailed primers that eliminates the stutter bands and accurately identifies even single nucleotide insertions and deletions. Using these protocols, we have generated multiple mutant alleles for seven genes, five of which were targeted with CompoZr ZFNs and two with CoDA ZFNs. Our data also revealed that at least five-fold higher mRNA dose was required to achieve mutagenesis with CoDA ZFNs than with CompoZr ZFNs, and their somatic lesion frequency was lower (<5% when compared to CopmoZr ZFNs (9-98%. This work provides high throughput protocols for efficient generation of zebrafish mutants using ZFNs and TALENs.

  12. PAN3 encodes a subunit of the Pab1p-dependent poly(A) nuclease in Saccharomyces cerevisiae.

    Science.gov (United States)

    Brown, C E; Tarun, S Z; Boeck, R; Sachs, A B

    1996-10-01

    The Pab1p-dependent poly(A) nuclease (PAN) from Saccharomyces cerevisiae copurifies with polypeptides of approximately 127 and 76 kDa. Previously, it was demonstrated that the 127-kDa Pan2 protein is required for PAN activity (R. Boeck, S. Tarun, M. Reiger, J. Deardorff, S. Müller-Auer, and A.B. Sachs, J. Biol. Chem. 271:432-438, 1996). Here we demonstrate that the 76-kDa protein, encoded by the nonessential PAN3 gene, is also required for enzymatic activity. Deletion of PAN3 resulted in the loss of PAN activity in yeast extracts, and immunodepletion of Pan3p from purified PAN fractions abolished enzymatic activity. We show by coimmunoprecipitation and directed two-hybrid studies that the Pan2 and Pan3 proteins physically interact. In addition, we demonstrate that a deletion of PAN2, PAN3, or both resulted in similar increases in mRNA poly(A) tail lengths in vivo. These data strongly suggest that both Pan2p and Pan3p are required subunits of the PAN enzyme and that PAN functions in vivo to shorten mRNA poly(A) tails.

  13. Structures of human exonuclease I DNA complexes suggest a unified mechanism for nuclease family

    Science.gov (United States)

    Orans, Jillian; McSweeney, Elizabeth A.; Iyer, Ravi R.; Hast, Michael A.; Hellinga, Homme W.; Modrich, Paul; Beese, Lorena S.

    2011-01-01

    Summary Human exonuclease 1 (hExo1) plays important roles in DNA repair and recombination processes that maintain genomic integrity. It is a member of the 5′ structure-specific nuclease family of exonucleases and endonucleases that includes FEN-1, XPG, and GEN1. We present structures of hExo1 in complex with a DNA substrate, followed by mutagenesis studies, and propose a common mechanism by which this nuclease family recognizes and processes diverse DNA structures. hExo1 induces a sharp bend in the DNA at nicks or gaps. Frayed 5′ ends of nicked duplexes resemble flap junctions, unifying the mechanisms of endo- and exo-nucleolytic processing. Conformational control of a mobile region in the catalytic site suggests a mechanism for allosteric regulation by binding to protein partners. The relative arrangement of substrate binding sites in these enzymes provides an elegant solution to a complex geometrical puzzle of substrate recognition and processing. PMID:21496642

  14. Structural basis for the substrate selectivity of Helicobacter pylori NucT nuclease activity.

    Directory of Open Access Journals (Sweden)

    Louisa Celma

    Full Text Available The Phospholipase D (PLD superfamily of proteins includes a group of enzymes with nuclease activity on various nucleic acid substrates. Here, with the aim of better understanding the substrate specificity determinants in this subfamily, we have characterised the enzymatic activity and the crystal structure of NucT, a nuclease implicated in Helicobacter pylori purine salvage and natural transformation and compared them to those of its bacterial and mammalian homologues. NucT exhibits an endonuclease activity with a strong preference for single stranded nucleic acids substrates. We identified histidine124 as essential for the catalytic activity of the protein. Comparison of the NucT crystal structure at 1.58 Å resolution reported here with those of other members of the sub-family suggests that the specificity of NucT for single-stranded nucleic acids is provided by the width of a positively charged groove giving access to the catalytic site.

  15. megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.

    Science.gov (United States)

    Boissel, Sandrine; Jarjour, Jordan; Astrakhan, Alexander; Adey, Andrew; Gouble, Agnès; Duchateau, Philippe; Shendure, Jay; Stoddard, Barry L; Certo, Michael T; Baker, David; Scharenberg, Andrew M

    2014-02-01

    Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e. rate of cleavage at 'off-target' sites), and efficient/facile means for delivery to desired target cells. Here, we describe the development of a single-chain rare-cleaving nuclease architecture, which we designate 'megaTAL', in which the DNA binding region of a transcription activator-like (TAL) effector is used to 'address' a site-specific meganuclease adjacent to a single desired genomic target site. This architecture allows the generation of extremely active and hyper-specific compact nucleases that are compatible with all current viral and nonviral cell delivery methods.

  16. Design principles for nuclease-deficient CRISPR-based transcriptional regulators

    DEFF Research Database (Denmark)

    Jensen, Michael K.

    2018-01-01

    The engineering of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated proteins (Cas) continues to expand the toolkit available for genome editing, reprogramming gene regulation, genome visualization, and epigenetic studies of living organisms. In this review...... the emerging design principles on the use of nuclease-deficient CRISPR-based reprogramming of gene expression will be presented. The review will focus on the designs implemented in yeast both at the level of CRISPR proteins and gRNA, but will lend due credits to the seminal studies performed in other species...... where relevant. In addition to design principles, this review also highlights applications benefitting from the use of CRISPR-mediated transcriptional regulation and discuss the future directions to further expand the toolkit for nuclease-deficient reprogramming of genomes. As such this review should...

  17. Dynamics and denaturation of a protein. Simulations and neutron scattering on staphylococcus nuclease

    International Nuclear Information System (INIS)

    Goupil-Lamy, Anne

    1997-01-01

    This research thesis reports simulations and experiments of inelastic scattering on the whole frequency spectrum to analyse the vibrations of the staphylococcus nuclease and its fragment, in order to study protein folding. Based on these experiments, information on eigenvectors which describe vibration modes can be directly obtained. Inelastic intensities are indeed fully determined by nuclear cross sections and the mean square displacement of each atom. Some experimentally noticed peaks are then explained by calculating a theoretical spectrum from an analysis of normal modes. The studied fragment is made of 136 c-terminal residues. The fragment structure obtained by molecular dynamics simulation is compared with available experimental data. Then, experiments of neutron scattering on the nuclease of staphylococcus and its fragment have been performed. Quasi elastic scattering spectra have been measured. The author then used simulations to try to reproduce the quasi-elastic spectrum. Experiments of inelastic scattering have then been performed [fr

  18. Phosphate binding in the active centre of tomato multifunctional nuclease TBN1 and analysis of superhelix formation by the enzyme

    Czech Academy of Sciences Publication Activity Database

    Stránský, J.; Koval, Tomáš; Podzimek, T.; Týcová, A.; Lipovová, P.; Matoušek, J.; Kolenko, Petr; Fejfarová, Karla; Dušková, J.; Skálová, T.; Hašek, J.; Dohnálek, Jan

    2015-01-01

    Roč. 71, č. 11 (2015), s. 1408-1415 ISSN 2053-230X R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) EE2.3.30.0029 Institutional support: RVO:61389013 Keywords : tomato multifunctional nuclease * TBN1 * type I nuclease Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.647, year: 2015

  19. Phosphate binding in the active centre of tomato multifunctional nuclease TBN1 and analysis of superhelix formation by the enzyme

    Czech Academy of Sciences Publication Activity Database

    Stránský, Jan; Koval, Tomáš; Podzimek, T.; Týcová, Anna; Lipovová, P.; Matoušek, Jaroslav; Kolenko, Petr; Fejfarová, Karla; Dušková, Jarmila; Skálová, Tereza; Hašek, Jindřich; Dohnálek, Jan

    2015-01-01

    Roč. 71, č. 11 (2015), s. 1408-1415 ISSN 2053-230X R&D Projects: GA MŠk LG14009; GA MŠk(CZ) EE2.3.30.0029; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 ; RVO:60077344 Keywords : tomato multifunctional nuclease * TBN1 * type I nuclease * superhelix Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.647, year: 2015

  20. A Novel Prokaryotic Green Fluorescent Protein Expression System for Testing Gene Editing Tools Activity Like Zinc Finger Nuclease

    OpenAIRE

    Sabzehei, Faezeh; Kouhpayeh, Shirin; Dastjerdeh, Mansoureh Shahbazi; Khanahmad, Hossein; Salehi, Rasoul; Naderi, Shamsi; Taghizadeh, Razieh; Rabiei, Parisa; Hejazi, Zahra; Shariati, Laleh

    2017-01-01

    Background: Gene editing technology has created a revolution in the field of genome editing. The three of the most famous tools in gene editing technology are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated systems. As their predictable nature, it is necessary to assess their efficiency. There are some methods for this purpose, but most of them are time labor and complicate...

  1. X-ray diffraction studies of red tomato nuclease TBN1

    Czech Academy of Sciences Publication Activity Database

    Kovaľ, Tomáš; Dohnálek, Jan; Lipovová, P.; Podzimek, T.; Matoušek, Jaroslav; Dušková, Jarmila; Skálová, Tereza; Štěpánková, Andrea; Kolenko, Petr; Hašek, Jindřich

    2010-01-01

    Roč. 17, 1a (2010), b40 ISSN 1211-5894. [Discussions in Structural Molecular Biology /8./. 18.03.2010-20.03.2010, Nové Hrady] R&D Projects: GA ČR GA202/06/0757 Institutional research plan: CEZ:AV0Z10100521; CEZ:AV0Z40500505 Keywords : plant bifunctional nuclease * crystallization * x-ray diffraction analysis Subject RIV: BM - Solid Matter Physics ; Magnetism

  2. Halophilic Nuclease of a Moderately Halophilic Bacillus sp.: Production, Purification, and Characterization

    OpenAIRE

    Onishi, Hiroshi; Mori, Tatsuro; Takeuchi, Setsuo; Tani, Keiko; Kobayashi, Takekazu; Kamekura, Masahiro

    1983-01-01

    A moderately halophilic bacterium, Bacillus sp., isolated from rotting wood on the seashore in Nauru, produced an extracellular nuclease when cultivated aerobically in media containing 1 to 2 M NaCl. The enzyme was purified from the culture filtrate to an electrophoretically homogeneous state by ethanol precipitation, DEAE-Sephadex A-50 column chromatography, and Sephadex G-200 gel filtration. The enzyme consisted of two charge isomers and showed both RNase and DNase activities. Molecular wei...

  3. Improved somatic mutagenesis in zebrafish using transcription activator-like effector nucleases (TALENs.

    Directory of Open Access Journals (Sweden)

    Finola E Moore

    Full Text Available Zinc Finger Nucleases (ZFNs made by Context-Dependent Assembly (CoDA and Transcription Activator-Like Effector Nucleases (TALENs provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%-76.8% compared to 1.1%-3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.

  4. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases

    DEFF Research Database (Denmark)

    Chen, Fuqiang; Pruett-Miller, Shondra M; Huang, Yuping

    2011-01-01

    Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point...... mutation, (ii) targeted genomic deletion of up to 100 kb and (iii) targeted insertion of small genetic elements concomitant with large genomic deletions....

  5. The history and market impact of CRISPR RNA-guided nucleases

    OpenAIRE

    van Erp, Paul BG; Bloomer, Gary; Wilkinson, Royce; Wiedenheft, Blake

    2015-01-01

    The interface between viruses and their hosts’ are hot spots for biological and biotechnological innovation. Bacteria use restriction endonucleases to destroy invading DNA, and industry has exploited these enzymes for molecular cut-and-paste reactions that are central to many recombinant DNA technologies. Today, another class of nucleases central to adaptive immune systems that protect bacteria and archaea from invading viruses and plasmids are blazing a similar path from basic science to pro...

  6. Zinc Finger Nuclease: A New Approach to Overcome Beta-Lactam Antibiotic Resistance

    OpenAIRE

    Shahbazi Dastjerdeh, Mansoureh; Kouhpayeh, Shirin; Sabzehei, Faezeh; Khanahmad, Hossein; Salehi, Mansour; Mohammadi, Zahra; Shariati, Laleh; Hejazi, Zahra; Rabiei, Parisa; Manian, Mostafa

    2016-01-01

    Background: The evolution of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) has been accelerated recently by the indiscriminate application of antibiotics. Antibiotic resistance has challenged the success of medical interventions and therefore is considered a hazardous threat to human health. Objectives: The present study aimed to describe the use of zinc finger nuclease (ZFN) technology to target and disrupt a plasmid-encoded ?-lactamase, which prevents horizontal...

  7. Plant Ribonucleases and Nucleases as Antiproliferative Agens Targeting Human Tumors Growing in Mice

    Czech Academy of Sciences Publication Activity Database

    Matoušek, Jaroslav; Matoušek, Josef

    2010-01-01

    Roč. 4, č. 1 (2010), s. 29-39 ISSN 1872-2156 R&D Projects: GA ČR GA521/06/1149; GA ČR GA521/09/1214 Institutional research plan: CEZ:AV0Z50510513; CEZ:AV0Z50450515 Keywords : antiproliferative cytotoxic * effect human * plant nuclease Subject RIV: EB - Genetics ; Molecular Biology

  8. Knockout of Myostatin by Zinc-finger Nuclease in Sheep Fibroblasts and Embryos

    OpenAIRE

    Zhang, Xuemei; Wang, Liqin; Wu, Yangsheng; Li, Wenrong; An, Jing; Zhang, Fuchun; Liu, Mingjun

    2016-01-01

    Myostatin (MSTN) can negatively regulate the growth and development of skeletal muscle, and natural mutations can cause “double-muscling” trait in animals. In order to block the inhibiting effect of MSTN on muscle growth, we transferred zinc-finger nucleases (ZFN) which targeted sheep MSTN gene into cultured fibroblasts. Gene targeted colonies were isolated from transfected fibroblasts by serial dilution culture and screened by sequencing. Two colonies were identified with mono-allele mutatio...

  9. Geminivirus-Mediated Genome Editing in Potato (Solanum tuberosum L.) Using Sequence-Specific Nucleases.

    Science.gov (United States)

    Butler, Nathaniel M; Baltes, Nicholas J; Voytas, Daniel F; Douches, David S

    2016-01-01

    Genome editing using sequence-specific nucleases (SSNs) is rapidly being developed for genetic engineering in crop species. The utilization of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated systems (CRISPR/Cas) for inducing double-strand breaks facilitates targeting of virtually any sequence for modification. Targeted mutagenesis via non-homologous end-joining (NHEJ) has been demonstrated extensively as being the preferred DNA repair pathway in plants. However, gene targeting via homologous recombination (HR) remains more elusive but could be a powerful tool for directed DNA repair. To overcome barriers associated with gene targeting, a geminivirus replicon (GVR) was used to deliver SSNs targeting the potato ACETOLACTATE SYNTHASE1 (ALS1) gene and repair templates designed to incorporate herbicide-inhibiting point mutations within the ALS1 locus. Transformed events modified with GVRs held point mutations that were capable of supporting a reduced herbicide susceptibility phenotype, while events transformed with conventional T-DNAs held no detectable mutations and were similar to wild-type. Regeneration of transformed events improved detection of point mutations that supported a stronger reduced herbicide susceptibility phenotype. These results demonstrate the use of geminiviruses for delivering genome editing reagents in plant species, and a novel approach to gene targeting in a vegetatively propagated species.

  10. Geminivirus-mediated genome editing in potato (Solanum tuberosum L. using sequence-specific nucleases

    Directory of Open Access Journals (Sweden)

    Nathaniel M Butler

    2016-07-01

    Full Text Available Genome editing using sequence-specific nucleases (SSNs is rapidly being developed for genetic engineering in crop species. The utilization of zinc finger nucleases (ZFNs, transcription activator-like effector nucleases (TALENs and CRISPR/Cas (clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated systems (Cas for inducing double-strand breaks facilitates targeting of virtually any sequence for modification. Targeted mutagenesis via nonhomologous end-joining has been demonstrated extensively as being the preferred DNA repair pathway in plants. However, gene targeting via homologous recombination remains more elusive but could be a powerful tool for directed DNA repair. To overcome barriers associated with gene targeting, a geminivirus replicon (GVR was used to deliver SSNs targeting the potato ACETOLACTATE SYNTHASE1 (ALS1 gene and repair templates designed to incorporate herbicide-inhibiting point mutations within the ALS1 locus. Transformed events modified with GVRs held both point mutations that were capable of supporting a reduced herbicide susceptibility phenotype, while events transformed with conventional T-DNAs held no detectable mutations and were similar to wild-type. Regeneration of transformed events improved detection of point mutations that supported a stronger reduced herbicide susceptibility phenotype. These results demonstrate the use of geminiviruses for delivering genome editing reagents in plant species, and an approach to gene targeting in a vegetatively propagated species.

  11. Applications of genome editing by programmable nucleases to the metabolic engineering of secondary metabolites.

    Science.gov (United States)

    Leitão, Ana Lúcia; Costa, Marina C; Enguita, Francisco J

    2017-01-10

    Genome engineering is a branch of modern biotechnology composed of a cohort of protocols designed to construct and modify a genotype with the main objective of giving rise to a desired phenotype. Conceptually, genome engineering is based on the so called genome editing technologies, a group of genetic techniques that allow either to delete or to insert genetic information in a particular genomic locus. Ten years ago, genome editing tools were limited to virus-driven integration and homologous DNA recombination. However, nowadays the uprising of programmable nucleases is rapidly changing this paradigm. There are two main families of modern tools for genome editing depending on the molecule that controls the specificity of the system and drives the editor machinery to its place of action. Enzymes such as Zn-finger and TALEN nucleases are protein-driven genome editors; while CRISPR system is a nucleic acid-guided editing system. Genome editing techniques are still not widely applied for the design of new compounds with pharmacological activity, but they are starting to be considered as promising tools for rational genome manipulation in biotechnology applications. In this review we will discuss the potential applications of programmable nucleases for the metabolic engineering of secondary metabolites with biological activity. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Plant genome editing by novel tools: TALEN and other sequence specific nucleases.

    Science.gov (United States)

    Sprink, Thorben; Metje, Janina; Hartung, Frank

    2015-04-01

    Genome editing technologies using sequence specific nucleases (SSNs) became a tremendously powerful and precise tool for reverse genetic approaches and applied biology. Transcription activator-like effector nucleases (TALENs) in particular, consisting of a free designable DNA binding domain and a nuclease, have been exploited today by a huge number of approaches in many different organisms. The convenience of designing the DNA binding domain and straightforward protocols for their assembly, as well as the broad number of applications in different scientific fields made it Natures method of the year 2011. TALENs act as molecular scissors by introducing double strand breaks (DSBs) to the DNA at a given location. The DSBs are subsequently repaired by the cell itself using different repair pathways such as non-homologous end joining (NHEJ) or homologous recombination (HR). These mechanisms can lead to deletions, insertions, replacements or larger chromosomal rearrangements. By offering a template DNA it is possible to channel the repair in direction of HR. In this article we review the recent findings in the field of SSN approaches with emphasis on plants. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. A 21-amino acid peptide from the cysteine cluster II of the family D DNA polymerase from Pyrococcus horikoshii stimulates its nuclease activity which is Mre11-like and prefers manganese ion as the cofactor.

    Science.gov (United States)

    Shen, Yulong; Tang, Xiao-Feng; Yokoyama, Hideshi; Matsui, Eriko; Matsui, Ikuo

    2004-01-01

    Family D DNA polymerase (PolD) is a new type of DNA polymerase possessing polymerization and 3'-5' exonuclease activities. Here we report the characterization of the nuclease activity of PolD from Pyrococcus horikoshii. By site-directed mutagenesis, we verified that the putative Mre11-like nuclease domain in the small subunit (DP1), predicted according to computer analysis and structure inference reported previously, is the catalytic domain. We show that D363, H365 and H454 are the essential residues, while D407, N453, H500, H563 and H565 are critical residues for the activity. We provide experimental evidence demonstrating that manganese, rather than magnesium, is the preferable metal ion for the nuclease activity of PolD. We also show that DP1 alone is insufficient to perform full catalysis, which additionally requires the formation of the PolD complex and manganese ion. We found that a 21 amino acid, subunit-interacting peptide of the sequence from cysteine cluster II of the large subunit (DP2) stimulates the exonuclease activity of DP1 and the internal deletion mutants of PolD lacking the 21-aa sequence. This indicates that the putative zinc finger motif of the cysteine cluster II is deeply involved in the nucleolytic catalysis.

  14. Selection-independent generation of gene knockout mouse embryonic stem cells using zinc-finger nucleases.

    Directory of Open Access Journals (Sweden)

    Anna Osiak

    Full Text Available Gene knockout in murine embryonic stem cells (ESCs has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs. Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.

  15. Use of designer nucleases for targeted gene and genome editing in plants.

    Science.gov (United States)

    Weeks, Donald P; Spalding, Martin H; Yang, Bing

    2016-02-01

    The ability to efficiently inactivate or replace genes in model organisms allowed a rapid expansion of our understanding of many of the genetic, biochemical, molecular and cellular mechanisms that support life. With the advent of new techniques for manipulating genes and genomes that are applicable not only to single-celled organisms, but also to more complex organisms such as animals and plants, the speed with which scientists and biotechnologists can expand fundamental knowledge and apply that knowledge to improvements in medicine, industry and agriculture is set to expand in an exponential fashion. At the heart of these advancements will be the use of gene editing tools such as zinc finger nucleases, modified meganucleases, hybrid DNA/RNA oligonucleotides, TAL effector nucleases and modified CRISPR/Cas9. Each of these tools has the ability to precisely target one specific DNA sequence within a genome and (except for DNA/RNA oligonucleotides) to create a double-stranded DNA break. DNA repair to such breaks sometimes leads to gene knockouts or gene replacement by homologous recombination if exogenously supplied homologous DNA fragments are made available. Genome rearrangements are also possible to engineer. Creation and use of such genome rearrangements, gene knockouts and gene replacements by the plant science community is gaining significant momentum. To document some of this progress and to explore the technology's longer term potential, this review highlights present and future uses of designer nucleases to greatly expedite research with model plant systems and to engineer genes and genomes in major and minor crop species for enhanced food production. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  16. A metal-free DNA nuclease based on a cyclic peptide scaffold

    Czech Academy of Sciences Publication Activity Database

    Alkhader, S.; Ezra, A.; Kašpárková, Jana; Brabec, Viktor; Yavin, E.

    2010-01-01

    Roč. 21, č. 8 (2010), s. 1425-1431 ISSN 1043-1802 R&D Projects: GA AV ČR(CZ) IAA400040803; GA MŠk(CZ) LC06030; GA MŠk(CZ) ME08017; GA MŠk(CZ) OC08003; GA AV ČR(CZ) KAN200200651 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : DNA * cleavage * chemical nuclease Subject RIV: BO - Biophysics Impact factor: 5.002, year: 2010

  17. Detection and characterization of verocytotoxin-producing Escherichia coli by automated 5 ' nuclease PCR assay

    DEFF Research Database (Denmark)

    Nielsen, Eva Møller; Andersen, Marianne Thorup

    2003-01-01

    In recent years increased attention has been focused on infections caused by isolates of verocytotoxin-producing Escherichia coli (VTEC) serotypes other than O157. These non-O157 VTEC isolates are commonly present in food and food production animals. Easy detection, isolation, and characterization...... of hydrophobic-grid membrane filters and DNA probe hybridization. Furthermore, we have developed 5' nuclease PCR assays for the detection of virulence factors typically present in VTEC isolates, including subtypes of three genes of the locus of enterocyte effacement (LEE) pathogenicity island. The 22 assays...

  18. The history and market impact of CRISPR RNA-guided nucleases.

    Science.gov (United States)

    van Erp, Paul Bg; Bloomer, Gary; Wilkinson, Royce; Wiedenheft, Blake

    2015-06-01

    The interface between viruses and their hosts' are hot spots for biological and biotechnological innovation. Bacteria use restriction endonucleases to destroy invading DNA, and industry has exploited these enzymes for molecular cut-and-paste reactions that are central to many recombinant DNA technologies. Today, another class of nucleases central to adaptive immune systems that protect bacteria and archaea from invading viruses and plasmids are blazing a similar path from basic science to profound biomedical and industrial applications. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Plant multifunctional nuclease TBN1 with unexpected phospholipase activity: structural study and reaction-mechanism analysis

    Czech Academy of Sciences Publication Activity Database

    Koval, Tomáš; Lipovová, P.; Podzimek, T.; Matoušek, Jaroslav; Dušková, Jarmila; Skálová, Tereza; Štěpánková, Andrea; Hašek, Jindřich; Dohnálek, Jan

    2013-01-01

    Roč. 69, č. 2 (2013), s. 213-226 ISSN 0907-4449 R&D Projects: GA MŠk EE2.3.30.0029; GA ČR GAP302/11/0855; GA ČR GA310/09/1407; GA ČR GA521/09/1214 Grant - others:AV ČR(CZ) AP0802 Program:Akademická prémie - Praemium Academiae Institutional support: RVO:61389013 ; RVO:60077344 Keywords : plant nucleases * catalytic zinc cluster * glycoproteins Subject RIV: CE - Biochemistry; EB - Genetics ; Molecular Biology (BC-A) Impact factor: 7.232, year: 2013

  20. Erasure of Tet-Oxidized 5-Methylcytosine by a SRAP Nuclease

    Directory of Open Access Journals (Sweden)

    Soo-Mi Kweon

    2017-10-01

    Full Text Available Enzymatic oxidation of 5-methylcytosine (5mC in DNA by the Tet dioxygenases reprograms genome function in embryogenesis and postnatal development. Tet-oxidized derivatives of 5mC such as 5-hydroxymethylcytosine (5hmC act as transient intermediates in DNA demethylation or persist as durable marks, yet how these alternative fates are specified at individual CpGs is not understood. Here, we report that the SOS response-associated peptidase (SRAP domain protein Srap1, the mammalian ortholog of an ancient protein superfamily associated with DNA damage response operons in bacteria, binds to Tet-oxidized forms of 5mC in DNA and catalyzes turnover of these bases to unmodified cytosine by an autopeptidase-coupled nuclease. Biallelic inactivation of murine Srap1 causes embryonic sublethality associated with widespread accumulation of ectopic 5hmC. These findings establish a function for a class of DNA base modification-selective nucleases and position Srap1 as a determinant of 5mC demethylation trajectories during mammalian embryonic development.

  1. Distinct Mechanisms of Nuclease-Directed DNA-Structure-Induced Genetic Instability in Cancer Genomes.

    Science.gov (United States)

    Zhao, Junhua; Wang, Guliang; Del Mundo, Imee M; McKinney, Jennifer A; Lu, Xiuli; Bacolla, Albino; Boulware, Stephen B; Zhang, Changsheng; Zhang, Haihua; Ren, Pengyu; Freudenreich, Catherine H; Vasquez, Karen M

    2018-01-30

    Sequences with the capacity to adopt alternative DNA structures have been implicated in cancer etiology; however, the mechanisms are unclear. For example, H-DNA-forming sequences within oncogenes have been shown to stimulate genetic instability in mammals. Here, we report that H-DNA-forming sequences are enriched at translocation breakpoints in human cancer genomes, further implicating them in cancer etiology. H-DNA-induced mutations were suppressed in human cells deficient in the nucleotide excision repair nucleases, ERCC1-XPF and XPG, but were stimulated in cells deficient in FEN1, a replication-related endonuclease. Further, we found that these nucleases cleaved H-DNA conformations, and the interactions of modeled H-DNA with ERCC1-XPF, XPG, and FEN1 proteins were explored at the sub-molecular level. The results suggest mechanisms of genetic instability triggered by H-DNA through distinct structure-specific, cleavage-based replication-independent and replication-dependent pathways, providing critical evidence for a role of the DNA structure itself in the etiology of cancer and other human diseases. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Crystallization and preliminary X-ray characterization of two thermostable DNA nucleases

    International Nuclear Information System (INIS)

    Kuettner, E. Bartholomeus; Pfeifer, Sven; Keim, Antje; Greiner-Stöffele, Thomas; Sträter, Norbert

    2006-01-01

    Two thermostable DNA nucleases from archaea were crystallized in different space groups; the crystals were suitable for X-ray analysis. Temperature-tolerant organisms are an important source to enhance the stability of enzymes used in biotechnological processes. The DNA-cleaving enzyme exonuclease III from Escherichia coli is used in several applications in gene technology. A thermostable variant could expand the applicability of the enzyme in these methods. Two homologous nucleases from Archaeoglobus fulgidus (ExoAf) and Methanothermobacter thermoautrophicus (ExoMt) were studied for this purpose. Both enzymes were crystallized in different space groups using (poly)ethylene glycols, 2,4-methyl pentandiol, dioxane, ethanol or 2-propanol as precipitants. The addition of a 10-mer DNA oligonucleotide was important to obtain monoclinic crystals of ExoAf and ExoMt that diffracted to resolutions better than 2 Å using synchrotron radiation. The crystal structures of the homologous proteins can serve as templates for genetic engineering of the E. coli exonuclease III and will aid in understanding the different catalytic properties of the enzymes

  3. Improving nuclease activity of copper(II)-terpyridine complex through solubilizing and charge effects of glycine.

    Science.gov (United States)

    Zhou, Wen; Wang, Xiaoyong; Hu, Ming; Guo, Zijian

    2013-04-01

    Copper complexes are potential metallonucleases that may find application in biotechnology and molecular biology. In this study, a ternary copper-terpyridine complex [Cu(ttpy)(Gly)(NO3)](NO3)·H2O (1) (ttpy=4'-p-tolyl-2,2':6,2″-terpyridine) is synthesized and characterized by X-ray crystallography and ESI-MS as an artificial nuclease. Glycine (Gly) is introduced into the complex to enhance the water-solubility and electrostatic affinity for the nucleic acid target. The interaction between complex 1 and DNA has been studied by spectroscopy and gel electrophoresis, using a structural analog [Cu(ttpy)(NO3)2] (2) as the reference. Complex 1 demonstrates an increased DNA binding ability and oxidative cleavage activity towards supercoiled pBR322 DNA as compared with complex 2. The enhanced water-solubility and positive charge of complex 1 may facilitate its access to DNA and formation of hydrogen bonds with the sugar-phosphate backbone. The results indicate that carefully positioned auxiliary groups in a copper complex can significantly affect the substrate binding or activation ability and consequently the nuclease efficiency of the complex. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Mung bean nuclease treatment increases capture specificity of microdroplet-PCR based targeted DNA enrichment.

    Directory of Open Access Journals (Sweden)

    Zhenming Yu

    Full Text Available Targeted DNA enrichment coupled with next generation sequencing has been increasingly used for interrogation of select sub-genomic regions at high depth of coverage in a cost effective manner. Specificity measured by on-target efficiency is a key performance metric for target enrichment. Non-specific capture leads to off-target reads, resulting in waste of sequencing throughput on irrelevant regions. Microdroplet-PCR allows simultaneous amplification of up to thousands of regions in the genome and is among the most commonly used strategies for target enrichment. Here we show that carryover of single-stranded template genomic DNA from microdroplet-PCR constitutes a major contributing factor for off-target reads in the resultant libraries. Moreover, treatment of microdroplet-PCR enrichment products with a nuclease specific to single-stranded DNA alleviates off-target load and improves enrichment specificity. We propose that nuclease treatment of enrichment products should be incorporated in the workflow of targeted sequencing using microdroplet-PCR for target capture. These findings may have a broad impact on other PCR based applications for which removal of template DNA is beneficial.

  5. Medium optimization for nuclease P1 production by Penicillium citrinum in solid-state fermentation using polyurethane foam as inert carrier

    NARCIS (Netherlands)

    Zhu, Y.; Knol, W.; Smits, J.P.; Bol, J.

    1996-01-01

    A solid-state fermentation system, using polyurethane foam as an inert carrier, was used for the production of nuclease P1 by Penicillium citrinum. Optimization of nuclease P1 production was carried out using a synthetic liquid medium. After a two-step medium optimization using a fractional

  6. Precise Genome Modification via Sequence-Specific Nucleases-Mediated Gene Targeting for Crop Improvement.

    Science.gov (United States)

    Sun, Yongwei; Li, Jingying; Xia, Lanqin

    2016-01-01

    Genome editing technologies enable precise modifications of DNA sequences in vivo and offer a great promise for harnessing plant genes in crop improvement. The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks by sequence-specific nucleases (SSNs) to initiate DNA repair reactions that are based on either non-homologous end joining (NHEJ) or homology-directed repair (HDR). While complete knock-outs and loss-of-function mutations generated by NHEJ are very valuable in defining gene functions, their applications in crop improvement are somewhat limited because many agriculturally important traits are conferred by random point mutations or indels at specific loci in either the genes' encoding or promoter regions. Therefore, genome modification through SSNs-mediated HDR for gene targeting (GT) that enables either gene replacement or knock-in will provide an unprecedented ability to facilitate plant breeding by allowing introduction of precise point mutations and new gene functions, or integration of foreign genes at specific and desired "safe" harbor in a predefined manner. The emergence of three programmable SSNs, such as zinc finger nucleases, transcriptional activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems has revolutionized genome modification in plants in a more controlled manner. However, while targeted mutagenesis is becoming routine in plants, the potential of GT technology has not been well realized for traits improvement in crops, mainly due to the fact that NHEJ predominates DNA repair process in somatic cells and competes with the HDR pathway, and thus HDR-mediated GT is a relative rare event in plants. Here, we review recent research findings mainly focusing on development and applications of precise GT in plants using three SSNs systems described above, and the potential mechanisms underlying HDR events in plant

  7. Recognition and repair of 2-aminofluorene- and 2-(acetylamino)fluorene-DNA adducts by UVRABC nuclease

    International Nuclear Information System (INIS)

    Pierce, J.R.; Case, R.; Tang, Moonshong

    1989-01-01

    Recognition of damage induced by N-hydroxy-2-aminofluorene (N-OH-AF) and N-acetoxy-2-(acetylamino)fluorene (NAAAF) in both φX174 RFI supercoiled DNA and a linear DNA fragment by purified UVRA, UVRB, and UVRC proteins was investigated. The authors have previously demonstrated that N-OH-AF and NAAAF treatments produce N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene (dG-C8-AAF), respectively, in DNA. Using a piperidine cleavage method and DNA sequence analysis, they have found that all guanine residues can be modified by N-OH-AF and NAAAF. These two kinds of adducts have different impacts on the DNA helix structure; while dG-C8-AF maintains the anti configuration, dG-C8-AAF is in the syn form. φX174 RF DNA-Escherichia coli transfection results indicate that while the uvrA, uvrB, and uvrC gene products are needed to repair dG-C8-AAF, the uvrC, but not the uvrA or uvrB gene products, is needed for repair of dG-C8-Af. However, they have found that in vitro the UVRA, UVRB, and UVRC proteins must work in concert to nick both dG-C8-AF and dG-C8-AAF. In general, the reactions of UVRABC nuclease toward dG-C8-AF are similar to those toward dG-C8-AAF; it incises seven to eight nucleotides from the 5' side and three to four nucleotides from the 3' side of the DNA adduct. Evidence is presented to suggest that hydrolysis on the 3' and 5' sides of the damaged base by UVRABC nuclease is not simultaneous and that at least occasionally hydrolysis occurs only on the 3' side or on the 5' side of the damage site. The possible mechanisms of UVRABC nuclease incision for AF-DNA are discussed

  8. Precise Genome Modification via Sequence-Specific Nucleases-Mediated Gene Targeting for Crop Improvement

    Science.gov (United States)

    Sun, Yongwei; Li, Jingying; Xia, Lanqin

    2016-01-01

    Genome editing technologies enable precise modifications of DNA sequences in vivo and offer a great promise for harnessing plant genes in crop improvement. The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks by sequence-specific nucleases (SSNs) to initiate DNA repair reactions that are based on either non-homologous end joining (NHEJ) or homology-directed repair (HDR). While complete knock-outs and loss-of-function mutations generated by NHEJ are very valuable in defining gene functions, their applications in crop improvement are somewhat limited because many agriculturally important traits are conferred by random point mutations or indels at specific loci in either the genes’ encoding or promoter regions. Therefore, genome modification through SSNs-mediated HDR for gene targeting (GT) that enables either gene replacement or knock-in will provide an unprecedented ability to facilitate plant breeding by allowing introduction of precise point mutations and new gene functions, or integration of foreign genes at specific and desired “safe” harbor in a predefined manner. The emergence of three programmable SSNs, such as zinc finger nucleases, transcriptional activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems has revolutionized genome modification in plants in a more controlled manner. However, while targeted mutagenesis is becoming routine in plants, the potential of GT technology has not been well realized for traits improvement in crops, mainly due to the fact that NHEJ predominates DNA repair process in somatic cells and competes with the HDR pathway, and thus HDR-mediated GT is a relative rare event in plants. Here, we review recent research findings mainly focusing on development and applications of precise GT in plants using three SSNs systems described above, and the potential mechanisms underlying HDR events in

  9. Precise genome modification via sequence-specific nucleases-mediated gene targeting for crop improvement

    Directory of Open Access Journals (Sweden)

    Yongwei Sun

    2016-12-01

    Full Text Available Genome editing technologies enable precise modifications of DNA sequences in vivo and offer a great promise for harnessing plant genes in crop improvement. The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks (DSBs by sequence-specific nucleases (SSNs to initiate DNA repair reactions that are based on either non-homologous end joining (NHEJ or homology-directed repair (HDR. While complete knock-outs and loss-of-function mutations generated by NHEJ are very valuable in defining gene functions, their applications in crop improvement are somewhat limited because many agriculturally important traits are conferred by random point mutations or indels at specific loci in either the genes’ encoding or promoter regions. Therefore, genome modification through SSNs-mediated HDR for gene targeting (GT that enables either gene replacement or knock-in will provide an unprecedented ability to facilitate plant breeding by allowing introduction of precise point mutations and new gene functions, or integration of foreign genes at specific and desired ‘safe’ harbor in a predefined manner. The emergence of three programmable SSNs such as zinc finger nucleases (ZFNs, transcriptional activator-like effector nucleases (TALENs, and the clustered regularly interspaced short palindromic repeat (CRISPR/CRISPR-associated protein 9 (Cas9 systems has revolutionized genome modification in plants in a more controlled manner. However, while targeted mutagenesis is becoming routine in plants, the potential of GT technology has not been well realized for traits improvement in crops, mainly due to the fact that NHEJ predominates DNA repair process in somatic cells and competes with the HDR pathway, and thus HDR-mediated GT is a relative rare event in plants. Here, we review recent research findings mainly focusing on development and applications of precise GT in plants using three SSNs systems described above, and the potential

  10. Oligonucleotides containing a piperazino-modified 2'-amino-LNA monomer exhibit very high duplex stability and remarkable nuclease resistance

    DEFF Research Database (Denmark)

    Lou, Chenguang; Vester, Birte; Wengel, Jesper

    2015-01-01

    Incorporation of a piperazino-modified 2'-amino-LNA monomer (PipLNA-T) into oligonucleotides conferred very high affinity and base-pairing selectivity towards complementary DNA and RNA strands. Furthermore, one PipLNA-T modification provided a robust nuclease resistance that safeguarded three...

  11. Rapid and Sensitive Detection of Breast Cancer Cells in Patient Blood with Nuclease-Activated Probe Technology

    Directory of Open Access Journals (Sweden)

    Sven Kruspe

    2017-09-01

    Full Text Available A challenge for circulating tumor cell (CTC-based diagnostics is the development of simple and inexpensive methods that reliably detect the diverse cells that make up CTCs. CTC-derived nucleases are one category of proteins that could be exploited to meet this challenge. Advantages of nucleases as CTC biomarkers include: (1 their elevated expression in many cancer cells, including cells implicated in metastasis that have undergone epithelial-to-mesenchymal transition; and (2 their enzymatic activity, which can be exploited for signal amplification in detection methods. Here, we describe a diagnostic assay based on quenched fluorescent nucleic acid probes that detect breast cancer CTCs via their nuclease activity. This assay exhibited robust performance in distinguishing breast cancer patients from healthy controls, and it is rapid, inexpensive, and easy to implement in most clinical labs. Given its broad applicability, this technology has the potential to have a substantive impact on the diagnosis and treatment of many cancers. Keywords: cancer, circulating tumor cells, diagnostic nucleic acids, nucleases, diagnostic markers, breast cancer, liquid biopsy

  12. Knockout of Myostatin by Zinc-finger Nuclease in Sheep Fibroblasts and Embryos

    Directory of Open Access Journals (Sweden)

    Xuemei Zhang

    2016-10-01

    Full Text Available Myostatin (MSTN can negatively regulate the growth and development of skeletal muscle, and natural mutations can cause “double-muscling” trait in animals. In order to block the inhibiting effect of MSTN on muscle growth, we transferred zinc-finger nucleases (ZFN which targeted sheep MSTN gene into cultured fibroblasts. Gene targeted colonies were isolated from transfected fibroblasts by serial dilution culture and screened by sequencing. Two colonies were identified with mono-allele mutation and one colony with bi-allelic deletion. Further, we introduced the MSTN-ZFN mRNA into sheep embryos by microinjection. Thirteen of thirty-seven parthenogenetic embryos were targeted by ZFN, with the efficiency of 35%. Our work established the technical foundation for generation of MSTN gene editing sheep by somatic cloning and microinjection ZFN into embryos.

  13. Knockout of Myostatin by Zinc-finger Nuclease in Sheep Fibroblasts and Embryos

    Science.gov (United States)

    Zhang, Xuemei; Wang, Liqin; Wu, Yangsheng; Li, Wenrong; An, Jing; Zhang, Fuchun; Liu, Mingjun

    2016-01-01

    Myostatin (MSTN) can negatively regulate the growth and development of skeletal muscle, and natural mutations can cause “double-muscling” trait in animals. In order to block the inhibiting effect of MSTN on muscle growth, we transferred zinc-finger nucleases (ZFN) which targeted sheep MSTN gene into cultured fibroblasts. Gene targeted colonies were isolated from transfected fibroblasts by serial dilution culture and screened by sequencing. Two colonies were identified with mono-allele mutation and one colony with bi-allelic deletion. Further, we introduced the MSTN-ZFN mRNA into sheep embryos by microinjection. Thirteen of thirty-seven parthenogenetic embryos were targeted by ZFN, with the efficiency of 35%. Our work established the technical foundation for generation of MSTN gene editing sheep by somatic cloning and microinjection ZFN into embryos. PMID:27189642

  14. Mm19, a Mycoplasma meleagridis Major Surface Nuclease that Is Related to the RE_AlwI Superfamily of Endonucleases.

    Directory of Open Access Journals (Sweden)

    Elhem Yacoub

    Full Text Available Mycoplasma meleagridis infection is widespread in turkeys, causing poor growth and feathering, airsacculitis, osteodystrophy, and reduction in hatchability. Like most mycoplasma species, M. meleagridis is characterized by its inability to synthesize purine and pyrimidine nucleotides de novo. Consistent with this intrinsic deficiency, we here report the cloning, expression, and characterization of a M. meleagridis gene sequence encoding a major surface nuclease, referred to as Mm19. Mm19 consists of a 1941-bp ORF encoding a 646-amino-acid polypeptide with a predicted molecular mass of 74,825 kDa. BLASTP analysis revealed a significant match with the catalytic/dimerization domain of type II restriction enzymes of the RE_AlwI superfamily. This finding is consistent with the genomic location of Mm19 sequence, which dispalys characteristics of a typical type II restriction-modification locus. Like intact M. meleagridis cells, the E. coli-expressed Mm19 fusion product was found to exhibit a nuclease activity against plasmid DNA, double-stranded DNA, single-stranded DNA, and RNA. The Mm19-associated nuclease activity was consistently enhanced with Mg2+ divalent cations, a hallmark of type II restriction enzymes. A rabbit hyperimmune antiserum raised against the bacterially expressed Mm19 strongly reacted with M. meleagridis intact cells and fully neutralized the surface-bound nuclease activity. Collectively, the results show that M. meleagridis expresses a strong surface-bound nuclease activity, which is the product of a single gene sequence that is related to the RE_AlwI superfamily of endonucleases.

  15. Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3.

    Science.gov (United States)

    Gong, Bei; Shin, Minsang; Sun, Jiali; Jung, Che-Hun; Bolt, Edward L; van der Oost, John; Kim, Jeong-Sun

    2014-11-18

    Mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas systems use a "Cascade" ribonucleoprotein complex to guide RNA specifically to complementary sequence in invader double-stranded DNA (dsDNA), a process called "interference." After target recognition by Cascade, formation of an R-loop triggers recruitment of a Cas3 nuclease-helicase, completing the interference process by destroying the invader dsDNA. To elucidate the molecular mechanism of CRISPR interference, we analyzed crystal structures of Cas3 from the bacterium Thermobaculum terrenum, with and without a bound ATP analog. The structures reveal a histidine-aspartate (HD)-type nuclease domain fused to superfamily-2 (SF2) helicase domains and a distinct C-terminal domain. Binding of ATP analog at the interface of the SF2 helicase RecA-like domains rearranges a motif V with implications for the enzyme mechanism. The HD-nucleolytic site contains two metal ions that are positioned at the end of a proposed nucleic acid-binding tunnel running through the SF2 helicase structure. This structural alignment suggests a mechanism for 3' to 5' nucleolytic processing of the displaced strand of invader DNA that is coordinated with ATP-dependent 3' to 5' translocation of Cas3 along DNA. In agreement with biochemical studies, the presented Cas3 structures reveal important mechanistic details on the neutralization of genetic invaders by type I CRISPR-Cas systems.

  16. Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification

    KAUST Repository

    Li, Lixin

    2012-01-22

    Transcription activator-like effectors (TALEs) can be used as DNA-targeting modules by engineering their repeat domains to dictate user-selected sequence specificity. TALEs have been shown to function as site-specific transcriptional activators in a variety of cell types and organisms. TALE nucleases (TALENs), generated by fusing the FokI cleavage domain to TALE, have been used to create genomic double-strand breaks. The identity of the TALE repeat variable di-residues, their number, and their order dictate the DNA sequence specificity. Because TALE repeats are nearly identical, their assembly by cloning or even by synthesis is challenging and time consuming. Here, we report the development and use of a rapid and straightforward approach for the construction of designer TALE (dTALE) activators and nucleases with user-selected DNA target specificity. Using our plasmid set of 100 repeat modules, researchers can assemble repeat domains for any 14-nucleotide target sequence in one sequential restriction-ligation cloning step and in only 24 h. We generated several custom dTALEs and dTALENs with new target sequence specificities and validated their function by transient expression in tobacco leaves and in vitro DNA cleavage assays, respectively. Moreover, we developed a web tool, called idTALE, to facilitate the design of dTALENs and the identification of their genomic targets and potential off-targets in the genomes of several model species. Our dTALE repeat assembly approach along with the web tool idTALE will expedite genome-engineering applications in a variety of cell types and organisms including plants. © 2012 Springer Science+Business Media B.V.

  17. Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease.

    Science.gov (United States)

    Doetschman, Thomas; Georgieva, Teodora

    2017-03-03

    Genetic engineering of model organisms and cultured cells has for decades provided important insights into the mechanisms underlying cardiovascular development and disease. In the past few years the development of several nuclease systems has broadened the range of model/cell systems that can be engineered. Of these, the CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has become the favorite for its ease of application. Here we will review this RNA-guided nuclease system for gene editing with respect to its usefulness for cardiovascular studies and with an eye toward potential therapy. Studies on its off-target activity, along with approaches to minimize this activity will be given. The advantages of gene editing versus gene targeting in embryonic stem cells, including the breadth of species and cell types to which it is applicable, will be discussed. We will also cover its use in iPSC for research and possible therapeutic purposes; and we will review its use in muscular dystrophy studies where considerable progress has been made toward dystrophin correction in mice. The CRISPR/Ca9s system is also being used for high-throughput screening of genes, gene regulatory regions, and long noncoding RNAs. In addition, the CRISPR system is being used for nongene-editing purposes such as activation and inhibition of gene expression, as well as for fluorescence tagging of chromosomal regions and individual mRNAs to track their cellular location. Finally, an approach to circumvent the inability of post-mitotic cells to support homologous recombination-based gene editing will be presented. In conclusion, applications of the CRISPR/Cas system are expanding at a breath-taking pace and are revolutionizing approaches to gain a better understanding of human diseases. © 2017 American Heart Association, Inc.

  18. Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

    International Nuclear Information System (INIS)

    Watanabe, Masahito; Umeyama, Kazuhiro; Matsunari, Hitomi; Takayanagi, Shuko; Haruyama, Erika; Nakano, Kazuaki; Fujiwara, Tsukasa; Ikezawa, Yuka; Nakauchi, Hiromitsu

    2010-01-01

    Research highlights: → EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. → ZFNs induced targeted mutations in porcine primary cultured cells. → Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

  19. De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks

    KAUST Repository

    Mahfouz, Magdy M.

    2011-01-24

    Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.

  20. TT2014 meeting report on the 12th Transgenic Technology meeting in Edinburgh: new era of transgenic technologies with programmable nucleases in the foreground

    Czech Academy of Sciences Publication Activity Database

    Beck, Inken; Sedláček, Radislav

    2015-01-01

    Roč. 24, č. 1 (2015), s. 179-183 ISSN 0962-8819 Institutional support: RVO:68378050 Keywords : Transgenic * Nuclease * Gene Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.054, year: 2015

  1. Genome Editing with Engineered Nucleases in Economically Important Animals and Plants: State of the Art in the Research Pipeline.

    Science.gov (United States)

    Sovová, Tereza; Kerins, Gerard; Demnerová, Kateřina; Ovesná, Jaroslava

    2017-01-01

    After induced mutagenesis and transgenesis, genome editing is the next step in the development of breeding techniques. Genome editing using site-directed nucleases - including meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system - is based on the mechanism of double strand breaks. The nuclease is directed to cleave the DNA at a specific place of the genome which is then repaired by natural repair mechanisms. Changes are introduced during the repair that are either accidental or can be targeted if a DNA template with the desirable sequence is provided. These techniques allow making virtually any change to the genome including specific DNA sequence changes, gene insertion, replacements or deletions with unprecedented precision and specificity while being less laborious and more straightforward compared to traditional breeding techniques or transgenesis. Therefore, the research in this field is developing quickly and, apart from model species, multiple studies have focused on economically important species and agronomically important traits that were the key subjects of this review. In plants, studies have been undertaken on disease resistance, herbicide tolerance, nutrient metabolism and nutritional value. In animals, the studies have mainly focused on disease resistance, meat production and allergenicity of milk. However, none of the promising studies has led to commercialization despite several patent applications. The uncertain legal status of genome-editing methods is one of the reasons for poor commercial development, as it is not clear whether the products would fall under the GMO regulation. We believe this issue should be clarified soon in order to allow promising methods to reach their full potential.

  2. Response surface optimization of carbon and nitrogen sources for nuclease P1 production by Penicillium citrinum F-5-5

    International Nuclear Information System (INIS)

    Liang Xinle; Huang Yingying; Zhang Hong; Chen Min; Liu Xuan

    2011-01-01

    Penicillium citrinum F-5-5, a nuclease P1 high-producing strain with 978.6 U/ml in potato glucose medium, was derived from the original Penicillium citrinum CICC 4011 with 60 Co γ-rays irradiation mutation and then protoplasts fusion treatment. Culture components were optimized for the nuclease P1 production, and response surface methodology was applied for the critical medium components(carbon and nitrogen sources) which were preselected by Plackett-Burman design approach. Glucose, soluble starch and corn steep powder showed significant effects on production of nuclease. Central composite design was used for the optimization levels by software Minitab 15, and it showed that, the optimal values for the concentration of glucose, soluble starch and corn steep powder were 30.89, 42.46 and 11.60 g/L, respectively. With this medium,an enzyme activity of 1687.16 U/ml could be obtained theoretically. Using this optimized medium, an experimental enzyme activity of 1672.6 U/ml was reached. (authors)

  3. Gene- and protein-delivered zinc finger-staphylococcal nuclease hybrid for inhibition of DNA replication of human papillomavirus.

    Science.gov (United States)

    Mino, Takashi; Mori, Tomoaki; Aoyama, Yasuhiro; Sera, Takashi

    2013-01-01

    Previously, we reported that artificial zinc-finger proteins (AZPs) inhibited virus DNA replication in planta and in mammalian cells by blocking binding of a viral replication protein to its replication origin. However, the replication mechanisms of viruses of interest need to be disentangled for the application. To develop more widely applicable methods for antiviral therapy, we explored the feasibility of inhibition of HPV-18 replication as a model system by cleaving its viral genome. To this end, we fused the staphylococcal nuclease cleaving DNA as a monomer to an AZP that binds to the viral genome. The resulting hybrid nuclease (designated AZP-SNase) cleaved its target DNA plasmid efficiently and sequence-specifically in vitro. Then, we confirmed that transfection with a plasmid expressing AZP-SNase inhibited HPV-18 DNA replication in transient replication assays using mammalian cells. Linker-mediated PCR analysis revealed that the AZP-SNase cleaved an HPV-18 ori plasmid around its binding site. Finally, we demonstrated that the protein-delivered AZP-SNase inhibited HPV-18 DNA replication as well and did not show any significant cytotoxicity. Thus, both gene- and protein-delivered hybrid nucleases efficiently inhibited HPV-18 DNA replication, leading to development of a more universal antiviral therapy for human DNA viruses.

  4. A Nuclease from Streptococcus mutans Facilitates Biofilm Dispersal and Escape from Killing by Neutrophil Extracellular Traps.

    Science.gov (United States)

    Liu, Jia; Sun, Luping; Liu, Wei; Guo, Lihong; Liu, Zhaohui; Wei, Xi; Ling, Junqi

    2017-01-01

    Streptococcus mutans is the primary etiologic agent of dental caries and occasionally infective endocarditis, with the ability to form biofilms and disperse cells into distal sites to exacerbate and spread infection. In this study, we identified a nuclease (DeoC) as a S. mutans biofilm dispersal modulating factor through microarray analysis. In vitro assays revealed a dispersal defect of a deoC deletion mutant, and functional studies with purified protein were indicative of the biofilm dispersal activity of DeoC. Neutrophils are a key host response factor restraining bacterial spreading through the formation of neutrophil extracellular traps (NETs), which consist of a nuclear DNA backbone associated with antimicrobial peptides. Therefore, we hypothesized that the dispersed S. mutans might utilize DeoC to degrade NETs and escape killing by the immune system. It was found that S. mutans induced NET formation upon contact with neutrophils, while the presence of NETs in turn enhanced the deoC expression of S. mutans . Fluorescence microscopy inspection showed that deoC deletion resulted in a decreased NET degradation ability of S. mutans and enhanced susceptibility to neutrophil killing. Data obtained from this study assigned two important roles for DeoC in S. mutans : contributing to the spread of infection through mediating biofilm dispersal, and facilitating the escape of S. mutans from neutrophil killing through NET degradation.

  5. Tudor staphylococcal nuclease is an evolutionarily conserved component of the programmed cell death degradome.

    Science.gov (United States)

    Sundström, Jens F; Vaculova, Alena; Smertenko, Andrei P; Savenkov, Eugene I; Golovko, Anna; Minina, Elena; Tiwari, Budhi S; Rodriguez-Nieto, Salvador; Zamyatnin, Andrey A; Välineva, Tuuli; Saarikettu, Juha; Frilander, Mikko J; Suarez, Maria F; Zavialov, Anton; Ståhl, Ulf; Hussey, Patrick J; Silvennoinen, Olli; Sundberg, Eva; Zhivotovsky, Boris; Bozhkov, Peter V

    2009-11-01

    Programmed cell death (PCD) is executed by proteases, which cleave diverse proteins thus modulating their biochemical and cellular functions. Proteases of the caspase family and hundreds of caspase substrates constitute a major part of the PCD degradome in animals. Plants lack close homologues of caspases, but instead possess an ancestral family of cysteine proteases, metacaspases. Although metacaspases are essential for PCD, their natural substrates remain unknown. Here we show that metacaspase mcII-Pa cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), during both developmental and stress-induced PCD. TSN knockdown leads to activation of ectopic cell death during reproduction, impairing plant fertility. Surprisingly, human TSN (also known as p100 or SND1), a multifunctional regulator of gene expression, is cleaved by caspase-3 during apoptosis. This cleavage impairs the ability of TSN to activate mRNA splicing, inhibits its ribonuclease activity and is important for the execution of apoptosis. Our results establish TSN as the first biological substrate of metacaspase and demonstrate that despite the divergence of plants and animals from a common ancestor about one billion years ago and their use of distinct PCD pathways, both have retained a common mechanism to compromise cell viability through the cleavage of the same substrate, TSN.

  6. DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair.

    Science.gov (United States)

    de Laat, W L; Appeldoorn, E; Sugasawa, K; Weterings, E; Jaspers, N G; Hoeijmakers, J H

    1998-08-15

    The human single-stranded DNA-binding replication A protein (RPA) is involved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3'- or 5'-protruding single-stranded arms, we found that hRPA binds ssDNA with a defined polarity; a strong ssDNA interaction domain of hRPA is positioned at the 5' side of its binding region, a weak ssDNA-binding domain resides at the 3' side. Polarity appears crucial for positioning of the excision repair nucleases XPG and ERCC1-XPF on the DNA. With the 3'-oriented side of hRPA facing a duplex ssDNA junction, hRPA interacts with and stimulates ERCC1-XPF, whereas the 5'-oriented side of hRPA at a DNA junction allows stable binding of XPG to hRPA. Our data pinpoint hRPA to the undamaged strand during nucleotide excision repair. Polarity of hRPA on ssDNA is likely to contribute to the directionality of other hRPA-dependent processes as well.

  7. Advances in genetic modification of farm animals using zinc-finger nucleases (ZFN).

    Science.gov (United States)

    Petersen, Bjoern; Niemann, Heiner

    2015-02-01

    Genome editing tools (GET), including zinc-finger nucleases (ZFN), transcription activator-like endonucleases (TALENS), and meganucleases possess long recognition sites and are thus capable of cutting DNA in a very specific manner. These genome editing tools mediate targeted genetic alterations by enhancing DNA mutation frequency via induction of double-strand breaks at a predetermined genomic site. Compared to conventional homologous recombination based gene targeting, GETs can increase gene targeting and gene disruption via mutagenic DNA repair more than 10,000-fold. Recently, a novel class of genome editing tools was described that uses RNAs to target a specific genomic site. The CRISPR/Cas9 system is capable of targeting even multiple genomic sites in one shot and thus could be superior to ZFNs or TALEN. Current results indicate that these tools can be successfully employed in a broad range of organisms which renders them useful for improving the understanding of complex physiological systems, producing transgenic animals, including creating large animal models for human diseases, creating specific cell lines, and plants, and even for treating human genetic diseases. This review provides an update on the use of ZFNs to modify the genome of farm animals, summarizes current knowledge on the underlying mechanism, and discusses new opportunities for generating genetically modified farm animals.

  8. Targeted Editing of Myostatin Gene in Sheep by Transcription Activator-like Effector Nucleases

    Directory of Open Access Journals (Sweden)

    Xinxia Zhao

    2016-03-01

    Full Text Available Myostatin (MSTN is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs in tandem with single-stranded DNA oligonucleotides (ssODNs. We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals.

  9. Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation

    Science.gov (United States)

    Vallerga, María Belén; Mansilla, Sabrina F.; Federico, María Belén; Bertolin, Agustina P.; Gottifredi, Vanesa

    2015-01-01

    After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates. PMID:26627254

  10. The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once.

    Science.gov (United States)

    Tsutakawa, Susan E; Lafrance-Vanasse, Julien; Tainer, John A

    2014-07-01

    To avoid genome instability, DNA repair nucleases must precisely target the correct damaged substrate before they are licensed to incise. Damage identification is a challenge for all DNA damage response proteins, but especially for nucleases that cut the DNA and necessarily create a cleaved DNA repair intermediate, likely more toxic than the initial damage. How do these enzymes achieve exquisite specificity without specific sequence recognition or, in some cases, without a non-canonical DNA nucleotide? Combined structural, biochemical, and biological analyses of repair nucleases are revealing their molecular tools for damage verification and safeguarding against inadvertent incision. Surprisingly, these enzymes also often act on RNA, which deserves more attention. Here, we review protein-DNA structures for nucleases involved in replication, base excision repair, mismatch repair, double strand break repair (DSBR), and telomere maintenance: apurinic/apyrimidinic endonuclease 1 (APE1), Endonuclease IV (Nfo), tyrosyl DNA phosphodiesterase (TDP2), UV Damage endonuclease (UVDE), very short patch repair endonuclease (Vsr), Endonuclease V (Nfi), Flap endonuclease 1 (FEN1), exonuclease 1 (Exo1), RNase T and Meiotic recombination 11 (Mre11). DNA and RNA structure-sensing nucleases are essential to life with roles in DNA replication, repair, and transcription. Increasingly these enzymes are employed as advanced tools for synthetic biology and as targets for cancer prognosis and interventions. Currently their structural biology is most fully illuminated for DNA repair, which is also essential to life. How DNA repair enzymes maintain genome fidelity is one of the DNA double helix secrets missed by James Watson and Francis Crick, that is only now being illuminated though structural biology and mutational analyses. Structures reveal motifs for repair nucleases and mechanisms whereby these enzymes follow the old carpenter adage: measure twice, cut once. Furthermore, to measure

  11. Nuclease activity of Legionella pneumophila Cas2 promotes intracellular infection of amoebal host cells.

    Science.gov (United States)

    Gunderson, Felizza F; Mallama, Celeste A; Fairbairn, Stephanie G; Cianciotto, Nicholas P

    2015-03-01

    Legionella pneumophila, the primary agent of Legionnaires' disease, flourishes in both natural and man-made environments by growing in a wide variety of aquatic amoebae. Recently, we determined that the Cas2 protein of L. pneumophila promotes intracellular infection of Acanthamoeba castellanii and Hartmannella vermiformis, the two amoebae most commonly linked to cases of disease. The Cas2 family of proteins is best known for its role in the bacterial and archeal clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) system that constitutes a form of adaptive immunity against phage and plasmid. However, the infection event mediated by L. pneumophila Cas2 appeared to be distinct from this function, because cas2 mutants exhibited infectivity defects in the absence of added phage or plasmid and since mutants lacking the CRISPR array or any one of the other cas genes were not impaired in infection ability. We now report that the Cas2 protein of L. pneumophila has both RNase and DNase activities, with the RNase activity being more pronounced. By characterizing a catalytically deficient version of Cas2, we determined that nuclease activity is critical for promoting infection of amoebae. Also, introduction of Cas2, but not its catalytic mutant form, into a strain of L. pneumophila that naturally lacks a CRISPR-Cas locus caused that strain to be 40- to 80-fold more infective for amoebae, unequivocally demonstrating that Cas2 facilitates the infection process independently of any other component encoded within the CRISPR-Cas locus. Finally, a cas2 mutant was impaired for infection of Willaertia magna but not Naegleria lovaniensis, suggesting that Cas2 promotes infection of most but not all amoebal hosts. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Efficacy of a marine bacterial nuclease against biofilm forming microorganisms isolated from chronic rhinosinusitis.

    Directory of Open Access Journals (Sweden)

    Robert C Shields

    Full Text Available BACKGROUND: The persistent colonization of paranasal sinus mucosa by microbial biofilms is a major factor in the pathogenesis of chronic rhinosinusitis (CRS. Control of microorganisms within biofilms is hampered by the presence of viscous extracellular polymers of host or microbial origin, including nucleic acids. The aim of this study was to investigate the role of extracellular DNA in biofilm formation by bacteria associated with CRS. METHODS/PRINCIPAL FINDINGS: Obstructive mucin was collected from patients during functional endoscopic sinus surgery. Examination of the mucous by transmission electron microscopy revealed an acellular matrix punctuated occasionally with host cells in varying states of degradation. Bacteria were observed in biofilms on mucosal biopsies, and between two and six different species were isolated from each of 20 different patient samples. In total, 16 different bacterial genera were isolated, of which the most commonly identified organisms were coagulase-negative staphylococci, Staphylococcus aureus and α-haemolytic streptococci. Twenty-four fresh clinical isolates were selected for investigation of biofilm formation in vitro using a microplate model system. Biofilms formed by 14 strains, including all 9 extracellular nuclease-producing bacteria, were significantly disrupted by treatment with a novel bacterial deoxyribonuclease, NucB, isolated from a marine strain of Bacillus licheniformis. Extracellular biofilm matrix was observed in untreated samples but not in those treated with NucB and extracellular DNA was purified from in vitro biofilms. CONCLUSION/SIGNIFICANCE: Our data demonstrate that bacteria associated with CRS form robust biofilms which can be reduced by treatment with matrix-degrading enzymes such as NucB. The dispersal of bacterial biofilms with NucB may offer an additional therapeutic target for CRS sufferers.

  13. Delivery of CdiA nuclease toxins into target cells during contact-dependent growth inhibition.

    Science.gov (United States)

    Webb, Julia S; Nikolakakis, Kiel C; Willett, Julia L E; Aoki, Stephanie K; Hayes, Christopher S; Low, David A

    2013-01-01

    Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CDI systems deploy a variety of distinct toxins, which are contained within the polymorphic C-terminal region (CdiA-CT) of CdiA proteins. Several CdiA-CTs are nucleases, suggesting that the toxins are transported into the target cell cytoplasm to interact with their substrates. To analyze CdiA transfer to target bacteria, we used the CDI system of uropathogenic Escherichia coli 536 (UPEC536) as a model. Antibodies recognizing the amino- and carboxyl-termini of CdiA(UPEC536) were used to visualize transfer of CdiA from CDI(UPEC536+) inhibitor cells to target cells using fluorescence microscopy. The results indicate that the entire CdiA(UPEC536) protein is deposited onto the surface of target bacteria. CdiA(UPEC536) transfer to bamA101 mutants is reduced, consistent with low expression of the CDI receptor BamA on these cells. Notably, our results indicate that the C-terminal CdiA-CT toxin region of CdiA(UPEC536) is translocated into target cells, but the N-terminal region remains at the cell surface based on protease sensitivity. These results suggest that the CdiA-CT toxin domain is cleaved from CdiA(UPEC536) prior to translocation. Delivery of a heterologous Dickeya dadantii CdiA-CT toxin, which has DNase activity, was also visualized. Following incubation with CDI(+) inhibitor cells targets became anucleate, showing that the D.dadantii CdiA-CT was delivered intracellularly. Together, these results demonstrate that diverse CDI toxins are efficiently translocated across target cell envelopes.

  14. Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.

    Directory of Open Access Journals (Sweden)

    Tatiana Flisikowska

    Full Text Available Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+ and IgG(+ B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.

  15. Zinc finger nuclease and homing endonuclease-mediated assembly of multigene plant transformation vectors.

    Science.gov (United States)

    Zeevi, Vardit; Liang, Zhuobin; Arieli, Uri; Tzfira, Tzvi

    2012-01-01

    Binary vectors are an indispensable component of modern Agrobacterium tumefaciens-mediated plant genetic transformation systems. A remarkable variety of binary plasmids have been developed to support the cloning and transfer of foreign genes into plant cells. The majority of these systems, however, are limited to the cloning and transfer of just a single gene of interest. Thus, plant biologists and biotechnologists face a major obstacle when planning the introduction of multigene traits into transgenic plants. Here, we describe the assembly of multitransgene binary vectors by using a combination of engineered zinc finger nucleases (ZFNs) and homing endonucleases. Our system is composed of a modified binary vector that has been engineered to carry an array of unique recognition sites for ZFNs and homing endonucleases and a family of modular satellite vectors. By combining the use of designed ZFNs and commercial restriction enzymes, multiple plant expression cassettes were sequentially cloned into the acceptor binary vector. Using this system, we produced binary vectors that carried up to nine genes. Arabidopsis (Arabidopsis thaliana) protoplasts and plants were transiently and stably transformed, respectively, by several multigene constructs, and the expression of the transformed genes was monitored across several generations. Because ZFNs can potentially be engineered to digest a wide variety of target sequences, our system allows overcoming the problem of the very limited number of commercial homing endonucleases. Thus, users of our system can enjoy a rich resource of plasmids that can be easily adapted to their various needs, and since our cloning system is based on ZFN and homing endonucleases, it may be possible to reconstruct other types of binary vectors and adapt our vectors for cloning on multigene vector systems in various binary plasmids.

  16. MCCE analysis of the pKas of introduced buried acids and bases in staphylococcal nuclease.

    Science.gov (United States)

    Gunner, M R; Zhu, Xuyu; Klein, Max C

    2011-12-01

    The pK(a)s of 96 acids and bases introduced into buried sites in the staphylococcal nuclease protein (SNase) were calculated using the multiconformation continuum electrostatics (MCCE) program and the results compared with experimental values. The pK(a)s are obtained by Monte Carlo sampling of coupled side chain protonation and position as a function of pH. The dependence of the results on the protein dielectric constant (ε(prot)) in the continuum electrostatics analysis and on the Lennard-Jones non-electrostatics parameters was evaluated. The pK(a)s of the introduced residues have a clear dependence on ε(prot,) whereas native ionizable residues do not. The native residues have electrostatic interactions with other residues in the protein favoring ionization, which are larger than the desolvation penalty favoring the neutral state. Increasing ε(prot) scales both terms, which for these residues leads to small changes in pK(a). The introduced residues have a larger desolvation penalty and negligible interactions with residues in the protein. For these residues, changing ε(prot) has a large influence on the calculated pK(a). An ε(prot) of 8-10 and a Lennard-Jones scaling of 0.25 is best here. The X-ray crystal structures of the mutated proteins are found to provide somewhat better results than calculations carried out on mutations made in silico. Initial relaxation of the in silico mutations by Gromacs and extensive side chain rotamer sampling within MCCE can significantly improve the match with experiment. Copyright © 2011 Wiley-Liss, Inc.

  17. Zinc Finger Nuclease: A New Approach to Overcome Beta-Lactam Antibiotic Resistance.

    Science.gov (United States)

    Shahbazi Dastjerdeh, Mansoureh; Kouhpayeh, Shirin; Sabzehei, Faezeh; Khanahmad, Hossein; Salehi, Mansour; Mohammadi, Zahra; Shariati, Laleh; Hejazi, Zahra; Rabiei, Parisa; Manian, Mostafa

    2016-01-01

    The evolution of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) has been accelerated recently by the indiscriminate application of antibiotics. Antibiotic resistance has challenged the success of medical interventions and therefore is considered a hazardous threat to human health. The present study aimed to describe the use of zinc finger nuclease (ZFN) technology to target and disrupt a plasmid-encoded β-lactamase, which prevents horizontal gene transfer-mediated evolution of ARBs. An engineered ZFN was designed to target a specific sequence in the ampicillin resistance gene (amp(R)) of the pTZ57R plasmid. The Escherichia coli bacteria already contained the pZFN kanamycin-resistant (kana(R)) plasmid as the case or the pP15A, kana(R) empty vector as the control, were transformed with the pTZ57R; the ability of the designed ZFN to disrupt the β-lactamase gene was evaluated with the subsequent disturbed ability of the bacteria to grow on ampicillin (amp) and ampicillin-kanamycin (amp-kana)-containing media. The effect of mild hypothermia on the ZFN gene targeting efficiency was also evaluated. The growth of bacteria in the case group on the amp and amp-kana-containing media was significantly lower compared with the control group at 37°C (P ampicillin resistance by the targeted disruption of the ampicillin resistance gene, which leads to inactivation of β-lactam synthesis. Therefore, ZFN technology could be engaged to decrease the antibiotic resistance issue with the construction of a ZFN archive against different ARGs. To tackle the resistance issue at the environmental level, recombinant phages expressing ZFNs against different ARGs could be constructed and released into both hospital and urban wastewater systems.

  18. Calculating the shift in pKa of the position 66 for an Staphylococcal nuclease mutant with the Replica Exchange Free Energy Perturbation method

    Science.gov (United States)

    Sabri Dashti, Danial; Roitberg, Adrian

    2011-03-01

    The Experimental pKa value of Glutamate66 in a hyperstable mutant of Staph Nuclease, which has been measured by Moreno et al., shows a large shift of around 5 pKa units with respect to a glutamate in solution. In order to reproduce the large experimental shift by single structure continuum solvent computational methods, it is required that the dielectric constant of the interior of the protein be set to around ten in the simulations. The physical reason behind this is not understood as of yet and hypotheses have been produced by the Moreno group regarding solvent penetration, protein reorganization etc. We tried to resolve this inconsistency between experimental and continuum methods by introducing a four-state thermodynamic cycle that has couples conformational states with protonation state of the side chain of E66. We propose that what the experimental methods, (which are mostly sensitive to configurational changes) are measuring is actually the equilibrium constant between the two configurational states rather than between the two protonation states. In this regard we applied our recently developed Replica Exchange method Free Energy Perturbation (REFEP) in implicit solvent to calculate the pKa value of E66 for each of the configurational states as well as the mixed configuration, and our results are in almost perfect agreement with the experiments of Moreno.

  19. Correction of dystrophin expression in cells from Duchenne muscular dystrophy patients through genomic excision of exon 51 by zinc finger nucleases.

    Science.gov (United States)

    Ousterout, David G; Kabadi, Ami M; Thakore, Pratiksha I; Perez-Pinera, Pablo; Brown, Matthew T; Majoros, William H; Reddy, Timothy E; Gersbach, Charles A

    2015-03-01

    Duchenne muscular dystrophy (DMD) is caused by genetic mutations that result in the absence of dystrophin protein expression. Oligonucleotide-induced exon skipping can restore the dystrophin reading frame and protein production. However, this requires continuous drug administration and may not generate complete skipping of the targeted exon. In this study, we apply genome editing with zinc finger nucleases (ZFNs) to permanently remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript. This approach can restore the dystrophin reading frame in ~13% of DMD patient mutations. Transfection of two ZFNs targeted to sites flanking the exon 51 splice acceptor into DMD patient myoblasts led to deletion of this genomic sequence. A clonal population was isolated with this deletion and following differentiation we confirmed loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin protein expression. Furthermore, transplantation of corrected cells into immunodeficient mice resulted in human dystrophin expression localized to the sarcolemmal membrane. Finally, we quantified ZFN toxicity in human cells and mutagenesis at predicted off-target sites. This study demonstrates a powerful method to restore the dystrophin reading frame and protein expression by permanently deleting exons.

  20. Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection.

    Science.gov (United States)

    Didigu, Chuka A; Wilen, Craig B; Wang, Jianbin; Duong, Jennifer; Secreto, Anthony J; Danet-Desnoyers, Gwenn A; Riley, James L; Gregory, Phillip D; June, Carl H; Holmes, Michael C; Doms, Robert W

    2014-01-02

    HIV-1 entry into CD4(+) T cells requires binding of the virus to CD4 followed by engagement of either the C-C chemokine receptor 5 (CCR5) or C-X-C chemokine receptor 4 (CXCR4) coreceptor. Pharmacologic blockade or genetic inactivation of either coreceptor protects cells from infection by viruses that exclusively use the targeted coreceptor. We have used zinc-finger nucleases to drive the simultaneous genetic modification of both ccr5 and cxcr4 in primary human CD4(+) T cells. These gene-modified cells proliferated normally and were resistant to both CCR5- and CXCR4-using HIV-1 in vitro. When introduced into a humanized mouse model of HIV-1 infection, these coreceptor negative cells engraft and traffic normally, and are protected from infection with CCR5- and CXCR4-using HIV-1 strains. These data suggest that simultaneous disruption of the HIV coreceptors may provide a useful approach for the long-term, drug-free treatment of established HIV-1 infections.

  1. 2'-O-methyl-modified anti-MDR1 fork-siRNA duplexes exhibiting high nuclease resistance and prolonged silencing activity.

    Science.gov (United States)

    Petrova Kruglova, Natalya S; Meschaninova, Mariya I; Venyaminova, Alya G; Zenkova, Marina A; Vlassov, Valentin V; Chernolovskaya, Elena L

    2010-12-01

    The thermodynamic asymmetry of siRNA duplexes determines their silencing activity. Favorable asymmetry can be achieved by incorporation of mismatches into the 3' part of the sense strand, providing fork-siRNAs, which exhibit higher silencing activity and higher sensitivity to nucleases. Recently, we found that selective 2'-O-methyl modifications of the nuclease-sensitive sites of siRNA significantly improve its nuclease resistance without substantial loss of silencing activity. Here, we examined the impact of nucleotide mismatches and the number and location of 2'-O-methyl modifications on the silencing activity and nuclease resistance of anti-MDR1 siRNAs. We found that both nonmodified and selectively modified fork-siRNAs with 4 mismatches at the 3' end of the sense strand suppress the expression of target gene at lower effective concentrations than the parent siRNAs with classical duplex design. The selective modification of nuclease-sensitive sites significantly improved the stability of fork-siRNAs in the presence of serum. The selectively modified fork-siRNA duplexes provided inhibitory effect over a period of 12 days posttransfection, whereas the gene silencing activity of the nonmodified analogs expired within 6 days. Thus, selective chemical modifications and structural alteration of siRNA duplexes improve their silencing properties and significantly prolong the duration of their silencing effect.

  2. Recognition and repair of the CC-1065-(N3-Adenine)-DNA adduct by the UVRABC nuclease

    International Nuclear Information System (INIS)

    Tang, M.; Lee, C.S.; Doisy, R.; Ross, L.; Needham-VanDevanter, D.R.; Hurley, L.H.

    1988-01-01

    The recognition and repair of the helix-stabilizing and relatively nondistortive CC-1065-(N3-adenine)-DNA adduct by UVRABC nuclease has been investigated both in vivo with phi X174RFI DNA by a transfection assay and in vitro by a site-directed adduct in a 117 base pair fragment from M13mp1. CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis which binds within the minor groove of DNA through N3 of adenine. In contrast to the helix-destabilizing and distortive modifications of DNA caused by ultraviolet light or N-acetoxy-2-(acetylamino)fluorene, CC-1065 increases the melting point of DNA and decreases the S1 nuclease activity. Using a viral DNA-Escherichia coli transfection system, the authors have found that the uvrA, uvrB, and uvrC genes, which code for the major excision repair proteins for UV- and NAAAF-induced DNA damage, are also involved in the repair of CC-1065-DNA adducts. In contrast, the uvrD gene product, which has been found to be involved in the repair of UV damage, has no effect in repairing CC-1065-DNA adducts. Purified UVRA, UVRB, and UVRC proteins must work in concert to incise the drug-modified phi X174RFI DNA. Using a site-directed and multiple CC-1065 modified (MspI-BstNI) 117 base pair fragment from M13mp1, they have found that UVRABC nuclease incises at the eight phosphodiester bond on the 5' side of the CC-1065-DNA adduct on the drug-modified strand. The enzymes do not cut the noncovalently modified strand. The DNA sequence and/or helix-stabilizing effect of multiple adducts may determine the recognition and/or incision of the drug-DNA adduct by UVRABC nuclease. These results are discussed in relation to the structure of the CC-1065-DNA adduct and the effect of drug binding on local DNA structure

  3. Characterization of the residual structure in the unfolded state of the Delta 131 Delta fragment of staphylococcal nuclease

    DEFF Research Database (Denmark)

    Francis, C. J.; Lindorff-Larsen, Kresten; Best, R. B.

    2006-01-01

    The determination of the conformational preferences in unfolded states of proteins constitutes an important challenge in structural biology. We use inter-residue distances estimated from site-directed spin-labeling NMR experimental measurements as ensemble-averaged restraints in all-atom molecular...... dynamics simulations to characterise the residual structure of the 131 fragment of staphylococcal nuclease under physiological conditions. Our findings indicate that 131 under these conditions shows a tendency to form transiently hydrophobic clusters similar to those present in the native state of wild...

  4. ZFNGenome: A comprehensive resource for locating zinc finger nuclease target sites in model organisms

    Directory of Open Access Journals (Sweden)

    Voytas Daniel F

    2011-01-01

    Full Text Available Abstract Background Zinc Finger Nucleases (ZFNs have tremendous potential as tools to facilitate genomic modifications, such as precise gene knockouts or gene replacements by homologous recombination. ZFNs can be used to advance both basic research and clinical applications, including gene therapy. Recently, the ability to engineer ZFNs that target any desired genomic DNA sequence with high fidelity has improved significantly with the introduction of rapid, robust, and publicly available techniques for ZFN design such as the Oligomerized Pool ENgineering (OPEN method. The motivation for this study is to make resources for genome modifications using OPEN-generated ZFNs more accessible to researchers by creating a user-friendly interface that identifies and provides quality scores for all potential ZFN target sites in the complete genomes of several model organisms. Description ZFNGenome is a GBrowse-based tool for identifying and visualizing potential target sites for OPEN-generated ZFNs. ZFNGenome currently includes a total of more than 11.6 million potential ZFN target sites, mapped within the fully sequenced genomes of seven model organisms; S. cerevisiae, C. reinhardtii, A. thaliana, D. melanogaster, D. rerio, C. elegans, and H. sapiens and can be visualized within the flexible GBrowse environment. Additional model organisms will be included in future updates. ZFNGenome provides information about each potential ZFN target site, including its chromosomal location and position relative to transcription initiation site(s. Users can query ZFNGenome using several different criteria (e.g., gene ID, transcript ID, target site sequence. Tracks in ZFNGenome also provide "uniqueness" and ZiFOpT (Zinc Finger OPEN Targeter "confidence" scores that estimate the likelihood that a chosen ZFN target site will function in vivo. ZFNGenome is dynamically linked to ZiFDB, allowing users access to all available information about zinc finger reagents, such as the

  5. The role of the N-terminal loop in the function of the colicin E7 nuclease domain

    DEFF Research Database (Denmark)

    Czene, Anikó; Németh, Eszter; Zóka, István G.

    2013-01-01

    Colicin E7 (ColE7) is a metallonuclease toxin of Escherichia coli belonging to the HNH superfamily of nucleases. It contains highly conserved amino acids in its HHX14NX8HX3H ββα-type metal ion binding C-terminal active centre. However, the proximity of the arginine at the N-terminus of the nuclease...... domain of ColE7 (NColE7, 446–576) is necessary for the hydrolytic activity. This poses a possibility of allosteric activation control in this protein. To obtain more information on this phenomenon, two protein mutants were expressed, i.e. four and 25 N-terminal amino acids were removed from NColE7....... The effect of the N-terminal truncation on the Zn2+ ion and DNA binding as well as on the activity was investigated in this study by mass spectrometry, synchrotron-radiation circular dichroism and fluorescence spectroscopy and agarose gel mobility shift assays. The dynamics of protein backbone movement...

  6. Affinity capillary electrophoresis with laser induced fluorescence detection for thrombin analysis using nuclease-resistant RNA aptamers.

    Science.gov (United States)

    Hao, Lihua; Bai, Yunlong; Wang, Hailin; Zhao, Qiang

    2016-12-09

    Aptamer affinity capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) combines the advantages of affinity aptamer, rapid CE separation, and high sensitivity detection. Here we reported an affinity CE-LIF assay for thrombin by using a fluorophore-labeled RNA aptamer containing 2'-fluoro modification in sugar rings of pyrimidine nucleotides (C and U) as affinity ligand. This RNA aptamer has high binding affinity, specificity and biostability. Thrombin at 0.2nM was successfully detected. This RNA aptamer allowed for the detection of thrombin spiked in diluted human serum sample due to the nuclease resistance. The RNA aptamer has comparable binding affinity to a 29-mer DNA aptamer for thrombin, and the binding site of the RNA aptamer on thrombin partially overlaps with the binding site of the 29-mer DNA aptamer on thrombin. It shows the nuclease-resistant RNA aptamers are promising in assays for thrombin. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs

    DEFF Research Database (Denmark)

    Duda, Katarzyna; Lonowski, Lindsey A; Kofoed-Nielsen, Michael

    2014-01-01

    Targeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing...... were minimal, and when occurring, our data suggest that they may be counteracted by selecting intermediate nuclease levels where off-target mutagenesis is low, but on-target mutagenesis remains relatively high. The method was also applicable to the CRISPR/Cas9 system, including CRISPR/Cas9 mutant...... nickase pairs, which exhibit low off-target mutagenesis compared to wild-type Cas9....

  8. Investigation of a redox-sensitive predictive model of mouse embryonic stem cells differentiation using quantitative nuclease protection assays and glutathione redox status

    Science.gov (United States)

    Investigation of a redox-sensitive predictive model of mouse embryonic stem cell differentiation via quantitative nuclease protection assays and glutathione redox status Chandler KJ,Hansen JM, Knudsen T,and Hunter ES 1. U.S. Environmental Protection Agency, Research Triangl...

  9. Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance

    Science.gov (United States)

    Romano Ibarra, Guillermo S; Paul, Biswajit; Sather, Blythe D; Younan, Patrick M; Sommer, Karen; Kowalski, John P; Hale, Malika; Stoddard, Barry; Jarjour, Jordan; Astrakhan, Alexander; Kiem, Hans-Peter; Rawlings, David J

    2016-01-01

    A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4+ T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4+ T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis. Gene-modified cells engrafted at levels equivalent to unmodified cells when transplanted into immunodeficient mice. Furthermore, genetically modified CD4+ cells were preferentially expanded during HIV-1 infection in vivo in an immunodeficient mouse model. Our results demonstrate the feasibility of targeting CCR5 in primary T cells using an engineered megaTAL nuclease, and the potential to use gene-modified cells to reconstitute a patient's immune system and provide protection from HIV infection. PMID:27741222

  10. Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system.

    Science.gov (United States)

    Sinkunas, Tomas; Gasiunas, Giedrius; Fremaux, Christophe; Barrangou, Rodolphe; Horvath, Philippe; Siksnys, Virginijus

    2011-04-06

    Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA.

  11. A tailored biocatalyst achieved by the rational anchoring of imidazole groups on a natural polymer: furnishing a potential artificial nuclease by sustainable materials engineering.

    Science.gov (United States)

    Ferreira, José G L; Grein-Iankovski, Aline; Oliveira, Marco A S; Simas-Tosin, Fernanda F; Riegel-Vidotti, Izabel C; Orth, Elisa S

    2015-04-11

    Foreseeing the development of artificial enzymes by sustainable materials engineering, we rationally anchored reactive imidazole groups on gum arabic, a natural biocompatible polymer. The tailored biocatalyst GAIMZ demonstrated catalytic activity (>10(5)-fold) in dephosphorylation reactions with recyclable features and was effective in cleaving plasmid DNA, comprising a potential artificial nuclease.

  12. Probing force-induced unfolding intermediates of a single staphylococcal nuclease molecule and the effect of ligand binding

    International Nuclear Information System (INIS)

    Ishii, Takaaki; Murayama, Yoshihiro; Katano, Atsuto; Maki, Kosuke; Kuwajima, Kunihiro; Sano, Masaki

    2008-01-01

    Single-molecule manipulation techniques have given experimental access to unfolding intermediates of proteins that are inaccessible in conventional experiments. A detailed characterization of the intermediates is a challenging problem that provides new possibilities for directly probing the energy landscape of proteins. We investigated single-molecule mechanical unfolding of a small globular protein, staphylococcal nuclease (SNase), using atomic force microscopy. The unfolding trajectories of the protein displayed sub-molecular and stochastic behavior with typical lengths corresponding to the size of the unfolded substructures. Our results support the view that the single protein unfolds along multiple pathways as suggested in recent theoretical studies. Moreover, we found the drastic change, caused by the ligand and inhibitor bindings, in the mechanical unfolding dynamics

  13. Functional characterization of a conserved archaeal viral operon revealing single-stranded DNA binding, annealing and nuclease activities

    DEFF Research Database (Denmark)

    Guo, Yang; Kragelund, Birthe Brandt; White, Malcolm F.

    2015-01-01

    encoding proteins of unknown function and forming an operon with ORF207 (gp19). SIRV2 gp17 was found to be a single-stranded DNA (ssDNA) binding protein different in structure from all previously characterized ssDNA binding proteins. Mutagenesis of a few conserved basic residues suggested a U......-shaped binding path for ssDNA. The recombinant gp18 showed an ssDNA annealing activity often associated with helicases and recombinases. To gain insight into the biological role of the entire operon, we characterized SIRV2 gp19 and showed it to possess a 5'→3' ssDNA exonuclease activity, in addition...... for rudiviruses and the close interaction among the ssDNA binding, annealing and nuclease proteins strongly point to a role of the gene operon in genome maturation and/or DNA recombination that may function in viral DNA replication/repair....

  14. Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference

    Energy Technology Data Exchange (ETDEWEB)

    Beloglazova, Natalia; Petit, Pierre; Flick, Robert; Brown, Greg; Savchenko, Alexei; Yakunin, Alexander F. (Toronto)

    2012-03-15

    Clustered regularly interspaced short palindromic repeats (CRISPRs) and Cas proteins represent an adaptive microbial immunity system against viruses and plasmids. Cas3 proteins have been proposed to play a key role in the CRISPR mechanism through the direct cleavage of invasive DNA. Here, we show that the Cas3 HD domain protein MJ0384 from Methanocaldococcus jannaschii cleaves endonucleolytically and exonucleolytically (3'-5') single-stranded DNAs and RNAs, as well as 3'-flaps, splayed arms, and R-loops. The degradation of branched DNA substrates by MJ0384 is stimulated by the Cas3 helicase MJ0383 and ATP. The crystal structure of MJ0384 revealed the active site with two bound metal cations and together with site-directed mutagenesis suggested a catalytic mechanism. Our studies suggest that the Cas3 HD nucleases working together with the Cas3 helicases can completely degrade invasive DNAs through the combination of endo- and exonuclease activities.

  15. Selection-free gene repair after adenoviral vector transduction of designer nucleases: rescue of dystrophin synthesis in DMD muscle cell populations.

    Science.gov (United States)

    Maggio, Ignazio; Stefanucci, Luca; Janssen, Josephine M; Liu, Jin; Chen, Xiaoyu; Mouly, Vincent; Gonçalves, Manuel A F V

    2016-02-18

    Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disorder caused by mutations in the 2.4 Mb dystrophin-encoding DMD gene. The integration of gene delivery and gene editing technologies based on viral vectors and sequence-specific designer nucleases, respectively, constitutes a potential therapeutic modality for permanently repairing defective DMD alleles in patient-derived myogenic cells. Therefore, we sought to investigate the feasibility of combining adenoviral vectors (AdVs) with CRISPR/Cas9 RNA-guided nucleases (RGNs) alone or together with transcriptional activator-like effector nucleases (TALENs), for endogenous DMD repair through non-homologous end-joining (NHEJ). The strategies tested involved; incorporating small insertions or deletions at out-of-frame sequences for reading frame resetting, splice acceptor knockout for DNA-level exon skipping, and RGN-RGN or RGN-TALEN multiplexing for targeted exon(s) removal. We demonstrate that genome editing based on the activation and recruitment of the NHEJ DNA repair pathway after AdV delivery of designer nuclease genes, is a versatile and robust approach for repairing DMD mutations in bulk populations of patient-derived muscle progenitor cells (up to 37% of corrected DMD templates). These results open up a DNA-level genetic medicine strategy in which viral vector-mediated transient designer nuclease expression leads to permanent and regulated dystrophin synthesis from corrected native DMD alleles. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. A Novel Prokaryotic Green Fluorescent Protein Expression System for Testing Gene Editing Tools Activity Like Zinc Finger Nuclease.

    Science.gov (United States)

    Sabzehei, Faezeh; Kouhpayeh, Shirin; Dastjerdeh, Mansoureh Shahbazi; Khanahmad, Hossein; Salehi, Rasoul; Naderi, Shamsi; Taghizadeh, Razieh; Rabiei, Parisa; Hejazi, Zahra; Shariati, Laleh

    2017-01-01

    Gene editing technology has created a revolution in the field of genome editing. The three of the most famous tools in gene editing technology are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated systems. As their predictable nature, it is necessary to assess their efficiency. There are some methods for this purpose, but most of them are time labor and complicated. Here, we introduce a new prokaryotic reporter system, which makes it possible to evaluate the efficiency of gene editing tools faster, cheaper, and simpler than previous methods. At first, the target sites of a custom ZFN, which is designed against a segment of ampicillin resistance gene, were cloned on both sides of green fluorescent protein (GFP) gene to construct pPRO-GFP. Then pPRO-GFP was transformed into Escherichia coli TOP10F' that contains pZFN (contains expression cassette of a ZFN against ampicillin resistant gene), or p15A-KanaR as a negative control. The transformed bacteria were cultured on three separate media that contained ampicillin, kanamycin, and ampicillin + kanamycin; then the resulted colonies were assessed by flow cytometry. The results of flow cytometry showed a significant difference between the case (bacteria contain pZFN) and control (bacteria contain p15A, KanaR) in MFI (Mean Fluorescence Intensity) ( P fluorescence. Our flow cytometry results showed that this ZFN could reduce the intensity of GFP color and colony count of bacteria in media containing amp + kana versus control sample.

  17. Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay.

    Science.gov (United States)

    Li, Chia-Lung; Yang, Wei-Zen; Shi, Zhonghao; Yuan, Hanna S

    2018-02-13

    Tudor staphylococcal nuclease (TSN) is an evolutionarily conserved ribonuclease in eukaryotes that is composed of five staphylococcal nuclease-like domains (SN1 to SN5) and a Tudor domain. TSN degrades hyper-edited double-stranded RNA, including primary miRNA precursors containing multiple I-U and U-I pairs, and mature miRNA during miRNA decay. However, how TSN binds and degrades its RNA substrates remains unclear. Here, we show that the C. elegans TSN (cTSN) is a monomeric Ca 2+ -dependent ribonuclease, cleaving RNA chains at the 5'-side of the phosphodiester linkage to produce degraded fragments with 5'-hydroxyl and 3'-phosphate ends. cTSN degrades single-stranded RNA and double-stranded RNA containing mismatched base pairs, but is not restricted to those containing multiple I-U and U-I pairs. cTSN has at least two catalytic active sites located in the SN1 and SN3 domains, since mutations of the putative Ca 2+ -binding residues in these two domains strongly impaired its ribonuclease activity. We further show by small-angle X-ray scattering that rice osTSN has a flexible two-lobed structure with open to closed conformations, indicating that TSN may change its conformation upon RNA binding. We conclude that TSN is a structure-specific ribonuclease targeting not only single-stranded RNA, but also unstructured regions of double-stranded RNA. This study provides the molecular basis for how TSN cooperates with RNA editing to eliminate duplex RNA in cell defense, and how TSN selects and degrades RNA during microRNA decay. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  18. DNA Immunization with the Gene Encoding P4 Nuclease of Leishmania amazonensis Protects Mice against Cutaneous Leishmaniasis

    Science.gov (United States)

    Campbell, Kimberly; Diao, Hong; Ji, Jiaxiang; Soong, Lynn

    2003-01-01

    Infection with the protozoan parasite Leishmania amazonensis can cause diverse clinical forms of leishmaniasis. Immunization with purified P4 nuclease protein has been shown to elicit a protective response in mice challenged with L. amazonensis and L. pifanoi. To explore the potential of a DNA-based vaccine, we tested the L. amazonensis gene encoding P4 nuclease as well as adjuvant constructs encoding murine interleukin-12 (IL-12) and L. amazonensis HSP70. Susceptible BALB/c mice were immunized with the DNA encoding P4 alone, P4/IL-12, or P4/HSP70 prior to challenge with L. amazonensis promastigotes. Mice given P4/IL-12 exhibited no lesion development and had a 3- to 4-log reduction in tissue parasite burdens compared to controls. This protection corresponded to significant increases in gamma interferon and tumor necrosis factor alpha production and a reduction in parasite-specific immunoglobulin G1, suggesting an enhancement in Th1 responses. Moreover, we immunized mice with the L. amazonensis vaccines to determine if this vaccine regimen could provide cross-protection against a genetically diverse species, L. major. While the P4/HSP70 vaccine led to self-healing lesions, the P4/IL-12 vaccine provided negligible protection against L. major infection. This is the first report of successful use of a DNA vaccine to induce protection against L. amazonensis infection. Additionally, our results indicate that different vaccine combinations, including DNA encoding P4, HSP70, or IL-12, can provide significant protection against both Old World and New World cutaneous leishmaniasis. PMID:14573646

  19. An over expression APP model for anti-Alzheimer disease drug screening created by zinc finger nuclease technology.

    Directory of Open Access Journals (Sweden)

    Xiaojing Zhang

    Full Text Available Zinc Finger Nucleases (ZFNs, famous for their ability to precisely and efficiently modify specific genomic loci, have been employed in numerous transgenic model organism and cell constructions. Here we employ the ZFNs technology, with homologous recombination (HR, to construct sequence-specific Amyloid Precursor Protein (APP knock-in cells. With the use of ZFNs, we established APP knock in cell lines with gene-modification efficiencies of about 7%. We electroporated DNA fragment containing the promoter and the protein coding regions of the zinc finger nucleases into cells, instead of the plasmids, to avoid problems associated with off target homologous recombination, and adopted a pair of mutated FokI cleavage domains to reduce the toxic effects of the ZFNs on cell growth. Since over-expression of APP, or a subdomain of it, might lead to an immediately lethal effect, we used the Cre-LoxP System to regulate APP expression. Our genetically transformed cell lines, w5c1 and s12c8, showed detectable APP and Amyloid β (Aβ production. The Swedish double mutation in the APP coding sequence enhanced APP and Aβ abundance. What is more, the activity of the three key secretases in Aβ formation could be modulated, indicating that these transgenic cells have potential for drug screening to modify amyloid metabolism in cells. Our transformed cells could readily be propagated in culture and should provide an excellent experimental medium for elucidating aspects of the molecular pathogenesis of Alzheimer's disease, especially those concerning the amyloidogenic pathways involving mutations in the APP coding sequence. The cellular models may also serve as a tool for deriving potentially useful therapeutic agents.

  20. Gene Editing in Human Lymphoid Cells: Role for Donor DNA, Type of Genomic Nuclease and Cell Selection Method

    Directory of Open Access Journals (Sweden)

    Anastasia Zotova

    2017-11-01

    Full Text Available Programmable endonucleases introduce DNA breaks at specific sites, which are repaired by non-homologous end joining (NHEJ or homology recombination (HDR. Genome editing in human lymphoid cells is challenging as these difficult-to-transfect cells may also inefficiently repair DNA by HDR. Here, we estimated efficiencies and dynamics of knockout (KO and knockin (KI generation in human T and B cell lines depending on repair template, target loci and types of genomic endonucleases. Using zinc finger nuclease (ZFN, we have engineered Jurkat and CEM cells with the 8.2 kb human immunodeficiency virus type 1 (HIV-1 ∆Env genome integrated at the adeno-associated virus integration site 1 (AAVS1 locus that stably produce virus particles and mediate infection upon transfection with helper vectors. Knockouts generated by ZFN or clustered regularly interspaced short palindromic repeats (CRISPR/Cas9 double nicking techniques were comparably efficient in lymphoid cells. However, unlike polyclonal sorted cells, gene-edited cells selected by cloning exerted tremendous deviations in functionality as estimated by replication of HIV-1 and human T cell leukemia virus type 1 (HTLV-1 in these cells. Notably, the recently reported high-fidelity eCas9 1.1 when combined to the nickase mutation displayed gene-dependent decrease in on-target activity. Thus, the balance between off-target effects and on-target efficiency of nucleases, as well as choice of the optimal method of edited cell selection should be taken into account for proper gene function validation in lymphoid cells.

  1. A Novel Prokaryotic Green Fluorescent Protein Expression System for Testing Gene Editing Tools Activity Like Zinc Finger Nuclease

    Directory of Open Access Journals (Sweden)

    Faezeh Sabzehei

    2017-01-01

    Full Text Available Background: Gene editing technology has created a revolution in the field of genome editing. The three of the most famous tools in gene editing technology are zinc finger nucleases (ZFNs, transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats (CRISPR, and CRISPR-associated systems. As their predictable nature, it is necessary to assess their efficiency. There are some methods for this purpose, but most of them are time labor and complicated. Here, we introduce a new prokaryotic reporter system, which makes it possible to evaluate the efficiency of gene editing tools faster, cheaper, and simpler than previous methods. Materials and Methods: At first, the target sites of a custom ZFN, which is designed against a segment of ampicillin resistance gene, were cloned on both sides of green fluorescent protein (GFP gene to construct pPRO-GFP. Then pPRO-GFP was transformed into Escherichia coli TOP10F' that contains pZFN (contains expression cassette of a ZFN against ampicillin resistant gene, or p15A-KanaR as a negative control. The transformed bacteria were cultured on three separate media that contained ampicillin, kanamycin, and ampicillin + kanamycin; then the resulted colonies were assessed by flow cytometry. Results: The results of flow cytometry showed a significant difference between the case (bacteria contain pZFN and control (bacteria contain p15A, KanaR in MFI (Mean Fluorescence Intensity (P < 0.0001. Conclusion: According to ZFN efficiency, it can bind and cut the target sites, the bilateral cutting can affect the intensity of GFP fluorescence. Our flow cytometry results showed that this ZFN could reduce the intensity of GFP color and colony count of bacteria in media containing amp + kana versus control sample.

  2. Proviral HIV-genome-wide and pol-gene specific zinc finger nucleases: usability for targeted HIV gene therapy.

    Science.gov (United States)

    Wayengera, Misaki

    2011-07-22

    Infection with HIV, which culminates in the establishment of a latent proviral reservoir, presents formidable challenges for ultimate cure. Building on the hypothesis that ex-vivo or even in-vivo abolition or disruption of HIV-gene/genome-action by target mutagenesis or excision can irreversibly abrogate HIV's innate fitness to replicate and survive, we previously identified the isoschizomeric bacteria restriction enzymes (REases) AcsI and ApoI as potent cleavers of the HIV-pol gene (11 and 9 times in HIV-1 and 2, respectively). However, both enzymes, along with others found to cleave across the entire HIV-1 genome, slice (SX) at palindromic sequences that are prevalent within the human genome and thereby pose the risk of host genome toxicity. A long-term goal in the field of R-M enzymatic therapeutics has thus been to generate synthetic restriction endonucleases with longer recognition sites limited in specificity to HIV. We aimed (i) to assemble and construct zinc finger arrays and nucleases (ZFN) with either proviral-HIV-pol gene or proviral-HIV-1 whole-genome specificity respectively, and (ii) to advance a model for pre-clinically testing lentiviral vectors (LV) that deliver and transduce either ZFN genotype. First, we computationally generated the consensus sequences of (a) 114 dsDNA-binding zinc finger (Zif) arrays (ZFAs or ZifHIV-pol) and (b) two zinc-finger nucleases (ZFNs) which, unlike the AcsI and ApoI homeodomains, possess specificity to >18 base-pair sequences uniquely present within the HIV-pol gene (ZifHIV-polFN). Another 15 ZFNs targeting >18 bp sequences within the complete HIV-1 proviral genome were constructed (ZifHIV-1FN). Second, a model for constructing lentiviral vectors (LVs) that deliver and transduce a diploid copy of either ZifHIV-polFN or ZifHIV-1FN chimeric genes (termed LV- 2xZifHIV-polFN and LV- 2xZifHIV-1FN, respectively) is proposed. Third, two preclinical models for controlled testing of the safety and efficacy of either of these

  3. A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Fichorova, Raina N., E-mail: rfichorova@rics.bwh.harvard.edu [Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA (United States); Mendonca, Kevin; Yamamoto, Hidemi S.; Murray, Ryan [Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA (United States); Chandra, Neelima; Doncel, Gustavo F. [CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA (United States)

    2015-06-15

    Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV + 2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P < 0.05), and decreased levels of TLR2 (P < 0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P < 0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product. - Highlights: • A transcriptome nuclease protection assay assessed microbicides for vaginal safety. • Biomarkers were

  4. Crystallization and preliminary crystallographic analysis of an Escherichia coli-selected mutant of the nuclease domain of the metallonuclease colicin E7

    International Nuclear Information System (INIS)

    Czene, Anikó; Tóth, Eszter; Gyurcsik, Béla; Otten, Harm; Poulsen, Jens-Christian N.; Lo Leggio, Leila; Larsen, Sine; Christensen, Hans E. M.; Nagata, Kyosuke

    2013-01-01

    An N-terminally truncated mutant of the colicin E7 nuclease domain was crystallized and diffraction data set was collected to 1.6 Å resolution. The metallonuclease colicin E7 is a member of the HNH family of endonucleases. It serves as a bacterial toxin in Escherichia coli, protecting the host cell from other related bacteria and bacteriophages by degradation of their chromosomal DNA under environmental stress. Its cell-killing activity is attributed to the nonspecific nuclease domain (NColE7), which possesses the catalytic ββα-type metal ion-binding HNH motif at its C-terminus. Mutations affecting the positively charged amino acids at the N-terminus of NColE7 (444–576) surprisingly showed no or significantly reduced endonuclease activity [Czene et al. (2013 ▶), J. Biol. Inorg. Chem.18, 309–321]. The necessity of the N-terminal amino acids for the function of the C-terminal catalytic centre poses the possibility of allosteric activation within the enzyme. Precise knowledge of the intramolecular interactions of these residues that affect the catalytic activity could turn NColE7 into a novel platform for artificial nuclease design. In this study, the N-terminal deletion mutant ΔN4-NColE7-C* of the nuclease domain of colicin E7 selected by E. coli was overexpressed and crystallized at room temperature by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 1.6 Å resolution and could be indexed and averaged in the trigonal space group P3 1 21 or P3 2 21, with unit-cell parameters a = b = 55.4, c = 73.1 Å. Structure determination by molecular replacement is in progress

  5. Nuclease Activity via Self-Activation and Anticancer Activity of Mononuclear Copper(II) Complex: Novel Role of Tertiary Butyl Group in the Ligand Frame

    OpenAIRE

    Ghosh, Kaushik; Kumar, Pramod; Mohan, Varun; Singh, Udai P.; Kasiri, Sahba; Mandal, Subhrangsu S.

    2012-01-01

    Copper complex [Cu(tBuPhimp)(Cl)] (1) derived from tridentate ligand tBuPhimpH having N2O donors was synthesized and molecular structure was determined. Phenoxyl radical complex was generated in solution at room temperature using Ce(IV) salt. Nuclease activity and anticancer activity of 1 was investigated. Roles of tert-butyl group and singlet oxygen in DNA cleavage activity were also discussed.

  6. Construction of a quadruple auxotrophic mutant of an industrial polyploid saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease.

    Science.gov (United States)

    Zhang, Guo-Chang; Kong, In Iok; Kim, Heejin; Liu, Jing-Jing; Cate, Jamie H D; Jin, Yong-Su

    2014-12-01

    Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3, trp1, leu2, and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  7. Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases

    Directory of Open Access Journals (Sweden)

    Jean-Baptiste Renaud

    2016-03-01

    Full Text Available Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homology-directed repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of single-stranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing.

  8. Substrate overlap and functional competition between human nucleotide excision repair and Escherichia coli photolyase and (A)BC excision nuclease

    International Nuclear Information System (INIS)

    Sibghat-Ullah; Sancar, Z.

    1990-01-01

    Human cell free extract prepared by the method of Manley et al. carries out repair synthesis on UV-irradiated DNA. Removal of pyrimidine dimers by photoreactivation with DNA photolyase reduces repair synthesis by about 50%. With excess enzyme in the reaction mixture photolyase reduced the repair signal by the same amount even in the absence of photoreactivating light, presumably by binding to pyrimidine dimers and interfering with the binding of human damage recognition protein. Similarly, the UvrB subunit of Escherichia coli (A)BC excinuclease when loaded onto UV-irradiated or psoralen-adducted DNA inhibited repair synthesis by cell-free extract by 75-80%. The opposite was true also as HeLa cell free extract specifically inhibited the photorepair of a thymine dimer by DNA photolyase and its removal by (A)BC excinuclease. Cell-free extracts from xeroderma pigmentosum (XP) complementation groups A and C were equally effective in blocking the E. coli repair proteins, while extracts from complementation groups D and E were ineffective in blocking the E. coli enzyme. These results suggest that XP-D and XP-E cells are defective in the damage recognition subunits(s) of human excision nuclease

  9. A Mismatch EndoNuclease Array-Based Methodology (MENA for Identifying Known SNPs or Novel Point Mutations

    Directory of Open Access Journals (Sweden)

    Josep M. Comeron

    2016-04-01

    Full Text Available Accurate and rapid identification or confirmation of single nucleotide polymorphisms (SNPs, point mutations and other human genomic variation facilitates understanding the genetic basis of disease. We have developed a new methodology (called MENA (Mismatch EndoNuclease Array pairing DNA mismatch endonuclease enzymology with tiling microarray hybridization in order to genotype both known point mutations (such as SNPs as well as identify previously undiscovered point mutations and small indels. We show that our assay can rapidly genotype known SNPs in a human genomic DNA sample with 99% accuracy, in addition to identifying novel point mutations and small indels with a false discovery rate as low as 10%. Our technology provides a platform for a variety of applications, including: (1 genotyping known SNPs as well as confirming newly discovered SNPs from whole genome sequencing analyses; (2 identifying novel point mutations and indels in any genomic region from any organism for which genome sequence information is available; and (3 screening panels of genes associated with particular diseases and disorders in patient samples to identify causative mutations. As a proof of principle for using MENA to discover novel mutations, we report identification of a novel allele of the beethoven (btv gene in Drosophila, which encodes a ciliary cytoplasmic dynein motor protein important for auditory mechanosensation.

  10. A Mismatch EndoNuclease Array-Based Methodology (MENA) for Identifying Known SNPs or Novel Point Mutations.

    Science.gov (United States)

    Comeron, Josep M; Reed, Jordan; Christie, Matthew; Jacobs, Julia S; Dierdorff, Jason; Eberl, Daniel F; Manak, J Robert

    2016-04-05

    Accurate and rapid identification or confirmation of single nucleotide polymorphisms (SNPs), point mutations and other human genomic variation facilitates understanding the genetic basis of disease. We have developed a new methodology (called MENA (Mismatch EndoNuclease Array)) pairing DNA mismatch endonuclease enzymology with tiling microarray hybridization in order to genotype both known point mutations (such as SNPs) as well as identify previously undiscovered point mutations and small indels. We show that our assay can rapidly genotype known SNPs in a human genomic DNA sample with 99% accuracy, in addition to identifying novel point mutations and small indels with a false discovery rate as low as 10%. Our technology provides a platform for a variety of applications, including: (1) genotyping known SNPs as well as confirming newly discovered SNPs from whole genome sequencing analyses; (2) identifying novel point mutations and indels in any genomic region from any organism for which genome sequence information is available; and (3) screening panels of genes associated with particular diseases and disorders in patient samples to identify causative mutations. As a proof of principle for using MENA to discover novel mutations, we report identification of a novel allele of the beethoven (btv) gene in Drosophila, which encodes a ciliary cytoplasmic dynein motor protein important for auditory mechanosensation.

  11. Generation of knockout rats with X-linked severe combined immunodeficiency (X-SCID) using zinc-finger nucleases.

    Science.gov (United States)

    Mashimo, Tomoji; Takizawa, Akiko; Voigt, Birger; Yoshimi, Kazuto; Hiai, Hiroshi; Kuramoto, Takashi; Serikawa, Tadao

    2010-01-25

    Although the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES) cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs) were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc. We report here on ZFN-induced gene targeting of the rat interleukin 2 receptor gamma (Il2rg) locus, where orthologous human and mouse mutations cause X-linked severe combined immune deficiency (X-SCID). Co-injection of mRNAs encoding custom-designed ZFNs into the pronucleus of fertilized oocytes yielded genetically modified offspring at rates greater than 20%, which possessed a wide variety of deletion/insertion mutations. ZFN-modified founders faithfully transmitted their genetic changes to the next generation along with the severe combined immune deficiency phenotype. The efficient and rapid generation of gene knockout rats shows that using ZFN technology is a new strategy for creating gene-targeted rat models of human diseases. In addition, the X-SCID rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies.

  12. Transcription activator-like effector nucleases mediated metabolic engineering for enhanced fatty acids production in Saccharomyces cerevisiae

    KAUST Repository

    Aouida, Mustapha

    2015-04-01

    Targeted engineering of microbial genomes holds much promise for diverse biotechnological applications. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/Cas9 systems are capable of efficiently editing microbial genomes, including that of Saccharomyces cerevisiae. Here, we demonstrate the use of TALENs to edit the genome of S.cerevisiae with the aim of inducing the overproduction of fatty acids. Heterodimeric TALENs were designed to simultaneously edit the FAA1 and FAA4 genes encoding acyl-CoA synthetases in S.cerevisiae. Functional yeast double knockouts generated using these TALENs over-produce large amounts of free fatty acids into the cell. This study demonstrates the use of TALENs for targeted engineering of yeast and demonstrates that this technology can be used to stimulate the enhanced production of free fatty acids, which are potential substrates for biofuel production. This proof-of-principle study extends the utility of TALENs as excellent genome editing tools and highlights their potential use for metabolic engineering of yeast and other organisms, such as microalgae and plants, for biofuel production. © 2015 The Society for Biotechnology, Japan.

  13. Tudor staphylococcal nuclease links formation of stress granules and processing bodies with mRNA catabolism in Arabidopsis.

    Science.gov (United States)

    Gutierrez-Beltran, Emilio; Moschou, Panagiotis N; Smertenko, Andrei P; Bozhkov, Peter V

    2015-03-01

    Tudor Staphylococcal Nuclease (TSN or Tudor-SN; also known as SND1) is an evolutionarily conserved protein involved in the transcriptional and posttranscriptional regulation of gene expression in animals. Although TSN was found to be indispensable for normal plant development and stress tolerance, the molecular mechanisms underlying these functions remain elusive. Here, we show that Arabidopsis thaliana TSN is essential for the integrity and function of cytoplasmic messenger ribonucleoprotein (mRNP) complexes called stress granules (SGs) and processing bodies (PBs), sites of posttranscriptional gene regulation during stress. TSN associates with SGs following their microtubule-dependent assembly and plays a scaffolding role in both SGs and PBs. The enzymatically active tandem repeat of four SN domains is crucial for targeting TSN to the cytoplasmic mRNA complexes and is sufficient for the cytoprotective function of TSN during stress. Furthermore, our work connects the cytoprotective function of TSN with its positive role in stress-induced mRNA decapping. While stress led to a pronounced increase in the accumulation of uncapped mRNAs in wild-type plants, this increase was abrogated in TSN knockout plants. Taken together, our results establish TSN as a key enzymatic component of the catabolic machinery responsible for the processing of mRNAs in the cytoplasmic mRNP complexes during stress. © 2015 American Society of Plant Biologists. All rights reserved.

  14. Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein

    Directory of Open Access Journals (Sweden)

    April Pawluk

    2017-12-01

    Full Text Available CRISPR (clustered regularly interspaced short palindromic repeat-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor.

  15. Generation of knockout rats with X-linked severe combined immunodeficiency (X-SCID using zinc-finger nucleases.

    Directory of Open Access Journals (Sweden)

    Tomoji Mashimo

    Full Text Available BACKGROUND: Although the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc. METHODOLOGY/PRINCIPAL FINDINGS: We report here on ZFN-induced gene targeting of the rat interleukin 2 receptor gamma (Il2rg locus, where orthologous human and mouse mutations cause X-linked severe combined immune deficiency (X-SCID. Co-injection of mRNAs encoding custom-designed ZFNs into the pronucleus of fertilized oocytes yielded genetically modified offspring at rates greater than 20%, which possessed a wide variety of deletion/insertion mutations. ZFN-modified founders faithfully transmitted their genetic changes to the next generation along with the severe combined immune deficiency phenotype. CONCLUSIONS AND SIGNIFICANCE: The efficient and rapid generation of gene knockout rats shows that using ZFN technology is a new strategy for creating gene-targeted rat models of human diseases. In addition, the X-SCID rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies.

  16. Targeted editing of goat genome with modular-assembly zinc finger nucleases based on activity prediction by computational molecular modeling.

    Science.gov (United States)

    Xiong, Kai; Li, Shanshan; Zhang, Hongxiao; Cui, Ye; Yu, Debing; Li, Yan; Sun, Wenxing; Fu, Yingying; Teng, Yun; Liu, Zhi; Zhou, Xiaolong; Xiao, Peng; Li, Juan; Liu, Honglin; Chen, Jie

    2013-07-01

    Zinc finger nuclease (ZFN) technology can mediate targeted genome modification to produce transgenic animals in a high-efficient and biological-safe way. Modular assembly is a rapid, convenient and open-source method for the synthesis of ZFNs. However, this biotechnology is hampered by multistep construction, low-efficiency editing and off-target cleavage. Here we synthesized and tested six pairs of three- or four-finger ZFNs to target one site in goat beta-lactoglobulin (BLG, a dominant allergen in goat milk) gene. Homology modeling was applied to build the structure model of ZFNs to predict their editing activities targeting at goat BLG gene. Goat fibroblast cells were transfected with plasmids that encoded ZFN pairs, and genomic DNA was isolated 72 h later for genome editing efficiency assay. The results of editing efficiency assay demonstrated that ZFNs with optimal interaction modes can edit goat BLG gene more efficiently, whereas ZFNs with unexpected interaction modes showed lower activities in editing BLG gene. We concluded that modular-assembly ZFNs can provide a rapid, public-available, and easy-to-practice platform for transgenic animal research and molecular modeling would help as a useful tool for ZFNs activity prediction.

  17. Efficient Reverse Transcription Using Locked Nucleic Acid Nucleotides towards the Evolution of Nuclease Resistant RNA Aptamers

    DEFF Research Database (Denmark)

    Crouzier, Lucile; Dubois, Camille; Edwards, Stacey L

    2012-01-01

    Modified nucleotides are increasingly being utilized in the de novo selection of aptamers for enhancing their drug-like character and abolishing the need for time consuming trial-and-error based post-selection modifications. Locked nucleic acid (LNA) is one of the most prominent and successful...... nucleic acid analogues because of its remarkable properties, and widely explored as building blocks in therapeutic oligonucleotides. Evolution of LNA-modified RNA aptamers requires an efficient reverse transcription method for PCR enrichment of the selected RNA aptamer candidates. Establishing this key...... step is a pre-requisite for performing LNA-modified RNA aptamer selection....

  18. Proviral HIV-genome-wide and pol-gene specific Zinc Finger Nucleases: Usability for targeted HIV gene therapy

    Directory of Open Access Journals (Sweden)

    Wayengera Misaki

    2011-07-01

    Full Text Available Abstract Background Infection with HIV, which culminates in the establishment of a latent proviral reservoir, presents formidable challenges for ultimate cure. Building on the hypothesis that ex-vivo or even in-vivo abolition or disruption of HIV-gene/genome-action by target mutagenesis or excision can irreversibly abrogate HIV's innate fitness to replicate and survive, we previously identified the isoschizomeric bacteria restriction enzymes (REases AcsI and ApoI as potent cleavers of the HIV-pol gene (11 and 9 times in HIV-1 and 2, respectively. However, both enzymes, along with others found to cleave across the entire HIV-1 genome, slice (SX at palindromic sequences that are prevalent within the human genome and thereby pose the risk of host genome toxicity. A long-term goal in the field of R-M enzymatic therapeutics has thus been to generate synthetic restriction endonucleases with longer recognition sites limited in specificity to HIV. We aimed (i to assemble and construct zinc finger arrays and nucleases (ZFN with either proviral-HIV-pol gene or proviral-HIV-1 whole-genome specificity respectively, and (ii to advance a model for pre-clinically testing lentiviral vectors (LV that deliver and transduce either ZFN genotype. Methods and Results First, we computationally generated the consensus sequences of (a 114 dsDNA-binding zinc finger (Zif arrays (ZFAs or ZifHIV-pol and (b two zinc-finger nucleases (ZFNs which, unlike the AcsI and ApoI homeodomains, possess specificity to >18 base-pair sequences uniquely present within the HIV-pol gene (ZifHIV-polFN. Another 15 ZFNs targeting >18 bp sequences within the complete HIV-1 proviral genome were constructed (ZifHIV-1FN. Second, a model for constructing lentiviral vectors (LVs that deliver and transduce a diploid copy of either ZifHIV-polFN or ZifHIV-1FN chimeric genes (termed LV- 2xZifHIV-polFN and LV- 2xZifHIV-1FN, respectively is proposed. Third, two preclinical models for controlled testing of

  19. Establishment of pten knockout medaka with transcription activator-like effector nucleases (TALENs as a model of PTEN deficiency disease.

    Directory of Open Access Journals (Sweden)

    Yuriko Matsuzaki

    Full Text Available Phosphatase and tensin homolog (PTEN is a lipid and protein phosphatase that antagonizes signaling by the phosphatidylinositol 3-kinase (PI3K-AKT signaling pathway. The PTEN gene is a major tumor suppressor, with mutations of this gene occurring frequently in tumors of humans and mice. We have now developed mutant medaka deficient in PTEN with the use of transcription activator-like effector nuclease (TALEN technology. Medaka possesses two pten genes, ptena and ptenb, similar to zebrafish. We established 16 ptena mutant lines and two ptenb mutant lines. Homozygous single pten mutants were found to be viable and fertile. In contrast, pten double-knockout (dko embryos manifested severe abnormalities in vasculogenesis, eye size, and tail development at 72 hours post fertilization(hpf and died before hatching. Immunoblot analysis revealed that the ratio of phosphorylated to total forms of AKT (pAKT/AKT in pten dko embryos was four times that in wild-type embryos, indicative of up-regulation of signaling by the PI3K-AKT pathway. Treatment of pten dko embryos with the PI3K inhibitor LY294002 reduced the pAKT/AKT ratio by about one-half and partially rescued the defect in vasculogenesis. Additional inhibitors of the PI3K-AKT pathway, including rapamycin and N-α-tosyl-L-phenylalanyl chloromethyl ketone, also partially restored vasculogenesis in the dko embryos. Our model system thus allows pten dko embryos to be readily distinguished from wild-type embryos at an early stage of development and is suitable for the screening of drugs able to compensate for PTEN deficiency.

  20. Heritable targeted inactivation of the rainbow trout (Oncorhynchus mykiss) master sex-determining gene using zinc-finger nucleases.

    Science.gov (United States)

    Yano, Ayaka; Nicol, Barbara; Jouanno, Elodie; Guiguen, Yann

    2014-04-01

    Gene targeting is a powerful tool for analyzing gene function. Recently, new technology for gene targeting using engineered zinc-finger nucleases (ZFNs) has been described in fish species. However, it has not yet been widely used for cold water and slow developing species, such as Salmonidae. Here, we present the results of successful ZFN-mediated disruption of the sex-determining gene sdY (sexually dimorphic on the Y chromosome) in rainbow trout (Oncorhynchus mykiss). Three pairs of ZFN mRNA targeted to different regions of the sdY gene were injected into fertilized rainbow trout eggs. Sperm from 1-year-old male founders (parental generation one or P1) carrying a ZFN-induced mutation in their germline were then used to produce F1 non-mosaic animals. In these F1 populations, we characterized 14 different mutations in the sdY gene, including one mutation leading to the deletion of leucine 43 (L43) and 13 mutations at other target sites that had different effects on the SdY protein, i.e., amino acid insertions, deletions, and frameshift mutations producing premature stop codons in the mRNA. The gonadal phenotype analysis of the F1-mutated animals revealed that the single L43 amino acid deletion did not lead to a male-to-female sex reversal, but all other mutations induced a clear ovarian phenotype. These results show that targeted gene disruption using ZFN is efficient in rainbow trout but depends on the ZFN design. We also characterized new sdY mutations resulting in male-to-female sex reversal, and we conclude that L43 seems dispensable for SdY function.

  1. Type II restriction endonuclease R.Eco29kI is a member of the GIY-YIG nuclease superfamily

    Directory of Open Access Journals (Sweden)

    Feder Marcin

    2007-07-01

    Full Text Available Abstract Background The majority of experimentally determined crystal structures of Type II restriction endonucleases (REases exhibit a common PD-(D/EXK fold. Crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI and half-pipe (R.PabI, and bioinformatics analyses supported by mutagenesis suggested that some REases belong to the HNH fold. Our previous bioinformatic analysis suggested that REase R.Eco29kI shares sequence similarities with one more unrelated nuclease superfamily, GIY-YIG, however so far no experimental data were available to support this prediction. The determination of a crystal structure of the GIY-YIG domain of homing endonuclease I-TevI provided a template for modeling of R.Eco29kI and prompted us to validate the model experimentally. Results Using protein fold-recognition methods we generated a new alignment between R.Eco29kI and I-TevI, which suggested a reassignment of one of the putative catalytic residues. A theoretical model of R.Eco29kI was constructed to illustrate its predicted three-dimensional fold and organization of the active site, comprising amino acid residues Y49, Y76, R104, H108, E142, and N154. A series of mutants was constructed to generate amino acid substitutions of selected residues (Y49A, R104A, H108F, E142A and N154L and the mutant proteins were examined for their ability to bind the DNA containing the Eco29kI site 5'-CCGCGG-3' and to catalyze the cleavage reaction. Experimental data reveal that residues Y49, R104, E142, H108, and N154 are important for the nuclease activity of R.Eco29kI, while H108 and N154 are also important for specific DNA binding by this enzyme. Conclusion Substitutions of residues Y49, R104, H108, E142 and N154 predicted by the model to be a part of the active site lead to mutant proteins with strong defects in the REase activity. These results are in very good agreement with the structural model presented in this work and with our

  2. Investigation of centers sensitive to S1-nuclease in the genoma of the yeast S. cerevisiae after in-vivo exposure to gamma radiation

    International Nuclear Information System (INIS)

    Geigl, E.M.

    1987-09-01

    The structure, distribution and repair of basal damage in DNS after exposure to 60 Co gamma radiation were investigated in S. cerevisiae cells. Small DNS regions with mispaired or unpaired bases of rather high stability were found whose rate of incidence and linear dose dependence appear to be similar to those of double strand breaks. In contrast to double strand breaks, they showed no statistical' distribution pattern across the genoma. Liquid holding experiments showed that centers sensitive to S1-nuclease will be repaired in S. cerevisiae by a combined process of recombination and postreplication repair; the gene products of the genes RAD50 and RAD18 are involved. (orig./AJ) [de

  3. In Vivo Zinc Finger Nuclease-mediated Targeted Integration of a Glucose-6-phosphatase Transgene Promotes Survival in Mice With Glycogen Storage Disease Type IA

    Science.gov (United States)

    Landau, Dustin J; Brooks, Elizabeth Drake; Perez-Pinera, Pablo; Amarasekara, Hiruni; Mefferd, Adam; Li, Songtao; Bird, Andrew; Gersbach, Charles A; Koeberl, Dwight D

    2016-01-01

    Glycogen storage disease type Ia (GSD Ia) is caused by glucose-6-phosphatase (G6Pase) deficiency in association with severe, life-threatening hypoglycemia that necessitates lifelong dietary therapy. Here we show that use of a zinc-finger nuclease (ZFN) targeted to the ROSA26 safe harbor locus and a ROSA26-targeting vector containing a G6PC donor transgene, both delivered with adeno-associated virus (AAV) vectors, markedly improved survival of G6Pase knockout (G6Pase-KO) mice compared with mice receiving the donor vector alone (P Ia, as compared with normal littermates, at 8 months following vector administration (P Ia. PMID:26865405

  4. Introduction of 2-O-benzyl abasic nucleosides to the 3'-overhang regions of siRNAs greatly improves nuclease resistance.

    Science.gov (United States)

    Nagaya, Yuki; Kitamura, Yoshiaki; Shibata, Aya; Ikeda, Masato; Akao, Yukihiro; Kitade, Yukio

    2017-12-15

    Chemically modified siRNAs containing 2-O-benzyl-1-deoxy-d-ribofuranose (R H OBn ) in their 3'-overhang region were significantly more resistant towards serum nucleases than siRNAs possessing the natural nucleoside in this region. The knockdown efficacies and binding affinities of these modified siRNAs to the recombinant human Argonaute protein 2 (hAgo2) PAZ domain were comparable with that of siRNA with a thymidine dimer at the 3'-end. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Influence of some exo nucleases in response to the induced genetic damage in Escherichia coli by alpha radiation

    International Nuclear Information System (INIS)

    Aguilar M, M.

    2005-01-01

    Within the strategies with those that E. coli counts to overcome to the genetic damage there is the SOS response, a group of genes that participate in repair and/or tolerance that it confers to the bacteria major opportunities of surviving. These genes are repressed and its only are expressed when it happens genetic damage. So that this system is activated it is necessary that DNA of a band exists and in this sense the double ruptures (RDB) its are not able to induce this response unless there is a previous processing. In stumps with defects in certain genes that have to do with repair of RDB (as recO, recJ and xonA) the activity of SOS is smaller than in a wild stump what suggests that these participate in the previous processes to the activation of the response. The ionizing radiation produce among other many lesions, RDB in greater or smaller proportion, depending on the ionization capacity. A parameter to evaluate this capacity is the lineal energy transfer (LET), defined as the average energy given by unit of distance travelled. In general the LET of the corpuscular radiations is a lot but high that of the electromagnetic one, for what produces bigger quantity of ionizations inside a restricted zone and it increases by this way the probability that RDB has been generated. This work has for object to infer the participation of xonA and recJ in this response and to evaluate the damage produced by ionizing radiation of different LET (alpha particles of different energies) in a stump with all the functional repair mechanisms. Its were considered two parameters: the survival and the activity of SOS evaluated by means of the chromo test. The results indicate that the activity of these exo nucleases is necessary for the repair of RDB as well as for the processing of lesions foresaw to the activation of SOS. As for the treatment with alphas of different energies is observed that so much the survival like the activity of SOS vary as the LET of the radiation changes

  6. The Tudor Staphylococcal Nuclease Protein ofEntamoeba histolyticaParticipates in Transcription Regulation and Stress Response.

    Science.gov (United States)

    Cázares-Apátiga, Javier; Medina-Gómez, Christian; Chávez-Munguía, Bibiana; Calixto-Gálvez, Mercedes; Orozco, Esther; Vázquez-Calzada, Carlos; Martínez-Higuera, Aarón; Rodríguez, Mario A

    2017-01-01

    Entamoeba histolytica is the protozoa parasite responsible of human amoebiasis, disease that causes from 40,000 to 100,000 deaths annually worldwide. However, few are known about the expression regulation of molecules involved in its pathogenicity. Transcription of some virulence-related genes is positively controlled by the cis-regulatory element named URE1. Previously we identified the transcription factor that binds to URE1, which displayed a nuclear and cytoplasmic localization. This protein belongs to the Tudor Staphyococcal nuclease (TSN) family, which in other systems participates in virtually all pathways of gene expression, suggesting that this amoebic transcription factor (EhTSN; former EhURE1BP) could also play multiple functions in E. histolytica . The aim of this study was to identify the possible cellular events where EhTSN is involved. Here, we found that EhTSN in nucleus is located in euchromatin and close to, but not into, heterochromatin. We also showed the association of EhTSN with proteins involved in transcription and that the knockdown of EhTSN provokes a diminishing in the mRNA level of the EhRabB gene, which in its promoter region contains the URE1 motif, confirming that EhTSN participates in transcription regulation. In cytoplasm, this protein was found linked to the membrane of small vesicles and to plasma membrane. Through pull-down assays and mass spectrometry we identity thirty two candidate proteins to interact with EhTSN. These proteins participate in transcription, metabolism, signaling, and stress response, among other cellular processes. Interaction of EhTSN with some candidate proteins involved in metabolism, and signaling was validated by co-immunoprecipitation or co-localization. Finally we showed the co-localization of EhTSN and HSP70 in putative stress granules during heat shock and that the knockdown of EhTSN increases the cell death during heat shock treatment, reinforcing the hypothesis that EhTSN has a role during stress

  7. Cell-penetrating peptides (CPPs): From delivery of nucleic acids and antigens to transduction of engineered nucleases for application in transgenesis.

    Science.gov (United States)

    Rádis-Baptista, Gandhi; Campelo, Iana S; Morlighem, Jean-Étienne R L; Melo, Luciana M; Freitas, Vicente J F

    2017-06-20

    Cell-penetrating peptides (CPPs) have been studied for their capacity to translocate across the lipid membrane of several cell types. In membrane translocation, these peptides can remarkably transport biologically active hydrophilic molecules, such as pharmaceuticals, nucleic acids (DNA and RNA) and even high-molecular-weight proteins, Fig. 3 into the cell cytoplasm and organelles. The development of CPPs as transduction agents includes the modification of gene and protein expression, the reprogramming and differentiation of induced pluripotent stem cells and the preparation of cellular vaccines. A relatively recent field of CPP application is the transduction of plasmid DNA vectors and CPP-fusion proteins to modify genomes and introduce new traits in cells and organisms. CPP-mediated transduction of components for genome editing is an advantageous alternative to viral DNA vectors. Engineered site-specific nucleases, such as Cre recombinase, ZFN, TALENs and CRISPR associated protein (Cas), have been coupled to CPPs, and the fused proteins have been used to permeate targeted cells and tissues. The functionally active fusion CPP-nucleases subsequently home to the nucleus, incise genomic DNA at specific sites and induce repair and recombination. This review has the objective of discussing CPPs and elucidating the prospective use of CPP-mediated transduction technology, particularly in genome modification and transgenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Mononuclear cells from patients recovered from cutaneous leishmaniasis respond to Leishmania major amastigote class I nuclease with a predominant Th1-like response

    Science.gov (United States)

    Farajnia, S; Mahboudi, F; Ajdari, S; Reiner, N E; Kariminia, A; Alimohammadian, M H

    2005-01-01

    The Leishmania major amastigote class I nuclease (LmaCIN) is a developmentally regulated protein that is highly expressed in the amastigote stage of L. major. This protein is homologous to the P4 nuclease of L. pifanoi, which has been shown to induce protective immune response in a murine model. To evaluate LmaCIN as a potential human vaccine candidate, cellular immune responses to recombinant LmaCIN were examined in individuals recovered from Old World cutaneous leishmaniasis. Peripheral blood mononuclear cells (PBMC) from patients recovered from L. major infection were cultured either with recombinant LmaCIN or autoclaved L. major (ALM) as control. rLmaCIN induced significant proliferation of PBMC from 90% of recovered patients. Phenotypic analysis of proliferating cells showed that CD8+ cells were the predominant cell type proliferating in response to rLmaC1N. Screening of culture supernatants for cytokines showed that rLmaCIN induced high levels of interferon (IFN)-γ (mean ± s.e.m.: 1398 ± 179 pg/ml) associated with little interleukin (IL)-10 and little or no IL-5 production. These findings show that LmaCIN is immunogenic in humans during L. major infection and that it can elicit immunological responses relevant to immunoprophylaxis of leishmaniasis. PMID:15730396

  9. Sensitive detection of microRNAs based on the conversion of colorimetric assay into electrochemical analysis with duplex-specific nuclease-assisted signal amplification.

    Science.gov (United States)

    Xia, Ning; Liu, Ke; Zhou, Yingying; Li, Yuanyuan; Yi, Xinyao

    2017-01-01

    miRNAs have emerged as new biomarkers for the detection of a wide variety of cancers. By employing duplex-specific nuclease for signal amplification and gold nanoparticles (AuNPs) as the carriers of detection probes, a novel electrochemical assay of miRNAs was performed. The method is based on conversion of the well-known colorimetric assay into electrochemical analysis with enhanced sensitivity. DNA capture probes immobilized on the electrode surface and ferrocene (Fc)-labeled DNA detection probes (denoted "Fc-DNA-Fc") presented in the solution induced the assembly of positively charged AuNPs on the electrode surface through the electrostatic interaction. As a result, a large number of Fc-DNA-Fc molecules were attached on the electrode surface, thus amplifying the electrochemical signal. When duplex-specific nuclease was added to recycle the process of miRNA-initiated digestion of the immobilized DNA probes, Fc-DNA-Fc-induced assembly of AuNPs on the electrode surface could not occur. This resulted in a significant fall in the oxidation current of Fc. The current was found to be inversely proportional to the concentration of miRNAs in the range of 0-25 fM, and a detection limit of 0.1 fM was achieved. Moreover, this work presents a new method for converting colorimetric assays into sensitive electrochemical analyses, and thus would be valuable for design of novel chemical/biosensors.

  10. Molecular Imaging of Human Embryonic Stem Cells Stably Expressing Human PET Reporter Genes After Zinc Finger Nuclease-Mediated Genome Editing.

    Science.gov (United States)

    Wolfs, Esther; Holvoet, Bryan; Ordovas, Laura; Breuls, Natacha; Helsen, Nicky; Schönberger, Matthias; Raitano, Susanna; Struys, Tom; Vanbilloen, Bert; Casteels, Cindy; Sampaolesi, Maurilio; Van Laere, Koen; Lambrichts, Ivo; Verfaillie, Catherine M; Deroose, Christophe M

    2017-10-01

    Molecular imaging is indispensable for determining the fate and persistence of engrafted stem cells. Standard strategies for transgene induction involve the use of viral vectors prone to silencing and insertional mutagenesis or the use of nonhuman genes. Methods: We used zinc finger nucleases to induce stable expression of human imaging reporter genes into the safe-harbor locus adeno-associated virus integration site 1 in human embryonic stem cells. Plasmids were generated carrying reporter genes for fluorescence, bioluminescence imaging, and human PET reporter genes. Results: In vitro assays confirmed their functionality, and embryonic stem cells retained differentiation capacity. Teratoma formation assays were performed, and tumors were imaged over time with PET and bioluminescence imaging. Conclusion: This study demonstrates the application of genome editing for targeted integration of human imaging reporter genes in human embryonic stem cells for long-term molecular imaging. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

  11. TT2014 meeting report on the 12th Transgenic Technology meeting in Edinburgh: new era of transgenic technologies with programmable nucleases in the foreground.

    Science.gov (United States)

    Beck, Inken M; Sedlacek, Radislav

    2015-02-01

    The 12th Transgenic Technology meeting was held in Edinburgh on 6th-8th October 2014 and interest to participate in the meeting overcame all expectations. The TT2014 was the largest meeting ever with more than 540 scientists, technicians, and students from all over the world. The meeting had an excellent scientific program that brought information on the latest ground-breaking technologies for gene targeting and genome editing using programmable nucleases into the foreground. These presentations were well balanced with several highlights over viewing topics in embryonic stem cell research, embryogenesis, disease models, and animals in agriculture. Ample space was reserved also for short talks presenting technical development and for highlighting posters contributions. A highlight of the meeting was the award of the 10th International Society of Transgenic Technologies Prize to Janet Rossant for her outstanding contributions in the field of mouse embryogenesis.

  12. TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE-Mediated Generation and Metabolic Analysis of Camalexin-Deficient cyp71a12 cyp71a13 Double Knockout Lines1

    Science.gov (United States)

    Müller, Teresa M.; Böttcher, Christoph; Morbitzer, Robert; Götz, Cornelia C.; Lehmann, Johannes; Lahaye, Thomas; Glawischnig, Erich

    2015-01-01

    In Arabidopsis (Arabidopsis thaliana), a number of defense-related metabolites are synthesized via indole-3-acetonitrile (IAN), including camalexin and indole-3-carboxylic acid (ICOOH) derivatives. Cytochrome P450 71A13 (CYP71A13) is a key enzyme for camalexin biosynthesis and catalyzes the conversion of indole-3-acetaldoxime (IAOx) to IAN. The CYP71A13 gene is located in tandem with its close homolog CYP71A12, also encoding an IAOx dehydratase. However, for CYP71A12, indole-3-carbaldehyde and cyanide were identified as major reaction products. To clarify CYP71A12 function in vivo and to better understand IAN metabolism, we generated two cyp71a12 cyp71a13 double knockout mutant lines. CYP71A12-specific transcription activator-like effector nucleases were introduced into the cyp71a13 background, and very efficient somatic mutagenesis was achieved. We observed stable transmission of the cyp71a12 mutation to the following generations, which is a major challenge for targeted mutagenesis in Arabidopsis. In contrast to cyp71a13 plants, in which camalexin accumulation is partially reduced, double mutants synthesized only traces of camalexin, demonstrating that CYP71A12 contributes to camalexin biosynthesis in leaf tissue. A major role of CYP71A12 was identified for the inducible biosynthesis of ICOOH. Specifically, the ICOOH methyl ester was reduced to 12% of the wild-type level in AgNO3-challenged cyp71a12 leaves. In contrast, indole-3-carbaldehyde derivatives apparently are synthesized via alternative pathways, such as the degradation of indole glucosinolates. Based on these results, we present a model for this surprisingly complex metabolic network with multiple IAN sources and channeling of IAOx-derived IAN into camalexin biosynthesis. In conclusion, transcription activator-like effector nuclease-mediated mutation is a powerful tool for functional analysis of tandem genes in secondary metabolism. PMID:25953104

  13. Stable enhanced green fluorescent protein expression after differentiation and transplantation of reporter human induced pluripotent stem cells generated by AAVS1 transcription activator-like effector nucleases.

    Science.gov (United States)

    Luo, Yongquan; Liu, Chengyu; Cerbini, Trevor; San, Hong; Lin, Yongshun; Chen, Guokai; Rao, Mahendra S; Zou, Jizhong

    2014-07-01

    Human induced pluripotent stem (hiPS) cell lines with tissue-specific or ubiquitous reporter genes are extremely useful for optimizing in vitro differentiation conditions as well as for monitoring transplanted cells in vivo. The adeno-associated virus integration site 1 (AAVS1) locus has been used as a "safe harbor" locus for inserting transgenes because of its open chromatin structure, which permits transgene expression without insertional mutagenesis. However, it is not clear whether targeted transgene expression at the AAVS1 locus is always protected from silencing when driven by various promoters, especially after differentiation and transplantation from hiPS cells. In this paper, we describe a pair of transcription activator-like effector nucleases (TALENs) that enable more efficient genome editing than the commercially available zinc finger nuclease at the AAVS1 site. Using these TALENs for targeted gene addition, we find that the cytomegalovirus-immediate early enhancer/chicken β-actin/rabbit β-globin (CAG) promoter is better than cytomegalovirus 7 and elongation factor 1α short promoters in driving strong expression of the transgene. The two independent AAVS1, CAG, and enhanced green fluorescent protein (EGFP) hiPS cell reporter lines that we have developed do not show silencing of EGFP either in undifferentiated hiPS cells or in randomly and lineage-specifically differentiated cells or in teratomas. Transplanting cardiomyocytes from an engineered AAVS1-CAG-EGFP hiPS cell line in a myocardial infarcted mouse model showed persistent expression of the transgene for at least 7 weeks in vivo. Our results show that high-efficiency targeting can be obtained with open-source TALENs and that careful optimization of the reporter and transgene constructs results in stable and persistent expression in vitro and in vivo. ©AlphaMed Press.

  14. Genome wide DNA methylation analysis of Haloferax volcanii H26 and identification of DNA methyltransferase related PD-(D/EXK nuclease family protein HVO_A0006

    Directory of Open Access Journals (Sweden)

    Matthew eOuellette

    2015-04-01

    Full Text Available Restriction-modification (RM systems have evolved to protect the cell from invading DNAs and are composed of two enzymes: a DNA methyltransferase and a restriction endonuclease. Although RM systems are present in both archaeal and bacterial genomes, DNA methylation in archaea has not been well defined. In order to characterize the function of RM systems in archaeal species, we have made use of the model haloarchaeon Haloferax volcanii. A genomic DNA methylation analysis of H. volcanii strain H26 was performed using PacBio single molecule real-time (SMRT sequencing. This analysis was also performed on a strain of H. volcanii in which an annotated DNA methyltransferase gene HVO_A0006 was deleted from the genome. Sequence analysis of H26 revealed two motifs which are modified in the genome: Cm4TAG and GCAm6BN6VTGC. Analysis of the ∆HVO_A0006 strain indicated that it exhibited reduced adenine methylation compared to the parental strain and altered the detected adenine motif. However, protein domain architecture analysis and amino acid alignments revealed that HVO_A0006 is homologous only to the N-terminal endonuclease region of Type IIG RM proteins and contains a PD-(D/EXK nuclease motif, suggesting that HVO_A0006 is a PD-(D/EXK nuclease family protein. Further bioinformatic analysis of the HVO_A0006 gene demonstrating that the gene is rare among the Halobacteria. It is surrounded by two transposition genes suggesting that HVO_A0006 is a fragment of a Type IIG RM gene, which has likely been acquired through gene transfer, and affects restriction-modification activity by interacting with another RM system component(s. Here, we present the first genome-wide characterization of DNA methylation in an archaeal species and examine the function of a DNA methyltransferase related gene HVO_A0006.

  15. mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5.

    Science.gov (United States)

    Mock, Ulrike; Machowicz, Rafał; Hauber, Ilona; Horn, Stefan; Abramowski, Pierre; Berdien, Belinda; Hauber, Joachim; Fehse, Boris

    2015-06-23

    Homozygosity for a natural deletion variant of the HIV-coreceptor molecule CCR5, CCR5Δ32, confers resistance toward HIV infection. Allogeneic stem cell transplantation from a CCR5Δ32-homozygous donor has resulted in the first cure from HIV ('Berlin patient'). Based thereon, genetic disruption of CCR5 using designer nucleases was proposed as a promising HIV gene-therapy approach. Here we introduce a novel TAL-effector nuclease, CCR5-Uco-TALEN that can be efficiently delivered into T cells by mRNA electroporation, a gentle and truly transient gene-transfer technique. CCR5-Uco-TALEN mediated high-rate CCR5 knockout (>90% in PM1 and >50% in primary T cells) combined with low off-target activity, as assessed by flow cytometry, next-generation sequencing and a newly devised, very convenient gene-editing frequency digital-PCR (GEF-dPCR). GEF-dPCR facilitates simultaneous detection of wild-type and gene-edited alleles with remarkable sensitivity and accuracy as shown for the CCR5 on-target and CCR2 off-target loci. CCR5-edited cells were protected from infection with HIV-derived lentiviral vectors, but also with the wild-type CCR5-tropic HIV-1BaL strain. Long-term exposure to HIV-1BaL resulted in almost complete suppression of viral replication and selection of CCR5-gene edited T cells. In conclusion, we have developed a novel TALEN for the targeted, high-efficiency knockout of CCR5 and a useful dPCR-based gene-editing detection method. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE-Mediated Generation and Metabolic Analysis of Camalexin-Deficient cyp71a12 cyp71a13 Double Knockout Lines.

    Science.gov (United States)

    Müller, Teresa M; Böttcher, Christoph; Morbitzer, Robert; Götz, Cornelia C; Lehmann, Johannes; Lahaye, Thomas; Glawischnig, Erich

    2015-07-01

    In Arabidopsis (Arabidopsis thaliana), a number of defense-related metabolites are synthesized via indole-3-acetonitrile (IAN), including camalexin and indole-3-carboxylic acid (ICOOH) derivatives. Cytochrome P450 71A13 (CYP71A13) is a key enzyme for camalexin biosynthesis and catalyzes the conversion of indole-3-acetaldoxime (IAOx) to IAN. The CYP71A13 gene is located in tandem with its close homolog CYP71A12, also encoding an IAOx dehydratase. However, for CYP71A12, indole-3-carbaldehyde and cyanide were identified as major reaction products. To clarify CYP71A12 function in vivo and to better understand IAN metabolism, we generated two cyp71a12 cyp71a13 double knockout mutant lines. CYP71A12-specific transcription activator-like effector nucleases were introduced into the cyp71a13 background, and very efficient somatic mutagenesis was achieved. We observed stable transmission of the cyp71a12 mutation to the following generations, which is a major challenge for targeted mutagenesis in Arabidopsis. In contrast to cyp71a13 plants, in which camalexin accumulation is partially reduced, double mutants synthesized only traces of camalexin, demonstrating that CYP71A12 contributes to camalexin biosynthesis in leaf tissue. A major role of CYP71A12 was identified for the inducible biosynthesis of ICOOH. Specifically, the ICOOH methyl ester was reduced to 12% of the wild-type level in AgNO3-challenged cyp71a12 leaves. In contrast, indole-3-carbaldehyde derivatives apparently are synthesized via alternative pathways, such as the degradation of indole glucosinolates. Based on these results, we present a model for this surprisingly complex metabolic network with multiple IAN sources and channeling of IAOx-derived IAN into camalexin biosynthesis. In conclusion, transcription activator-like effector nuclease-mediated mutation is a powerful tool for functional analysis of tandem genes in secondary metabolism. © 2015 American Society of Plant Biologists. All Rights Reserved.

  17. Crystallization and preliminary crystallographic analysis of an Escherichia coli-selected mutant of the nuclease domain of the metallonuclease colicin E7

    DEFF Research Database (Denmark)

    Czene, Aniko; Toth, Eszter; Gyurcsik, Bela

    2013-01-01

    The metallonuclease colicin E7 is a member of the HNH family of endonucleases. It serves as a bacterial toxin in Escherichia coli, protecting the host cell from other related bacteria and bacteriophages by degradation of their chromosomal DNA under environmental stress. Its cell-killing activity ....... X-ray diffraction data were collected to 1.6 Å resolution and could be indexed and averaged in the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 55.4, c = 73.1 Å. Structure determination by molecular replacement is in progress.......The metallonuclease colicin E7 is a member of the HNH family of endonucleases. It serves as a bacterial toxin in Escherichia coli, protecting the host cell from other related bacteria and bacteriophages by degradation of their chromosomal DNA under environmental stress. Its cell-killing activity...... is attributed to the nonspecific nuclease domain (NColE7), which possesses the catalytic ββα-type metal ion-binding HNH motif at its C-terminus. Mutations affecting the positively charged amino acids at the N-terminus of NColE7 (444-576) surprisingly showed no or significantly reduced endonuclease activity...

  18. Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells*♦

    Science.gov (United States)

    Canver, Matthew C.; Bauer, Daniel E.; Dass, Abhishek; Yien, Yvette Y.; Chung, Jacky; Masuda, Takeshi; Maeda, Takahiro; Paw, Barry H.; Orkin, Stuart H.

    2014-01-01

    The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements. PMID:24907273

  19. In Vivo Zinc Finger Nuclease-mediated Targeted Integration of a Glucose-6-phosphatase Transgene Promotes Survival in Mice With Glycogen Storage Disease Type IA.

    Science.gov (United States)

    Landau, Dustin J; Brooks, Elizabeth Drake; Perez-Pinera, Pablo; Amarasekara, Hiruni; Mefferd, Adam; Li, Songtao; Bird, Andrew; Gersbach, Charles A; Koeberl, Dwight D

    2016-04-01

    Glycogen storage disease type Ia (GSD Ia) is caused by glucose-6-phosphatase (G6Pase) deficiency in association with severe, life-threatening hypoglycemia that necessitates lifelong dietary therapy. Here we show that use of a zinc-finger nuclease (ZFN) targeted to the ROSA26 safe harbor locus and a ROSA26-targeting vector containing a G6PC donor transgene, both delivered with adeno-associated virus (AAV) vectors, markedly improved survival of G6Pase knockout (G6Pase-KO) mice compared with mice receiving the donor vector alone (P < 0.04). Furthermore, transgene integration has been confirmed by sequencing in the majority of the mice treated with both vectors. Targeted alleles were 4.6-fold more common in livers of mice with GSD Ia, as compared with normal littermates, at 8 months following vector administration (P < 0.02). This suggests a selective advantage for vector-transduced hepatocytes following ZFN-mediated integration of the G6Pase vector. A short-term experiment also showed that 3-month-old mice receiving the ZFN had significantly-improved biochemical correction, in comparison with mice that received the donor vector alone. These data suggest that the use of ZFNs to drive integration of G6Pase at a safe harbor locus might improve vector persistence and efficacy, and lower mortality in GSD Ia.

  20. Clinical Scale Zinc Finger Nuclease-mediated Gene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatment of Metastatic Melanoma.

    Science.gov (United States)

    Beane, Joal D; Lee, Gary; Zheng, Zhili; Mendel, Matthew; Abate-Daga, Daniel; Bharathan, Mini; Black, Mary; Gandhi, Nimisha; Yu, Zhiya; Chandran, Smita; Giedlin, Martin; Ando, Dale; Miller, Jeff; Paschon, David; Guschin, Dmitry; Rebar, Edward J; Reik, Andreas; Holmes, Michael C; Gregory, Philip D; Restifo, Nicholas P; Rosenberg, Steven A; Morgan, Richard A; Feldman, Steven A

    2015-08-01

    Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9-84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500-2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted.

  1. Antioxidant, DNA binding and nuclease activities of heteroleptic copper(II) complexes derived from 2-((2-(piperazin-1-yl)ethylimino)methyl)-4-substituted phenols and diimines

    Science.gov (United States)

    Ravichandran, J.; Gurumoorthy, P.; Imran Musthafa, M. A.; Kalilur Rahiman, A.

    2014-12-01

    A series of heteroleptic copper(II) complexes of the type [CuL1-4(diimine)](ClO4)2 (1-8) [L1-4 = 2-((2-(piperazin-1-yl)ethylimino)methyl)-4-substituted phenols, and diimine = 2,2‧-bipyridyl (bpy) or 1,10-phenanthroline (phen)], have been synthesized and characterized by spectroscopic methods. The IR spectra of complexes indicate the presence of uncoordinated perchlorate anions and the electronic spectra revealed the square pyramidal geometry with N4O coordination environment around copper(II) nuclei. Electrochemical studies of the mononuclear complexes evidenced one-electron irreversible reduction wave in the cathodic region. The EPR spectra of complexes with g|| (2.206-2.214) and A|| (154-172 × 10-4 cm-1) values support the square-based CuN3O coordination chromophore and the presence of unpaired electron localized in dx-y ground state. Antioxidant studies against DPPH revealed effective radical scavenging properties of the synthesized complexes. Binding studies suggest that the heteroleptic copper(II) complexes interact with calf thymus DNA (CT-DNA) through minor-groove and electrostatic interaction, and all the complexes display pronounced nuclease activity against supercoiled pBR322 DNA.

  2. Characterization of genomic deletion efficiency mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells.

    Science.gov (United States)

    Canver, Matthew C; Bauer, Daniel E; Dass, Abhishek; Yien, Yvette Y; Chung, Jacky; Masuda, Takeshi; Maeda, Takahiro; Paw, Barry H; Orkin, Stuart H

    2014-08-01

    The clustered regularly interspaced short [corrected] palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3).

    Science.gov (United States)

    Mulepati, Sabin; Bailey, Scott

    2011-09-09

    RNA transcribed from clustered regularly interspaced short palindromic repeats (CRISPRs) protects many prokaryotes from invasion by foreign DNA such as viruses, conjugative plasmids, and transposable elements. Cas3 (CRISPR-associated protein 3) is essential for this CRISPR protection and is thought to mediate cleavage of the foreign DNA through its N-terminal histidine-aspartate (HD) domain. We report here the 1.8 Å crystal structure of the HD domain of Cas3 from Thermus thermophilus HB8. Structural and biochemical studies predict that this enzyme binds two metal ions at its active site. We also demonstrate that the single-stranded DNA endonuclease activity of this T. thermophilus domain is activated not by magnesium but by transition metal ions such as manganese and nickel. Structure-guided mutagenesis confirms the importance of the metal-binding residues for the nuclease activity and identifies other active site residues. Overall, these results provide a framework for understanding the role of Cas3 in the CRISPR system.

  4. Mapping of Complete Set of Ribose and Base Modifications of Yeast rRNA by RP-HPLC and Mung Bean Nuclease Assay.

    Directory of Open Access Journals (Sweden)

    Jun Yang

    Full Text Available Ribosomes are large ribonucleoprotein complexes that are fundamental for protein synthesis. Ribosomes are ribozymes because their catalytic functions such as peptidyl transferase and peptidyl-tRNA hydrolysis depend on the rRNA. rRNA is a heterogeneous biopolymer comprising of at least 112 chemically modified residues that are believed to expand its topological potential. In the present study, we established a comprehensive modification profile of Saccharomyces cerevisiae's 18S and 25S rRNA using a high resolution Reversed-Phase High Performance Liquid Chromatography (RP-HPLC. A combination of mung bean nuclease assay, rDNA point mutants and snoRNA deletions allowed us to systematically map all ribose and base modifications on both rRNAs to a single nucleotide resolution. We also calculated approximate molar levels for each modification using their UV (254nm molar response factors, showing sub-stoichiometric amount of modifications at certain residues. The chemical nature, their precise location and identification of partial modification will facilitate understanding the precise role of these chemical modifications, and provide further evidence for ribosome heterogeneity in eukaryotes.

  5. Seamless Requirements

    OpenAIRE

    Naumchev, Alexandr; Meyer, Bertrand

    2017-01-01

    Popular notations for functional requirements specifications frequently ignore developers' needs, target specific development models, or require translation of requirements into tests for verification; the results can give out-of-sync or downright incompatible artifacts. Seamless Requirements, a new approach to specifying functional requirements, contributes to developers' understanding of requirements and to software quality regardless of the process, while the process itself becomes lighter...

  6. Cofactor requirement of HpyAV restriction endonuclease.

    Directory of Open Access Journals (Sweden)

    Siu-Hong Chan

    Full Text Available BACKGROUND: Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M systems in microorganisms. PRINCIPAL FINDINGS: We have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg(++. The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity. CONCLUSIONS/SIGNIFICANCE: Some HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms.

  7. Cofactor requirement of HpyAV restriction endonuclease.

    Science.gov (United States)

    Chan, Siu-Hong; Opitz, Lars; Higgins, Lauren; O'loane, Diana; Xu, Shuang-Yong

    2010-02-05

    Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M) systems in microorganisms. We have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg(++). The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity. Some HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms.

  8. Zinc finger nuclease-expressing baculoviral vectors mediate targeted genome integration of reprogramming factor genes to facilitate the generation of human induced pluripotent stem cells.

    Science.gov (United States)

    Phang, Rui-Zhe; Tay, Felix Chang; Goh, Sal-Lee; Lau, Cia-Hin; Zhu, Haibao; Tan, Wee-Kiat; Liang, Qingle; Chen, Can; Du, Shouhui; Li, Zhendong; Tay, Johan Chin-Kang; Wu, Chunxiao; Zeng, Jieming; Fan, Weimin; Toh, Han Chong; Wang, Shu

    2013-12-01

    Integrative gene transfer using retroviruses to express reprogramming factors displays high efficiency in generating induced pluripotent stem cells (iPSCs), but the value of the method is limited because of the concern over mutagenesis associated with random insertion of transgenes. Site-specific integration into a preselected locus by engineered zinc-finger nuclease (ZFN) technology provides a potential way to overcome the problem. Here, we report the successful reprogramming of human fibroblasts into a state of pluripotency by baculoviral transduction-mediated, site-specific integration of OKSM (Oct3/4, Klf4, Sox2, and c-myc) transcription factor genes into the AAVS1 locus in human chromosome 19. Two nonintegrative baculoviral vectors were used for cotransduction, one expressing ZFNs and another as a donor vector encoding the four transcription factors. iPSC colonies were obtained at a high efficiency of 12% (the mean value of eight individual experiments). All characterized iPSC clones carried the transgenic cassette only at the ZFN-specified AAVS1 locus. We further demonstrated that when the donor cassette was flanked by heterospecific loxP sequences, the reprogramming genes in iPSCs could be replaced by another transgene using a baculoviral vector-based Cre recombinase-mediated cassette exchange system, thereby producing iPSCs free of exogenous reprogramming factors. Although the use of nonintegrating methods to generate iPSCs is rapidly becoming a standard approach, methods based on site-specific integration of reprogramming factor genes as reported here hold the potential for efficient generation of genetically amenable iPSCs suitable for future gene therapy applications.

  9. [In vitro study of joint intervention of E-cad and Bmi-1 mediated by transcription activator-like effector nuclease in nasopharyngeal carcinoma].

    Science.gov (United States)

    Luo, Tingting; Yan, Aifen; Liu, Lian; Jiang, Hong; Feng, Cuilan; Liu, Guannan; Liu, Fang; Tang, Dongsheng; Zhou, Tianhong

    2018-03-28

    To explore the effect of intervention of E-cadherin (E-cad) and B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) mediated by transcription activator-like effector nuclease (TALEN) on the biological behaviors of nasopharyngeal carcinoma cells.
 Methods: Multi-locus gene targeting vectors pUC-DS1-CMV-E-cad-2A-Neo-DS2 and pUC-DS1-Bmi-1 shRNA-Zeo-DS2 were constructed, and the E-cad and Bmi-1 targeting vectors were transferred with TALEN plasmids to CNE-2 cells individually or simultaneously. The integration of target genes were detected by PCR, the expressions of E-cad and Bmi-1 were detected by Western blot. The changes of cell proliferation were detected by cell counting kit-8 (CCK-8) assay. The cell cycle and apoptosis were detected by flow cytometry. The cell migration and invasion were detected by Transwell assay.
 Results: The E-cad and Bmi-1 shRNA expression elements were successfully integrated into the genome of CNE-2 cells, the protein expression level of E-cad was up-regulated, and the protein expression level of Bmi-1 was down-regulated. The intervention of E-cad and Bmi-1 didn't affect the proliferation, cell cycle and apoptosis of CNE-2 cells, but it significantly inhibited the migration and invasion ability of CNE-2 cells. Furthermore, the intervention of E-cad and Bmi-1 together significantly inhibited the migration ability of nasopharyngeal carcinoma cells compared with the intervention of E-cad or Bmi-1 alone (all Pcad and Bmi-1 mediated by TALEN can effectively inhibit the migration and invasion of nasopharyngeal carcinoma cells in vitro, which may lay the preliminary experimental basis for gene therapy of human cancer.

  10. Editing of the Luteinizing Hormone Gene to Sterilize Channel Catfish, Ictalurus punctatus, Using a Modified Zinc Finger Nuclease Technology with Electroporation.

    Science.gov (United States)

    Qin, Zhenkui; Li, Yun; Su, Baofeng; Cheng, Qi; Ye, Zhi; Perera, Dayan A; Fobes, Michael; Shang, Mei; Dunham, Rex A

    2016-04-01

    Channel catfish (Ictalurus punctatus) is the most important freshwater aquaculture species in the USA. Genetically enhanced fish that are sterile could both profit the catfish industry and reduce potential environmental and ecological risks. As the first step to generate sterile channel catfish, three sets of zinc finger nuclease (ZFN) plasmids targeting the luteinizing hormone (LH) gene were designed and electroporated into one-cell embryos, different concentrations were introduced, and the Cel-I assay was conducted to detect mutations. Channel catfish carrying the mutated LH gene were sterile, as confirmed by DNA sequencing and mating experiments. The overall mutation rate was 19.7 % for 66 channel catfish, and the best treatment was ZFN set 1 at the concentration 25 μg/ml. To our knowledge, this is the first instance of gene editing of fish via plasmid introduction instead of mRNA microinjection. The introduction of the ZFN plasmids may have reduced mosaicism, as mutated individuals were gene edited in every tissue evaluated. Apparently, the plasmids were eventually degraded without integration, as they were not detectable in mutated individuals using PCR. Carp pituitary extract failed to induce spawning and restoration of fertility, indicating the need for developing other hormone therapies to achieve reversal of sterility upon demand. This is the first sterilization achieved using ZFN technology in an aquaculture species and the first successful gene editing of channel catfish. Our results will help understand the roles of the LH gene, purposeful sterilization of teleost fishes, and is a step towards control of domestic, hybrid, exotic, invasive, and transgenic fishes.

  11. Synthesis, structural elucidation, microbial, antioxidant and nuclease activities of some novel divalent M(II complexes derived from 5-fluorouracil and l-tyrosine

    Directory of Open Access Journals (Sweden)

    Jeyaprakash Dharmaraja

    2017-01-01

    Full Text Available Novel N2O2 sequence of mononuclear amino acid metal(II complexes (1a–1e was synthesized from 5-fluorouracil (5-FU: A and l-tyrosine (tyr: B with Mn(II, Co(II, Ni(II, Cu(II and Zn(II ions. The synthesized complexes were structurally characterized by analytical, spectral (FT-IR, UV–vis, 1H NMR, FAB-Mass, TGA/DTA and EPR as well as molar conductance and magnetic studies. From spectral studies, both the ligands act as bidentate and they bind metal(II ions through deprotonated-N3 and C4O atoms and amino-N and deprotonated carboxylato-O atoms, respectively, to form a stable metal chelate. The observed low molar conductance values suggest a non-electrolytic nature. Calculated g tensor values of Cu(II complex (1d at 77 and 300 K confirm their geometry. Thermal behavior of metal(II complexes (1a–1c shows loss of coordinated water molecules in the first step followed by decomposition of ligand moieties in a respective manner and leads to form air stable metal oxide as final residues. Powder X-ray diffraction and SEM studies illustrate that all the complexes have uniform microcrystalline with homogenous morphology. Mn(II, Ni(II and Cu(II complexes show significant in vitro antimicrobial and antioxidant activities than 5-fluorouracil(A. Moreover, the nuclease studies of Ni(II and Cu(II complexes (1c and 1d show considerable DNA binding and oxidative DNA cleavage activities than other complexes.

  12. Global analysis of double-strand break processing reveals in vivo properties of the helicase-nuclease complex AddAB.

    Science.gov (United States)

    Badrinarayanan, Anjana; Le, Tung B K; Spille, Jan-Hendrik; Cisse, Ibrahim I; Laub, Michael T

    2017-05-01

    In bacteria, double-strand break (DSB) repair via homologous recombination is thought to be initiated through the bi-directional degradation and resection of DNA ends by a helicase-nuclease complex such as AddAB. The activity of AddAB has been well-studied in vitro, with translocation speeds between 400-2000 bp/s on linear DNA suggesting that a large section of DNA around a break site is processed for repair. However, the translocation rate and activity of AddAB in vivo is not known, and how AddAB is regulated to prevent excessive DNA degradation around a break site is unclear. To examine the functions and mechanistic regulation of AddAB inside bacterial cells, we developed a next-generation sequencing-based approach to assay DNA processing after a site-specific DSB was introduced on the chromosome of Caulobacter crescentus. Using this assay we determined the in vivo rates of DSB processing by AddAB and found that putative chi sites attenuate processing in a RecA-dependent manner. This RecA-mediated regulation of AddAB prevents the excessive loss of DNA around a break site, limiting the effects of DSB processing on transcription. In sum, our results, taken together with prior studies, support a mechanism for regulating AddAB that couples two key events of DSB repair-the attenuation of DNA-end processing and the initiation of homology search by RecA-thereby helping to ensure that genomic integrity is maintained during DSB repair.

  13. Lateral flow nucleic acid biosensor for sensitive detection of microRNAs based on the dual amplification strategy of duplex-specific nuclease and hybridization chain reaction.

    Directory of Open Access Journals (Sweden)

    Na Ying

    Full Text Available MicroRNAs (miRNAs constitute novel biomarkers for various diseases. Accurate and quantitative analysis of miRNA expression is critical for biomedical research and clinical theranostics. In this study, a method was developed for sensitive and specific detection of miRNAs via dual signal amplification based on duplex specific nuclease (DSN and hybridization chain reaction (HCR. A reporter probe (RP, comprising recognition sequence (3' end modified with biotin for a target miRNA of miR-21 and capture sequence (5' end modified with Fam for HCR product, was designed and synthesized. HCR was initiated by partial sequence of initiator probe (IP, the other part of which can hybridize with capture sequence of RP, and was assembled by hairpin probes modified with biotin (H1-bio and H2-bio. A miR-21 triggered cyclical DSN cleavage of RP, which was immobilized to a streptavidin (SA coated magnetic bead (MB. The released Fam labeled capture sequence then hybridized with the HCR product to generate a detectable dsDNA. This polymer was then dropped on lateral flow strip and positive result was observed. The proposed method allowed quantitative sequence-specific detection of miR-21 (with a detection limit of 2.1 fM, S/N = 3 in a dynamic range from 100 fM to 100 pM, with an excellent ability to discriminate differences in miRNAs. The method showed acceptable testing recoveries for the determination of miRNAs in serum.

  14. Bombyx mori DNA/RNA non-specific nuclease: expression of isoforms in insect culture cells, subcellular localization and functional assays.

    Science.gov (United States)

    Liu, Jisheng; Swevers, Luc; Iatrou, Kostas; Huvenne, Hanneke; Smagghe, Guy

    2012-08-01

    A DNA/RNA non-specific alkaline nuclease (BmdsRNase) was isolated from the digestive juice of Bombyx mori. While originally reported to be produced by the midgut only, in this project it was found that the mRNA of this enzyme was also expressed in the epidermis, fat body, gut, thoracic muscles, Malpighian tubules, brain, and silk glands of 5th instar larvae, indicating additional functions to its reported role in nucleic acid digestion in the midgut. In order to study the functional properties of BmdsRNase, three pEA-BmdsRNase expression constructs were generated, characterized by presence or absence of a signal peptide and a propeptide, and used for expression in lepidopteran Hi5 tissue culture cells. Western blot indicated that these different forms of BmdsRNase protein were not secreted into the growth medium, while they were detected in the pellets and supernatants of Hi5 cell extracts. Nucleic acids cleavage experiments indicated that full-length BmdsRNase could digest dsRNA and that the processed form (absence of signal peptide and propeptide) of BmdsRNase could degrade both DNA and dsRNA in Hi5 cell culture. Using a reporter assay targeted by transfected homologous dsRNA, it was shown that the digestive property of the processed form could interfere with the RNAi response. Immunostaining of processed BmdsRNase protein showed asymmetric localization in the cellular cytoplasm and co-localization with Flag-tagged Dicer-2 was also observed. In conclusion, our in vitro studies indicated that intracellular protein isoforms of BmdsRNase can be functional and involved in the regulation of nucleic acid metabolism in the cytoplasm. In particular, because of its propensity to degrade dsRNA, the enzyme might be involved in the innate immune response against invading nucleic acids such as RNA viruses. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Gene editing using a zinc-finger nuclease mimicking the CCR5Δ32 mutation induces resistance to CCR5-using HIV-1.

    Science.gov (United States)

    Badia, Roger; Riveira-Muñoz, Eva; Clotet, Bonaventura; Esté, José A; Ballana, Ester

    2014-07-01

    To characterize a new zinc-finger nuclease (ZFN) that targets close to the sequence of the 32 bp deletion polymorphism in the CCR5 gene, and to generate cells resistant to HIV-1 strains that use CCR5. CCR5Δ32 is a naturally occurring deletion that provides genetic resistance to R5-tropic HIV-1. The specificity and efficacy of a newly identified target for CCR5 gene editing, near the CCR5Δ32 sequence (ZFNCCR5Δ32), was assessed as well as its ability to generate cells resistant to HIV infection with reduced off-target effects. ZFNCCR5Δ32 activity was evaluated by heteroduplex formation in human K562 cells. Assessment of ZFNCCR5Δ32 specificity was analysed in silico. The yield of ZFNCCR5Δ32 in cell culture was improved by fluorescence-activated cell sorting, and the anti-HIV potency of ZFNCCR5Δ32 was measured in vitro in TZM-bl cells against HIV-1 strains. ZFNCCR5Δ32 effectively recognized the CCR5Δ32 region, inducing a frameshift of the CCR5 coding region that resulted in the complete absence of CCR5 expression of mRNA and of protein at the cell surface. CCR5 knockout cells were refractory to HIV-1 infection by the R5-using strain BaL. Unlike previous CCR5 ZFN studies, the new ZFN has no detectable off-target activity. ZFNCCR5Δ32 is a specific and efficient tool for the generation of CCR5 knockouts. Its ability to mimic the natural CCR5Δ32 phenotype in the absence of relevant off-site cutting events suggests that ZFNCCR5Δ32 might be safe in clinical research. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. The 3'-end region of the human PDGFR-β core promoter nuclease hypersensitive element forms a mixture of two unique end-insertion G-quadruplexes.

    Science.gov (United States)

    Onel, Buket; Carver, Megan; Agrawal, Prashansa; Hurley, Laurence H; Yang, Danzhou

    2018-04-01

    While the most stable G-quadruplex formed in the human PDGFR-β promoter nuclease hypersensitive element (NHE) is the 5'-mid G-quadruplex, the 3'-end sequence that contains a 3'-GGA run forms a less stable G-quadruplex. Recently, the 3'-end G-quadruplex was found to be a transcriptional repressor and can be selectively targeted by a small molecule for PDGFR-β downregulation. We use 1D and 2D high-field NMR, in combination with Dimethylsulfate Footprinting, Circular Dichroism Spectroscopy, and Electrophoretic Mobility Shift Assay. We determine that the PDGFR-β extended 3'-end NHE sequence forms two novel end-insertion intramolecular G-quadruplexes that co-exist in equilibrium under physiological salt conditions. One G-quadruplex has a 3'-non-adjacent flanking guanine inserted into the 3'-external tetrad (3'-insertion-G4), and another has a 5'-non-adjacent flanking guanine inserted into the 5'-external tetrad (5'-insertion-G4). The two guanines in the GGA-run move up or down within the G-quadruplex to accommodate the inserted guanine. Each end-insertion G-quadruplex has a low thermal stability as compared to the 5'-mid G-quadruplex, but the selective stabilization of GSA1129 shifts the equilibrium toward the 3'-end G-quadruplex in the PDGFR-β NHE. An equilibrium mixture of two unique end-insertion intramolecular G-quadruplexes forms in the PDGFR-β NHE 3'-end sequence that contains a GGA-run and non-adjacent guanines in both the 3'- and 5'- flanking segments; the novel end-insertion structures of the 3'-end G-quadruplex are selectively stabilized by GSA1129. We show for the first time that an equilibrium mixture of two unusual end-insertion G-quadruplexes forms in a native promoter sequence and appears to be the molecular recognition for PDGFR-β downregulation. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Modeling of the human alveolar rhabdomyosarcoma Pax3-Foxo1 chromosome translocation in mouse myoblasts using CRISPR-Cas9 nuclease.

    Directory of Open Access Journals (Sweden)

    Irina V Lagutina

    Full Text Available Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13(q36.1;q14.1 in human alveolar rhabdomyosarcoma (A-RMS creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation. To circumvent this problem, we irreversibly inverted the orientation of a 4.9 Mb syntenic fragment on chromosome 3, encompassing Foxo1, by using Cre-mediated recombination of two pairs of unrelated oppositely oriented LoxP sites situated at the borders of the syntenic region. We tested if spatial proximity of the Pax3 and Foxo1 loci in myoblasts of mice homozygous for the inversion facilitated Pax3-Foxo1 fusion gene formation upon induction of targeted CRISPR-Cas9 nuclease-induced DNA double strand breaks in Pax3 and Foxo1. Fluorescent in situ hybridization indicated that fore limb myoblasts show a higher frequency of Pax3/Foxo1 co-localization than hind limb myoblasts. Indeed, more fusion genes were generated in fore limb myoblasts via a reciprocal t(1;3, which expressed correctly spliced Pax3-Foxo1 mRNA encoding Pax3-Foxo1 fusion protein. We conclude that locus proximity facilitates chromosome translocation upon induction of DNA double strand breaks. Given that the Pax3-Foxo1 fusion gene will contain all the regulatory sequences necessary for precise regulation of its expression, we propose that CRISPR-Cas9 provides a novel means to faithfully model human diseases caused by chromosome translocation in mice.

  18. NM23-H2 may play an indirect role in transcriptional activation of c-myc gene expression but does not cleave the nuclease hypersensitive element III1

    International Nuclear Information System (INIS)

    Dexheimer, Thomas S.; Carey, Steven S.; Zuohe, Song; Gokhale, Vijay M.; Hu, Xiaohui; Murata, Lauren B.; Maes, Estelle M.; Weichsel, Andrzej; Sun, Daekyu; Meuillet, Emmanuelle J.; Montfort, William R.; Hurley, Laurence H.

    2009-01-01

    The formation of G-quadruplex structures within the nuclease hypersensitive element (NHE) III 1 region of the c-myc promoter and the ability of these structures to repress c-myc transcription have been well established. However, just how these extremely stable DNA secondary structures are transformed to activate c-myc transcription is still unknown. NM23-H2/nucleoside diphosphate kinase B has been recognized as an activator of c-myc transcription via interactions with the NHE III 1 region of the c-myc gene promoter. Through the use of RNA interference, we confirmed the transcriptional regulatory role of NM23-H2. In addition, we find that further purification of NM23-H2 results in loss of the previously identified DNA strand cleavage activity, but retention of its DNA binding activity. NM23-H2 binds to both single-stranded guanine- and cytosine-rich strands of the c-myc NHE III 1 and, to a lesser extent, to a random single-stranded DNA template. However, it does not bind to or cleave the NHE III 1 in duplex form. Significantly, potassium ions and compounds that stabilize the G-quadruplex and i-motif structures have an inhibitory effect on NM23-H2 DNA-binding activity. Mutation of Arg 88 to Ala 88 (R88A) reduced both DNA and nucleotide binding but had minimal effect on the NM23-H2 crystal structure. On the basis of these data and molecular modeling studies, we have proposed a stepwise trapping-out of the NHE III 1 region in a single-stranded form, thus allowing single-stranded transcription factors to bind and activate c-myc transcription. Furthermore, this model provides a rationale for how the stabilization of the G-quadruplex or i-motif structures formed within the c-myc gene promoter region can inhibit NM23-H2 from activating c-myc gene expression.

  19. Copper complexes as chemical nucleases

    Indian Academy of Sciences (India)

    Unknown

    hydrogen are as well expected to oxidize guanine or other nucleobases. Current efforts are on to ..... recording the absorption intensity at 260 nm with a molar extinction coefficient value. 6600 dm3 mol–1 cm–1 37. ... (H2A, 100 µM) followed by dilution with the Tris-HCl buffer to a total volume of 20 µL. The samples were ...

  20. Requirements Engineering

    CERN Document Server

    Hull, Elizabeth; Dick, Jeremy

    2011-01-01

    Written for those who want to develop their knowledge of requirements engineering process, whether practitioners or students.Using the latest research and driven by practical experience from industry, Requirements Engineering gives useful hints to practitioners on how to write and structure requirements. It explains the importance of Systems Engineering and the creation of effective solutions to problems. It describes the underlying representations used in system modeling and introduces the UML2, and considers the relationship between requirements and modeling. Covering a generic multi-layer r

  1. Software requirements

    CERN Document Server

    Wiegers, Karl E

    2003-01-01

    Without formal, verifiable software requirements-and an effective system for managing them-the programs that developers think they've agreed to build often will not be the same products their customers are expecting. In SOFTWARE REQUIREMENTS, Second Edition, requirements engineering authority Karl Wiegers amplifies the best practices presented in his original award-winning text?now a mainstay for anyone participating in the software development process. In this book, you'll discover effective techniques for managing the requirements engineering process all the way through the development cy

  2. Type II restriction endonuclease R.Hpy188I belongs to the GIY-YIG nuclease superfamily, but exhibits an unusual active site.

    Science.gov (United States)

    Kaminska, Katarzyna H; Kawai, Mikihiko; Boniecki, Michal; Kobayashi, Ichizo; Bujnicki, Janusz M

    2008-11-14

    a non-homologous position in sequence that nonetheless allows for spatial conservation of the guanidino group potentially involved in phosphate binding. The present study eliminates a significant "white spot" on the structural map of REases. It also provides important insight into sequence-structure-function relationships in the GIY-YIG nuclease superfamily. Our results reveal that in the case of proteins with no or few detectable homologs in the standard "non-redundant" database, it is useful to expand this database by adding the metagenomic sequences, which may provide evolutionary linkage to detect more remote homologs.

  3. Type II restriction endonuclease R.Hpy188I belongs to the GIY-YIG nuclease superfamily, but exhibits an unusual active site

    Directory of Open Access Journals (Sweden)

    Kobayashi Ichizo

    2008-11-01

    have the otherwise invariant Arg residue in a non-homologous position in sequence that nonetheless allows for spatial conservation of the guanidino group potentially involved in phosphate binding. Conclusion The present study eliminates a significant "white spot" on the structural map of REases. It also provides important insight into sequence-structure-function relationships in the GIY-YIG nuclease superfamily. Our results reveal that in the case of proteins with no or few detectable homologs in the standard "non-redundant" database, it is useful to expand this database by adding the metagenomic sequences, which may provide evolutionary linkage to detect more remote homologs.

  4. Identity of zinc finger nucleases with specificity to herpes simplex virus type II genomic DNA: novel HSV-2 vaccine/therapy precursors

    Directory of Open Access Journals (Sweden)

    Wayengera Misaki

    2011-06-01

    Full Text Available Abstract Background Herpes simplex type II (HSV-2 is a member of the family herpesviridae. Human infection with this double stranded linear DNA virus causes genital ulcerative disease and existing treatment options only serve to resolve the symptomatology (ulcers associated with active HSV-2 infection but do not eliminate latent virus. As a result, infection with HSV-2 follows a life-long relapsing (active versus latent course. On the basis of a primitive bacterium anti-phage DNA defense, the restriction modification (R-M system, we previously identified the Escherichia coli restriction enzyme (REase EcoRII as a novel peptide to excise or irreversibly disrupt latent HSV-2 DNA from infected cells. However, sequences of the site specificity palindrome of EcoRII 5'-CCWGG-3' (W = A or T are equally present within the human genome and are a potential source of host-genome toxicity. This feature has limited previous HSV-2 EcoRII based therapeutic models to microbicides only, and highlights the need to engineer artificial REases (zinc finger nucleases-ZFNs with specificity to HSV-2 genomic-DNA only. Herein, the therapeutic-potential of zinc finger arrays (ZFAs and ZFNs is identified and modeled, with unique specificity to the HSV-2 genome. Methods and results Using the whole genome of HSV-2 strain HG52 (Dolan A et al.,, and with the ZFN-consortium's CoDA-ZiFiT software pre-set at default, more than 28,000 ZFAs with specificity to HSV-2 DNA were identified. Using computational assembly (through in-silico linkage to the Flavobacterium okeanokoites endonuclease Fok I of the type IIS class, 684 ZFNs with specificity to the HSV-2 genome, were constructed. Graphic-analysis of the HSV-2 genome-cleavage pattern using the afore-identified ZFNs revealed that the highest cleavage-incidence occurred within the 30,950 base-pairs (~between the genomic context coordinates 0.80 and 1.00 at the 3' end of the HSV-2 genome. At approximately 3,095 bp before and after the

  5. Closure requirements

    International Nuclear Information System (INIS)

    Hutchinson, I.P.G.; Ellison, R.D.

    1992-01-01

    Closure of a waste management unit can be either permanent or temporary. Permanent closure may be due to: economic factors which make it uneconomical to mine the remaining minerals; depletion of mineral resources; physical site constraints that preclude further mining and beneficiation; environmental, regulatory or other requirements that make it uneconomical to continue to develop the resources. Temporary closure can occur for a period of several months to several years, and may be caused by factors such as: periods of high rainfall or snowfall which prevent mining and waste disposal; economic circumstances which temporarily make it uneconomical to mine the target mineral; labor problems requiring a cessation of operations for a period of time; construction activities that are required to upgrade project components such as the process facilities and waste management units; and mine or process plant failures that require extensive repairs. Permanent closure of a mine waste management unit involves the provision of durable surface containment features to protect the waters of the State in the long-term. Temporary closure may involve activities that range from ongoing maintenance of the existing facilities to the installation of several permanent closure features in order to reduce ongoing maintenance. This paper deals with the permanent closure features

  6. Coordinateendonucleolytic 5' and 3' trimming of terminally blocked blunt DNA double-strand break ends by Artemis nuclease and DNA-dependent protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Povirk, Lawrence; Yannone, Steven M.; Khan, Imran S.; Zhou, Rui-Zhe; Zhou, Tong; Valerie, Kristoffer; F., Lawrence

    2008-02-18

    Previous work showed that, in the presence of DNA-PK, Artemis slowly trims 3'-phosphoglycolate-terminated blunt ends. To examine the trimming reaction in more detail, long internally labeled DNA substrates were treated with Artemis. In the absence of DNA-PK, Artemis catalyzed extensive 5' {yields} 3' exonucleolytic resection of double-stranded DNA. This resection required a 5'-phosphate but did not require ATP, and was accompanied by endonucleolytic cleavage of the resulting 3' overhang. In the presence of DNA-PK, Artemis-mediated trimming was more limited, was ATP-dependent, and did not require a 5'-phosphate. For a blunt end with either a 3'-phosphoglycolate or 3'-hydroxyl terminus, endonucleolytic trimming of 2-4 nucleotides from the 3'-terminal strand was accompanied by trimming of 6 nucleotides from the 5'-terminal strand. The results suggest that autophosphorylated DNA-PK suppresses the exonuclease activity of Artemis toward blunt-ended DNA, and promotes slow and limited endonucleolytic trimming of the 5'-terminal strand, resulting in short 3' overhangs that are trimmed endonucleolytically. Thus, Artemis and DNA-PK can convert terminally blocked DNA ends of diverse geometry and chemical structure to a form suitable for polymerase mediated patching and ligation, with minimal loss of terminal sequence. Such processing could account for the very small deletions often found at DNA double-strand break repair sites.

  7. Synthesis, structures, nuclease activity, cytotoxicity, DFT and molecular docking studies of two nitrato bridged homodinuclear (Cu-Cu, Zn-Zn) complexes containing 2,2'-bipyridine and a chalcone derivative.

    Science.gov (United States)

    Gaur, Ruchi; Choubey, Diksha Kumari; Usman, Mohammad; Ward, Benzamin D; Roy, Jagat Kumar; Mishra, Lallan

    2017-08-01

    Nitrato briged dinuclear complexes of type [Cu 2 (L) 2 (bpy) 2 (NO 3 )](NO 3 )·4H 2 O, 1 and [Zn 2 (L) 2 (bpy) 2 (NO 3 )](NO 3 )·4H 2 O, 2 (L=deprotonated form of free ligand LH, [1-(2-hydroxyphenyl)-3-(9-anthracenyl) propenone; bpy=2,2'bipyridine] are synthesized and characterized using a battery of physicochemical techniques and X-ray crystallography. A distorted square pyramidal geometry is assigned to them with N 2 O 3 coordination core around the metal ion. The co-ligand L binds the metal ions through its O,O' atoms in anti-syn mode. The metal centers in complexes 1 and 2 are separated via bridging nitrato group at a distance of 6.073Å and 5.635Å respectively. Their structures and absorption spectra are supported by the computational studies using density functional theory (DFT) and TD-DFT. Both complexes exhibit nuclease activity and cleave supercoiled (form I) DNA. The complex 1 preferentially binds major groove of DNA and follows an oxidative pathway whereas complex 2 binds with minor groove of DNA via hydrolytic pathway. Both complexes inhibit topoisomerase I relaxation activity with IC 50 values of 7 and 35μM. Molecular docking studies support the groove binding and topoisomerase I binding of the complexes. The complex 1 showed a significant cytotoxicity against HeLa cell lines (a cervical cancer cell lines) in vitro with IC 50 value calculated as 2.9±0.021μM as compared to 28.2±0. 044μΜ for complex 2. Complex 2 induces the cell apoptosis at a later-stage as compared to complex 1. The cell apoptosis and topoisomerase inhibition by complexes enable them to be potential candidates as future anticancer drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Accumulation of amplified target DNAs using thiol/biotin labeling, S1 nuclease, and ferrocene–streptavidin–magnetic system and a direct detection of specific DNA signals with screen printed gold electrode

    Directory of Open Access Journals (Sweden)

    Piyasak Chaumpluk et al

    2007-01-01

    Full Text Available Combinations of PCR-based amplification platform using 5' thiolated and biotinylated specific primers, S1 nuclease–PCR products treatment, ferrocene–streptavidin (Fc–Stv–magnetic binding for DNA accumulation, and screen printed gold electrode for the DNA allocation, were applied to Hoechst 33258-induced DNA aggregation and signals induction system for direct signals detection and DNA quantification in food samples. Thiolated and biotinylated at each 5' terminus enabled DNA purification through S1 nuclease treatment for primers and non-specific DNA elimination and enabled DNA trapping with a ferrocene–streptavidin–magnetic system. This facilitated the accumulation of target DNAs at higher concentration, resulting in enhanced signals. After allocation of DNA on the surface of gold electrode via thiol binding, intensity of DNA signals through these treatments could be measured directly after being induced by Hoechst 33258. Wider amplitude changes in anodic current peaks between negative and positive samples (increasing from 3.70 to 10.10 μA compared with those applied with no treatment combinations (decreasing from 3.92 to 1.23 μA were observed. This enhancement of the signals allowed a greater efficiency of DNA quantification. When this combination was used for GMOs content estimation in reference samples, results revealed an improved accuracy from 66% to 96%. The combined biosensor system, although more costly than the standard Hoechst 33258/carbon electrode system, provided an alternative choice for DNA quantification, offering labor-free immobilization of probe onto electrode surface, easy test administration, and efficient semi-quantitative test without expensive instruments.

  9. Feed tank transfer requirements

    International Nuclear Information System (INIS)

    Freeman-Pollard, J.R.

    1998-01-01

    This document presents a definition of tank turnover; DOE responsibilities; TWRS DST permitting requirements; TWRS Authorization Basis (AB) requirements; TWRS AP Tank Farm operational requirements; unreviewed safety question (USQ) requirements; records and reporting requirements, and documentation which will require revision in support of transferring a DST in AP Tank Farm to a privatization contractor for use during Phase 1B

  10. Influence of some exo nucleases in response to the induced genetic damage in Escherichia coli by alpha radiation; Influencia de algunas exonucleasas en respuesta al dano genetico inducido en Escherichia coli por radiacion alfa

    Energy Technology Data Exchange (ETDEWEB)

    Aguilar M, M

    2005-07-01

    Within the strategies with those that E. coli counts to overcome to the genetic damage there is the SOS response, a group of genes that participate in repair and/or tolerance that it confers to the bacteria major opportunities of surviving. These genes are repressed and its only are expressed when it happens genetic damage. So that this system is activated it is necessary that DNA of a band exists and in this sense the double ruptures (RDB) its are not able to induce this response unless there is a previous processing. In stumps with defects in certain genes that have to do with repair of RDB (as recO, recJ and xonA) the activity of SOS is smaller than in a wild stump what suggests that these participate in the previous processes to the activation of the response. The ionizing radiation produce among other many lesions, RDB in greater or smaller proportion, depending on the ionization capacity. A parameter to evaluate this capacity is the lineal energy transfer (LET), defined as the average energy given by unit of distance travelled. In general the LET of the corpuscular radiations is a lot but high that of the electromagnetic one, for what produces bigger quantity of ionizations inside a restricted zone and it increases by this way the probability that RDB has been generated. This work has for object to infer the participation of xonA and recJ in this response and to evaluate the damage produced by ionizing radiation of different LET (alpha particles of different energies) in a stump with all the functional repair mechanisms. Its were considered two parameters: the survival and the activity of SOS evaluated by means of the chromo test. The results indicate that the activity of these exo nucleases is necessary for the repair of RDB as well as for the processing of lesions foresaw to the activation of SOS. As for the treatment with alphas of different energies is observed that so much the survival like the activity of SOS vary as the LET of the radiation changes

  11. NM23-H2 may play an indirect role in transcriptional activation of c-myc gene expression but does not cleave the nuclease hypersensitive element III[subscript 1

    Energy Technology Data Exchange (ETDEWEB)

    Dexheimer, Thomas S.; Carey, Steven S.; Zuohe, Song; Gokhale, Vijay M.; Hu, Xiaohui; Murata, Lauren B.; Maes, Estelle M.; Weichsel, Andrzej; Sun, Daekyu; Meuillet, Emmanuelle J.; Montfort, William R.; Hurley, Laurence H. (Ariz)

    2009-05-13

    The formation of G-quadruplex structures within the nuclease hypersensitive element (NHE) III{sub 1} region of the c-myc promoter and the ability of these structures to repress c-myc transcription have been well established. However, just how these extremely stable DNA secondary structures are transformed to activate c-myc transcription is still unknown. NM23-H2/nucleoside diphosphate kinase B has been recognized as an activator of c-myc transcription via interactions with the NHE III{sub 1} region of the c-myc gene promoter. Through the use of RNA interference, we confirmed the transcriptional regulatory role of NM23-H2. In addition, we find that further purification of NM23-H2 results in loss of the previously identified DNA strand cleavage activity, but retention of its DNA binding activity. NM23-H2 binds to both single-stranded guanine- and cytosine-rich strands of the c-myc NHE III{sub 1} and, to a lesser extent, to a random single-stranded DNA template. However, it does not bind to or cleave the NHE III{sub 1} in duplex form. Significantly, potassium ions and compounds that stabilize the G-quadruplex and i-motif structures have an inhibitory effect on NM23-H2 DNA-binding activity. Mutation of Arg{sup 88} to Ala{sup 88} (R88A) reduced both DNA and nucleotide binding but had minimal effect on the NM23-H2 crystal structure. On the basis of these data and molecular modeling studies, we have proposed a stepwise trapping-out of the NHE III{sub 1} region in a single-stranded form, thus allowing single-stranded transcription factors to bind and activate c-myc transcription. Furthermore, this model provides a rationale for how the stabilization of the G-quadruplex or i-motif structures formed within the c-myc gene promoter region can inhibit NM23-H2 from activating c-myc gene expression.

  12. Tudor Staphylococcal Nuclease (Tudor-SN), a Novel Regulator Facilitating G1/S Phase Transition, Acting as a Co-activator of E2F-1 in Cell Cycle Regulation*

    Science.gov (United States)

    Su, Chao; Zhang, Chunyan; Tecle, Adiam; Fu, Xue; He, Jinyan; Song, Juan; Zhang, Wei; Sun, Xiaoming; Ren, Yuanyuan; Silvennoinen, Olli; Yao, Zhi; Yang, Xi; Wei, Minxin; Yang, Jie

    2015-01-01

    Tudor staphylococcal nuclease (Tudor-SN) is a multifunctional protein implicated in a variety of cellular processes. In the present study, we identified Tudor-SN as a novel regulator in cell cycle. Tudor-SN was abundant in proliferating cells whereas barely expressed in terminally differentiated cells. Functional analysis indicated that ectopic overexpression of Tudor-SN promoted the G1/S transition, whereas knockdown of Tudor-SN caused G1 arrest. Moreover, the live-cell time-lapse experiment demonstrated that the cell cycle of MEF−/− (knock-out of Tudor-SN in mouse embryonic fibroblasts) was prolonged compared with wild-type MEF+/+. We noticed that Tudor-SN was constantly expressed in every cell cycle phase, but was highly phosphorylated in the G1/S border. Further study revealed that Tudor-SN was a potential substrate of Cdk2/4/6, supportively, we found the physical interaction of endogenous Tudor-SN with Cdk4/6 in G1 and the G1/S border, and with Cdk2 in the G1/S border and S phase. In addition, roscovitine (Cdk1/2/5 inhibitor) or CINK4 (Cdk4/6 inhibitor) could inhibit the phosphorylation of Tudor-SN, whereas ectopic overexpression of Cdk2/4/6 increased the Tudor-SN phosphorylation. The underlying molecular mechanisms indicated that Tudor-SN could physically interact with E2F-1 in vivo, and could enhance the physical association of E2F-1 with GCN5 (a cofactor of E2F-1, which possesses histone acetyltransferase activity), and promote the binding ability of E2F-1 to the promoter region of its target genes CYCLIN A and E2F-1, and as a result, facilitate the gene transcriptional activation. Taken together, Tudor-SN is identified as a novel co-activator of E2F-1, which could facilitate E2F-1-mediated gene transcriptional activation of target genes, which play essential roles in G1/S transition. PMID:25627688

  13. The EH-domain-containing protein Pan1 is required for normal organization of the actin cytoskeleton in Saccharomyces cerevisiae.

    Science.gov (United States)

    Tang, H Y; Cai, M

    1996-09-01

    Normal cell growth and division in the yeast Saccharomyces cerevisiae involve dramatic and frequent changes in the organization of the actin cytoskeleton. Previous studies have suggested that the reorganization of the actin cytoskeleton in accordance with cell cycle progression is controlled, directly or indirectly, by the cyclin-dependent kinase Cdc28. Here we report that by isolating rapid-death mutants in the background of the Start-deficient cdc28-4 mutation, the essential yeast gene PAN1, previously thought to encode the yeast poly(A) nuclease, is identified as a new factor required for normal organization of the actin cytoskeleton. We show that at restrictive temperature, the pan1 mutant exhibited abnormal bud growth, failed to maintain a proper distribution of the actin cytoskeleton, was unable to reorganize actin the cytoskeleton during cell cycle, and was defective in cytokinesis. The mutant also displayed a random pattern of budding even at permissive temperature. Ectopic expression of PAN1 by the GAL promoter caused abnormal distribution of the actin cytoskeleton when a single-copy vector was used. Immunofluorescence staining revealed that the Pan1 protein colocalized with the cortical actin patches, suggesting that it may be a filamentous actin-binding protein. The Pan1 protein contains an EF-hand calcium-binding domain, a putative Src homology 3 (SH3)-binding domain, a region similar to the actin cytoskeleton assembly control protein Sla1, and two repeats of a newly identified protein motif known as the EH domain. These findings suggest that Pan1, recently recognized as not responsible for the poly(A) nuclease activity (A. B. Sachs and J. A. Deardorff, erratum, Cell 83:1059, 1995; R. Boeck, S. Tarun, Jr., M. Rieger, J. A. Deardorff, S. Muller-Auer, and A. B. Sachs, J. Biol. Chem. 271:432-438, 1996), plays an important role in the organization of the actin cytoskeleton in S. cerevisiae.

  14. The construction of the zinc finger nucleases

    Indian Academy of Sciences (India)

    Michel Morange

    In three previous contributions, I tried to show how complex and tortuous had been the historical process ... First, we must describe two other lines of research and groups of researchers whose results were necessary ... precise position in the mitochondrial genome (Jacquier and. Dujon 1985). One of these endonucleases, ...

  15. Synthesis, characterisation, nuclease and cytotoxic activity of ...

    Indian Academy of Sciences (India)

    GULZAR A BHAT

    2018-02-07

    Feb 7, 2018 ... tumor incidence, tumor volume, degree of malignancy and recurrence in various human cancers including sar- coma, leukemia, Hodgkin's lymphoma, brain tumours and cancer of the cervix, breast, liver and lung; sup- porting the hypothesis that copper could be used as a potential cancer-specific target.3 ...

  16. Ribonucleases, nucleases and antiangiogenins in antiproliferative activities

    Czech Academy of Sciences Publication Activity Database

    Matoušek, Josef

    2011-01-01

    Roč. 6, č. 3 (2011), s. 363-382 ISSN 1574-3624 R&D Projects: GA ČR GA521/06/1149; GA ČR GA521/09/1214 Institutional research plan: CEZ:AV0Z50450515 Keywords : Angiogenin * Anticancer * Antiproliferative Subject RIV: CE - Biochemistry Impact factor: 0.500, year: 2011

  17. The construction of the zinc finger nucleases

    Indian Academy of Sciences (India)

    Michel Morange

    shown to be involved in the immunity against bacteriophages, and the slow development of highly specific ... vasan Chandrasegaran, had the major role. In three articles published between 1992 and 1996 in the .... road was now open to adapt the system to any DNA sequence. 5. The end of the story. The road was not as ...

  18. Feed tank transfer requirements

    Energy Technology Data Exchange (ETDEWEB)

    Freeman-Pollard, J.R.

    1998-09-16

    This document presents a definition of tank turnover. Also, DOE and PC responsibilities; TWRS DST permitting requirements; TWRS Authorization Basis (AB) requirements; TWRS AP Tank Farm operational requirements; unreviewed safety question (USQ) requirements are presented for two cases (i.e., tank modifications occurring before tank turnover and tank modification occurring after tank turnover). Finally, records and reporting requirements, and documentation which will require revision in support of transferring a DST in AP Tank Farm to a privatization contractor are presented.

  19. Discovering system requirements

    Energy Technology Data Exchange (ETDEWEB)

    Bahill, A.T.; Bentz, B. [Univ. of Arizona, Tucson, AZ (United States). Systems and Industrial Engineering; Dean, F.F. [Sandia National Labs., Albuquerque, NM (United States)

    1996-07-01

    Cost and schedule overruns are often caused by poor requirements that are produced by people who do not understand the requirements process. This report provides a high-level overview of the system requirements process, explaining types, sources, and characteristics of good requirements. System requirements, however, are seldom stated by the customer. Therefore, this report shows ways to help you work with your customer to discover the system requirements. It also explains terminology commonly used in the requirements development field, such as verification, validation, technical performance measures, and the various design reviews.

  20. Postmarket Requirements and Commitments

    Data.gov (United States)

    U.S. Department of Health & Human Services — Provides information to the public on postmarket requirements and commitments. The phrase postmarket requirements and commitments refers to studies and clinical...

  1. Requirements for existing buildings

    DEFF Research Database (Denmark)

    Thomsen, Kirsten Engelund; Wittchen, Kim Bjarne

    This report collects energy performance requirements for existing buildings in European member states by June 2012.......This report collects energy performance requirements for existing buildings in European member states by June 2012....

  2. PIT Coating Requirements Analysis

    Energy Technology Data Exchange (ETDEWEB)

    MINTEER, D.J.

    2000-10-20

    This study identifies the applicable requirements for procurement and installation of a coating intended for tank farm valve and pump pit interior surfaces. These requirements are intended to be incorporated into project specification documents and design media. This study also evaluates previously recommended coatings and identifies requirement-compliant coating products.

  3. Future Home Network Requirements

    DEFF Research Database (Denmark)

    Charbonnier, Benoit; Wessing, Henrik; Lannoo, Bart

    This paper presents the requirements for future Home Area Networks (HAN). Firstly, we discuss the applications and services as well as their requirements. Then, usage scenarios are devised to establish a first specification for the HAN. The main requirements are an increased bandwidth (towards 1...

  4. PIT Coating Requirements Analysis

    International Nuclear Information System (INIS)

    MINTEER, D.J.

    2000-01-01

    This study identifies the applicable requirements for procurement and installation of a coating intended for tank farm valve and pump pit interior surfaces. These requirements are intended to be incorporated into project specification documents and design media. This study also evaluates previously recommended coatings and identifies requirement-compliant coating products

  5. User Requirements for Wireless

    DEFF Research Database (Denmark)

    In most IT system development processes, the identification or elicitation of user requirements is recognized as a key building block. In practice, the identification of user needs and wants is a challenge and inadequate or faulty identifications in this step of an IT system development can cause...... involvement and requirements elicitation Usable security requirements for design of privacy...

  6. Groupware requirements evolution patterns

    NARCIS (Netherlands)

    Pumareja, D.T.

    2013-01-01

    This study is an empirical investigation of requirements evolution for groupware systems in use by means of case studies. Its goal is to contribute to the development of a theory of requirements evolution. A conceptual framework offering an integrated view of requirements as a collection of domains

  7. TRANSPORTATION SYSTEM REQUIREMENTS DOCUMENT

    International Nuclear Information System (INIS)

    2004-01-01

    This document establishes the Transportation system requirements for the U.S. Department of Energy's (DOE's) Civilian Radioactive Waste Management System (CRWMS). These requirements are derived from the Civilian Radioactive Waste Management System Requirements Document (CRD). The Transportation System Requirements Document (TSRD) was developed in accordance with LP-3.1Q-OCRWM, Preparation, Review, and Approval of Office of National Transportation Level-2 Baseline Requirements. As illustrated in Figure 1, the TSRD forms a part of the DOE Office of Civilian Radioactive Waste Management (OCRWM) Technical Baseline

  8. Transportation System Requirements Document

    Energy Technology Data Exchange (ETDEWEB)

    1993-09-01

    This Transportation System Requirements Document (Trans-SRD) describes the functions to be performed by and the technical requirements for the Transportation System to transport spent nuclear fuel (SNF) and high-level radioactive waste (HLW) from Purchaser and Producer sites to a Civilian Radioactive Waste Management System (CRWMS) site, and between CRWMS sites. The purpose of this document is to define the system-level requirements for Transportation consistent with the CRWMS Requirement Document (CRD). These requirements include design and operations requirements to the extent they impact on the development of the physical segments of Transportation. The document also presents an overall description of Transportation, its functions, its segments, and the requirements allocated to the segments and the system-level interfaces with Transportation. The interface identification and description are published in the CRWMS Interface Specification.

  9. A fluorescent, genetically engineered microorganism that degrades organophosphates and commits suicide when required.

    Science.gov (United States)

    Li, Qin; Wu, Yi-Jun

    2009-03-01

    One way to reduce the potential risk of genetically engineered microorganisms (GEMs) to the environment is to use a containment system that does not interfere with the performance of the GEM until activated. Such a system can be created by inserting a suicide cassette consisting of a toxin-encoding gene controlled by an inducible promoter. We constructed a GEM that can degrade organophosphorus compounds, emit green fluorescence, and commit suicide when required by putting the genes that control these different functions under different promoters. The genes for enhanced green fluorescent protein (EGFP) and organophosphorus hydrolase (OPH) were cloned downstream of the lambda PL promoter in the plasmid pBV220. These genes could be expressed freely as long as the GEM was metabolizing because the repressor sequence cIts857 had been deleted. The extracellular nuclease gene of Serratia marcescens, without its leader-coding sequence, provided the suicide mechanism. This was put under the control of the T7 promoter to form a suicide cassette activated by the presence of an environmental signal, in this case, arabinose. To improve the reliability of this containment system, the suicide cassette was duplicated within the conditional suicide plasmid. The plasmid carrying the EGFP and OPH fusion genes and that containing the suicide cassette were compatible and coexisted in the same host.

  10. Semantic Requirements Engineering

    Science.gov (United States)

    Saeki, Motoshi

    Requirements engineering (RE) techniques play a crucial role in information systems development processes. There are many excellent techniques of RE to assist requirements analysts and stakeholders in producing requirements specification of higher quality, and some of them are put into practice in industry. However, one of the issues of these RE techniques is that they do not handle semantic aspects of requirements. If we can deal with the meaning of requirements by using automated techniques, we can get more effective RE techniques to produce requirements specifications of higher quality. In this chapter, we consider an ontology as a semantic domain so as to provide the meaning for requirements, and discuss the potentials of the RE techniques using an ontology as a semantic basis. Especially, we illustrate an extension of goal-oriented requirements analysis where this idea is embedded, i.e. we provide the semantics for goal descriptions written in natural language using a mapping from them to an ontology. The inference mechanisms of the ontology allow us to decompose a goal into sub-goals and to find missing goals. Furthermore, in this chapter we discuss the possibilities of the techniques to support the other activities of RE processes using this ontological technique, e.g. measuring quality metrics and controlling versions of requirements from a semantic view. Due to similarity to Semantic Web techniques, we call a family of these engineering techniques Semantic Requirements Engineering in this chapter.

  11. Environmental Requirements Management

    Energy Technology Data Exchange (ETDEWEB)

    Cusack, Laura J.; Bramson, Jeffrey E.; Archuleta, Jose A.; Frey, Jeffrey A.

    2015-01-08

    CH2M HILL Plateau Remediation Company (CH2M HILL) is the U.S. Department of Energy (DOE) prime contractor responsible for the environmental cleanup of the Hanford Site Central Plateau. As part of this responsibility, the CH2M HILL is faced with the task of complying with thousands of environmental requirements which originate from over 200 federal, state, and local laws and regulations, DOE Orders, waste management and effluent discharge permits, Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) response and Resource Conservation and Recovery Act (RCRA) corrective action documents, and official regulatory agency correspondence. The challenge is to manage this vast number of requirements to ensure they are appropriately and effectively integrated into CH2M HILL operations. Ensuring compliance with a large number of environmental requirements relies on an organization’s ability to identify, evaluate, communicate, and verify those requirements. To ensure that compliance is maintained, all changes need to be tracked. The CH2M HILL identified that the existing system used to manage environmental requirements was difficult to maintain and that improvements should be made to increase functionality. CH2M HILL established an environmental requirements management procedure and tools to assure that all environmental requirements are effectively and efficiently managed. Having a complete and accurate set of environmental requirements applicable to CH2M HILL operations will promote a more efficient approach to: • Communicating requirements • Planning work • Maintaining work controls • Maintaining compliance

  12. Creativity in Requirement Identification

    DEFF Research Database (Denmark)

    Sørensen, Lene Tolstrup; Olesen, Henning

    Traditional requirements engineering focuses mainly on analysis and elicitation. However, current trends in new system, device and software are towards involving all stakeholders in the early stages of the engineering process to define the user requirements. Creativity is here seen as a major...

  13. Navigating the Requirements Jungle

    Science.gov (United States)

    Langer, Boris; Tautschnig, Michael

    Research on validation and verification of requirements specifications has thus far focused on functional properties. Yet, in embedded systems, functional requirements constitute only a small fraction of the properties that must hold to guarantee proper and safe operation of the system under design.

  14. Writing testable software requirements

    Energy Technology Data Exchange (ETDEWEB)

    Knirk, D. [Sandia National Labs., Albuquerque, NM (United States)

    1997-11-01

    This tutorial identifies common problems in analyzing requirements in the problem and constructing a written specification of what the software is to do. It deals with two main problem areas: identifying and describing problem requirements, and analyzing and describing behavior specifications.

  15. User Requirements for Wireless

    DEFF Research Database (Denmark)

    In most IT system development processes, the identification or elicitation of user requirements is recognized as a key building block. In practice, the identification of user needs and wants is a challenge and inadequate or faulty identifications in this step of an IT system development can cause...... huge problems with the final product. The elicitation of user requirements as such changes according to age groups;, to gender,; to cultural settings,; and into time; and experience in the use of the system/software. User requirements, therefore, cannot be used between projects, IT systems......, and different software. That makes the elicitation of user requirements an inherent part of any software development project and a resourceful activity as well. This book provides insights to the process of identifying user requirements and to different types by describing varying case studies in which...

  16. Requirements in engineering projects

    CERN Document Server

    Fernandes, João M

    2016-01-01

    This book focuses on various topics related to engineering and management of requirements, in particular elicitation, negotiation, prioritisation, and documentation (whether with natural languages or with graphical models). The book provides methods and techniques that help to characterise, in a systematic manner, the requirements of the intended engineering system.  It was written with the goal of being adopted as the main text for courses on requirements engineering, or as a strong reference to the topics of requirements in courses with a broader scope. It can also be used in vocational courses, for professionals interested in the software and information systems domain.   Readers who have finished this book will be able to: - establish and plan a requirements engineering process within the development of complex engineering systems; - define and identify the types of relevant requirements in engineering projects; - choose and apply the most appropriate techniques to elicit the requirements of a giv...

  17. Characterization of a TUTase/RNase complex required forDrosophilagametogenesis.

    Science.gov (United States)

    Lin, Ching-Jung; Wen, Jiayu; Bejarano, Fernando; Hu, Fuqu; Bortolamiol-Becet, Diane; Kan, Lijuan; Sanfilippo, Piero; Kondo, Shu; Lai, Eric C

    2017-03-01

    Post-transcriptional regulatory strategies that involve coupling between terminal uridyltransferase (TUTase) and exoribonuclease enzymes have recently been characterized in diverse species. Of note, the 3' exoribonuclease Dis3L2 has received substantial attention as a factor that metabolizes uridylated substrates in contexts such as general mRNA degradation, turnover of specific miRNAs, and quality control of noncoding RNAs. To date, most studies of Dis3L2 have focused on fungi and mammalian cells. Here, we introduce Drosophila as a system that permits analysis of molecular mechanisms as well as the ability to interrogate organismal phenotypes. We started with a structure-function analysis of the Drosophila TUTase Tailor, which we recently identified to inhibit biogenesis of splicing-derived miRNA hairpins. Next, we show that Tailor/Dis3L2 form a complex via N-terminal domains in the respective proteins that are distinct from their catalytic domains. In vitro, Dis3L2 has nuclease activity, but substrate oligouridylation by Tailor stimulates their degradation by Dis3L2, especially for structured substrates. We analyzed mutants of Tailor and Dis3L2 , which are viable and lack overt morphological defects. Instead, these mutants exhibit defects in female and male fertility, implying specific requirements in the germline. Dis3L2 defects are more severe than Tailor , and their requirements appear stronger in males than in females. In particular, loss of Dis3L2 completely blocks productive spermatogenesis, causing male sterility. RNA-seq analysis from single- and double-mutant testes reveals aberrant gene expression programs and suggests that noncoding RNAs may be preferentially affected by Dis3L2. Overall, our studies of a new tailing/trimming complex reveal unexpectedly specific requirements during gametogenesis. © 2017 Lin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  18. TWRSview system requirements specification

    International Nuclear Information System (INIS)

    Caldwell, J.A.; Lee, A.K.

    1995-12-01

    This document provides the system requirements specification for the TWRSview software system. The TWRSview software system is being developed to integrate electronic data supporting the development of the TWRS technical baseline

  19. Turning Desirements into Requirements

    Science.gov (United States)

    2015-09-01

    27 Defense AT&L: September–October 2015 Turning “Desirements” into Requirements Charles Court Court is the Requirements Center Director at the...Defense Systems Management College at the Defense Acquisition University at Fort Belvoir, Virginia. He is a former Wild Weasel Electronic Warfare Officer...a Mustang would do much better. (The author wanted the Aston Martin from the movie “Goldfinger.” You know: The one with the ejection seat, automated

  20. Automatic requirements traceability

    OpenAIRE

    Andžiulytė, Justė

    2017-01-01

    This paper focuses on automatic requirements traceability and algorithms that automatically find recommendation links for requirements. The main objective of this paper is the evaluation of these algorithms and preparation of the method defining algorithms to be used in different cases. This paper presents and examines probabilistic, vector space and latent semantic indexing models of information retrieval and association rule mining using authors own implementations of these algorithms and o...

  1. CLIC Detector Power Requirements

    CERN Document Server

    Gaddi, A

    2013-01-01

    An estimate for the CLIC detector power requirements is outlined starting from the available data on power consumptions of the four LHC experiments and considering the differences between a typical LHC Detector (CMS) and the CLIC baseline detector concept. In particular the impact of the power pulsing scheme for the CLIC Detector electronics on the overall detector consumption is considered. The document will be updated with the requirements of the sub-detector electronics once they are more defined.

  2. BRD usability requirements

    Energy Technology Data Exchange (ETDEWEB)

    Deshpande, Alina [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2015-03-12

    This document describes the usability requirements for the Biosurveillance resource directory (BRD); that is, who will be using the tool and what tasks they will be using it for. It does not include information on technical implementation (e.g., whether specific information is contained in the database or pulled on demand from other sources). It also avoids specific design ideas (such as widget descriptions) unless they are necessary to illustrate a requirement.

  3. Utility requirements for fusion

    International Nuclear Information System (INIS)

    Vondrasek, R.J.

    1982-02-01

    This report describes work done and results obtained during performance of Task 1 of a study of Utility Requirements and Criteria for Fusion Options. The work consisted of developing a list of utility requirements for fusion optics containing definition of the requirements and showing their relative importance to the utility industry. The project team members developed a preliminary list which was refined by discussions and literature searches. The refined list was recast as a questionnaire which was sent to a substantial portion of the utility industry in this country. Forty-three questionnaire recipients responded including thirty-two utilities. A workshop was held to develop a revised requirements list using the survey responses as a major input. The list prepared by the workshop was further refined by a panel consisting of vice presidents of the three project team firms. The results of the study indicate that in addition to considering the cost of energy for a power plant, utilities consider twenty-three other requirements. Four of the requirements were judged to be vital to plant acceptability: Plant Capital Cost, Financial Liability, Plant Safety and Licensability

  4. Range Flight Safety Requirements

    Science.gov (United States)

    Loftin, Charles E.; Hudson, Sandra M.

    2018-01-01

    The purpose of this NASA Technical Standard is to provide the technical requirements for the NPR 8715.5, Range Flight Safety Program, in regards to protection of the public, the NASA workforce, and property as it pertains to risk analysis, Flight Safety Systems (FSS), and range flight operations. This standard is approved for use by NASA Headquarters and NASA Centers, including Component Facilities and Technical and Service Support Centers, and may be cited in contract, program, and other Agency documents as a technical requirement. This standard may also apply to the Jet Propulsion Laboratory or to other contractors, grant recipients, or parties to agreements to the extent specified or referenced in their contracts, grants, or agreements, when these organizations conduct or participate in missions that involve range flight operations as defined by NPR 8715.5.1.2.2 In this standard, all mandatory actions (i.e., requirements) are denoted by statements containing the term “shall.”1.3 TailoringTailoring of this standard for application to a specific program or project shall be formally documented as part of program or project requirements and approved by the responsible Technical Authority in accordance with NPR 8715.3, NASA General Safety Program Requirements.

  5. NP Science Network Requirements

    Energy Technology Data Exchange (ETDEWEB)

    Dart, Eli [Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Rotman, Lauren [Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Tierney, Brian [Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

    2011-08-26

    The Energy Sciences Network (ESnet) is the primary provider of network connectivity for the U.S. Department of Energy (DOE) Office of Science (SC), the single largest supporter of basic research in the physical sciences in the United States. To support SC programs, ESnet regularly updates and refreshes its understanding of the networking requirements of the instruments, facilities, scientists, and science programs it serves. This focus has helped ESnet to be a highly successful enabler of scientific discovery for over 20 years. In August 2011, ESnet and the Office of Nuclear Physics (NP), of the DOE SC, organized a workshop to characterize the networking requirements of the programs funded by NP. The requirements identified at the workshop are summarized in the Findings section, and are described in more detail in the body of the report.

  6. Quantifying requirements volatility effects

    NARCIS (Netherlands)

    Kulk, G.P.; Verhoef, C.

    2008-01-01

    In an organization operating in the bancassurance sector we identified a low-risk IT subportfolio of 84 IT projects comprising together 16,500 function points, each project varying in size and duration, for which we were able to quantify its requirements volatility. This representative portfolio

  7. Data Crosscutting Requirements Review

    Energy Technology Data Exchange (ETDEWEB)

    Kleese van Dam, Kerstin [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Shoshani, Arie [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Plata, Charity [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2013-04-01

    In April 2013, a diverse group of researchers from the U.S. Department of Energy (DOE) scientific community assembled to assess data requirements associated with DOE-sponsored scientific facilities and large-scale experiments. Participants in the review included facilities staff, program managers, and scientific experts from the offices of Basic Energy Sciences, Biological and Environmental Research, High Energy Physics, and Advanced Scientific Computing Research. As part of the meeting, review participants discussed key issues associated with three distinct aspects of the data challenge: 1) processing, 2) management, and 3) analysis. These discussions identified commonalities and differences among the needs of varied scientific communities. They also helped to articulate gaps between current approaches and future needs, as well as the research advances that will be required to close these gaps. Moreover, the review provided a rare opportunity for experts from across the Office of Science to learn about their collective expertise, challenges, and opportunities. The "Data Crosscutting Requirements Review" generated specific findings and recommendations for addressing large-scale data crosscutting requirements.

  8. Requirements for Xenon International

    Energy Technology Data Exchange (ETDEWEB)

    Hayes, James C. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Ely, James H. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Haas, Derek A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Harper, Warren W. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Heimbigner, Tom R. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Hubbard, Charles W. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Humble, Paul H. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Madison, Jill C. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Morris, Scott J. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Panisko, Mark E. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Ripplinger, Mike D. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Stewart, Timothy L. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2015-12-30

    This document defines the requirements for the new Xenon International radioxenon system. The output of this project will be a Pacific Northwest National Laboratory (PNNL) developed prototype and a manufacturer-developed production prototype. The two prototypes are intended to be as close to matching as possible; this will be facilitated by overlapping development cycles and open communication between PNNL and the manufacturer.

  9. Requirements for Xenon International

    Energy Technology Data Exchange (ETDEWEB)

    Hayes, James C.; Ely, James H.

    2013-09-26

    This document defines the requirements for the new Xenon International radioxenon system. The output of this project will be a Pacific Northwest National Laboratory (PNNL) developed prototype and a manufacturer-developed production prototype. The two prototypes are intended to be as close to matching as possible; this will be facilitated by overlapping development cycles and open communication between PNNL and the manufacturer.

  10. Next Generation Microbiology Requirements

    Science.gov (United States)

    Ott, C. M.; Oubre, C. M.; Elliott, T. F.; Castro, V. A.; Pierson, D. L.

    2012-01-01

    As humans continue to explore deep into space, microorganisms will travel with them. The primary means to mitigate the risk of infectious disease are a combination of prudent spacecraft design and rigorous operational controls. The effectiveness of these methods are evaluated by microbiological monitoring of spacecraft, food, water, and the crew that is performed preflight, in-flight, and post-flight. Current NASA requirements associated with microbiological monitoring are based on culture-based methodology where microorganisms are grown on a semi-solid growth medium and enumerated. Subsequent identification of the organisms requires specialized labor and large equipment, which historically has been performed on Earth. Requirements that rely strictly on culture-based units limit the use of non-culture based monitoring technology. Specifically, the culture-based "measurement criteria" are Colony Forming Units (CFU, representing the growth of one microorganism at a single location on the agar medium) per a given volume, area, or sample size. As the CFU unit by definition is culture-based, these requirements limit alternative technologies for spaceflight applications. As spaceflight missions such as those to Mars extend further into space, culture-based technology will become difficult to implement due to the (a) limited shelf life of the culture media, (b) mass/volume necessary to carry these consumables, and (c) problems associated with the production of biohazardous material in the habitable volume of the spacecraft. In addition, an extensive amount of new knowledge has been obtained during the Space Shuttle, NASA-Mir, and International Space Station Programs, which gave direction for new or modified microbial control requirements for vehicle design and mission operations. The goal of this task is to develop and recommend a new set of requirements for vehicle design and mission operations, including microbiological monitoring, based upon "lessons learned" and new

  11. Entrepreneurial learning requires action

    DEFF Research Database (Denmark)

    Brink, Tove; Madsen, Svend Ole

    2014-01-01

    apply in industry. The findings of this study show that SME managers employ a practice-shaped holistic multi- and cross-disciplinary approach to learning. This learning approach is supported by theory dissemination, business challenge applications, and organisational prerequisites. Diversified learning......This paper reveals how managers of small- and medium-sized enterprises (SMEs) can utilise their participation in research-based training. Empirical research from a longitudinal study of 10 SMEs managers in the wind turbine industry is provided to describe a learning approach that SME managers can...... that is enhanced by essential large-scale industry players and other SME managers are required to create action and value in learning. An open-mindedness to new learning approaches by SME managers and an open-mindedness to multi- and cross-disciplinary collaboration with SME managers by facilitators is required....

  12. Ecodesign requirements for televisions

    DEFF Research Database (Denmark)

    Huulgaard, Rikke Dorothea; Dalgaard, Randi; Merciai, Stefano

    2013-01-01

    phase. This is not in line with the scientific understanding of ecodesign, where attention should be put on all life cycle phases and all relevant environmental impact categories. This study focuses on the requirements for televisions (TV). A life cycle assessment (LCA) is carried out on two TVs...... to analyse if other environmental hotspots and life cycle phases should be included in the requirements in the IM of the Ecodesign Directive besides energy consumption in the use phase analysis. Methods The consequential approach is used. The data for the LCA have been gathered from two manufacturers of TVs....... In one case, the data were delivered in Excel spreadsheets; in the other case, the authors of this paper together with the manufacturer disassembled a TV and collected the data manually. Results and discussion When applying the consequential approach, the production phase has the highest environmental...

  13. Preanalytical requirements of urinalysis

    Science.gov (United States)

    Delanghe, Joris; Speeckaert, Marijn

    2014-01-01

    Urine may be a waste product, but it contains an enormous amount of information. Well-standardized procedures for collection, transport, sample preparation and analysis should become the basis of an effective diagnostic strategy for urinalysis. As reproducibility of urinalysis has been greatly improved due to recent technological progress, preanalytical requirements of urinalysis have gained importance and have become stricter. Since the patients themselves often sample urine specimens, urinalysis is very susceptible to preanalytical issues. Various sampling methods and inappropriate specimen transport can cause important preanalytical errors. The use of preservatives may be helpful for particular analytes. Unfortunately, a universal preservative that allows a complete urinalysis does not (yet) exist. The preanalytical aspects are also of major importance for newer applications (e.g. metabolomics). The present review deals with the current preanalytical problems and requirements for the most common urinary analytes. PMID:24627718

  14. LHCb Online Networking Requirements

    CERN Document Server

    Jost, B

    2003-01-01

    This document describes the networking requirements of the LHCb online installation. It lists both quantitative aspects such as the number of required switch ports, as well as some qualitative features of the equipment, such as minimum buffer sizes in switches. The document comprises both the data acquisition network and the controls/general-purpose network. While the numbers represent our best current knowledge and are intended to give (in particular) network equipment manufacturers an overview of our needs, this document should not be confused with a market survey questionnaire or a formal tendering document. However the information contained in this document will be the input of any such document. A preliminary schedule for procurement and installation is also given.

  15. Utility requirements for HTGRs

    International Nuclear Information System (INIS)

    Nicholls, D.R.

    1997-01-01

    Eskom, the state utility of South Africa, is currently evaluating the technical and economic feasibility of the helium cooled Pebble Bed Modular Reactor with a closed cycle gas turbine power conversion system for future power generating additions to its electric system. This paper provides an overview of the Eskom system including the needs of the utility for future generation capacity and the key performance requirements necessary for incorporation of this gas cooled reactor plant. (author)

  16. BER Science Network Requirements

    Energy Technology Data Exchange (ETDEWEB)

    Alapaty, Kiran; Allen, Ben; Bell, Greg; Benton, David; Brettin, Tom; Canon, Shane; Dart, Eli; Cotter, Steve; Crivelli, Silvia; Carlson, Rich; Dattoria, Vince; Desai, Narayan; Egan, Richard; Tierney, Brian; Goodwin, Ken; Gregurick, Susan; Hicks, Susan; Johnston, Bill; de Jong, Bert; Kleese van Dam, Kerstin; Livny, Miron; Markowitz, Victor; McGraw, Jim; McCord, Raymond; Oehmen, Chris; Regimbal, Kevin; Shipman, Galen; Strand, Gary; Flick, Jeff; Turnbull, Susan; Williams, Dean; Zurawski, Jason

    2010-11-01

    The Energy Sciences Network (ESnet) is the primary provider of network connectivity for the US Department of Energy Office of Science, the single largest supporter of basic research in the physical sciences in the United States. In support of the Office of Science programs, ESnet regularly updates and refreshes its understanding of the networking requirements of the instruments, facilities, scientists, and science programs that it serves. This focus has helped ESnet to be a highly successful enabler of scientific discovery for over 20 years. In April 2010 ESnet and the Office of Biological and Environmental Research, of the DOE Office of Science, organized a workshop to characterize the networking requirements of the science programs funded by BER. The requirements identified at the workshop are summarized and described in more detail in the case studies and the Findings section. A number of common themes emerged from the case studies and workshop discussions. One is that BER science, like many other disciplines, is becoming more and more distributed and collaborative in nature. Another common theme is that data set sizes are exploding. Climate Science in particular is on the verge of needing to manage exabytes of data, and Genomics is on the verge of a huge paradigm shift in the number of sites with sequencers and the amount of sequencer data being generated.

  17. LEGACY MANAGEMENT REQUIRES INFORMATION

    International Nuclear Information System (INIS)

    CONNELL, C.W.; HILDEBRAND, R.D.

    2006-01-01

    ''Legacy Management Requires Information'' describes the goal(s) of the US Department of Energy's Office of Legacy Management (LM) relative to maintaining critical records and the way those goals are being addressed at Hanford. The paper discusses the current practices for document control, as well as the use of modern databases for both storing and accessing the data to support cleanup decisions. In addition to the information goals of LM, the Hanford Federal Facility Agreement and Consent Order, known as the ''Tri-Party Agreement'' (TPA) is one of the main drivers in documentation and data management. The TPA, which specifies discrete milestones for cleaning up the Hanford Site, is a legally binding agreement among the US Department of Energy (DOE), the Washington State Department of Ecology (Ecology), and the US Environmental Protection Agency (EPA). The TPA requires that DOE provide the lead regulatory agency with the results of analytical laboratory and non-laboratory tests/readings to help guide them in making decisions. The Agreement also calls for each signatory to preserve--for at least ten years after the Agreement has ended--all of the records in its or its contractors, possession related to sampling, analysis, investigations, and monitoring conducted. The tools used at Hanford to meet TPA requirements are also the tools that can satisfy the needs of LM

  18. LEGACY MANAGEMENT REQUIRES INFORMATION

    Energy Technology Data Exchange (ETDEWEB)

    CONNELL, C.W.; HILDEBRAND, R.D.

    2006-12-14

    ''Legacy Management Requires Information'' describes the goal(s) of the US Department of Energy's Office of Legacy Management (LM) relative to maintaining critical records and the way those goals are being addressed at Hanford. The paper discusses the current practices for document control, as well as the use of modern databases for both storing and accessing the data to support cleanup decisions. In addition to the information goals of LM, the Hanford Federal Facility Agreement and Consent Order, known as the ''Tri-Party Agreement'' (TPA) is one of the main drivers in documentation and data management. The TPA, which specifies discrete milestones for cleaning up the Hanford Site, is a legally binding agreement among the US Department of Energy (DOE), the Washington State Department of Ecology (Ecology), and the US Environmental Protection Agency (EPA). The TPA requires that DOE provide the lead regulatory agency with the results of analytical laboratory and non-laboratory tests/readings to help guide them in making decisions. The Agreement also calls for each signatory to preserve--for at least ten years after the Agreement has ended--all of the records in its or its contractors, possession related to sampling, analysis, investigations, and monitoring conducted. The tools used at Hanford to meet TPA requirements are also the tools that can satisfy the needs of LM.

  19. TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells

    Science.gov (United States)

    Pedersen, Rune Troelsgaard; Kruse, Thomas; Nilsson, Jakob

    2015-01-01

    Genome integrity is critically dependent on timely DNA replication and accurate chromosome segregation. Replication stress delays replication into G2/M, which in turn impairs proper chromosome segregation and inflicts DNA damage on the daughter cells. Here we show that TopBP1 forms foci upon mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin recruitment of SLX4 and by facilitating unscheduled DNA synthesis. PMID:26283799

  20. SLX-1 is required for maintaining genomic integrity and promoting meiotic noncrossovers in the Caenorhabditis elegans germline.

    Directory of Open Access Journals (Sweden)

    Takamune T Saito

    2012-08-01

    Full Text Available Although the SLX4 complex, which includes structure-specific nucleases such as XPF, MUS81, and SLX1, plays important roles in the repair of several kinds of DNA damage, the function of SLX1 in the germline remains unknown. Here we characterized the endonuclease activities of the Caenorhabditis elegans SLX-1-HIM-18/SLX-4 complex co-purified from human 293T cells and determined SLX-1 germline function via analysis of slx-1(tm2644 mutants. SLX-1 shows a HIM-18/SLX-4-dependent endonuclease activity toward replication forks, 5'-flaps, and Holliday junctions. slx-1 mutants exhibit hypersensitivity to UV, nitrogen mustard, and camptothecin, but not gamma irradiation. Consistent with a role in DNA repair, recombination intermediates accumulate in both mitotic and meiotic germ cells in slx-1 mutants. Importantly, meiotic crossover distribution, but not crossover frequency, is altered on chromosomes in slx-1 mutants compared to wild type. This alteration is not due to changes in either the levels or distribution of double-strand breaks (DSBs along chromosomes. We propose that SLX-1 is required for repair at stalled or collapsed replication forks, interstrand crosslink repair, and nucleotide excision repair during mitosis. Moreover, we hypothesize that SLX-1 regulates the crossover landscape during meiosis by acting as a noncrossover-promoting factor in a subset of DSBs.

  1. Mixed ligand complexes of Cu(II)/Zn(II) ions containing (m-)/(p-) carboxylato phenyl azo pentane 2,4-dione and 2,2‧-bipyridine/1,10 phenanthroline: Synthesis, characterization, DNA binding, nuclease and topoisomerase I inhibitory activity

    Science.gov (United States)

    Hasan, Md. Amin; Kumari, Niraj; Singh, Kanhaiya; Singh, Kiran; Mishra, Lallan

    2016-01-01

    Metal complexes of type [Cu(L1H)2(bpy)] (1), [Zn(L1H)2(bpy)] (2), [Cu(L2H)2(bpy)] (3) and [Cu(L2H)2(Phen)] (4) (L1H2 = 3-[N‧-(1-acetyl-2-oxo-propylidene)-hydrazino]-benzoic acid, L2H2 = 4-[N‧-(1-acetyl-2-oxo-propylidene)-hydrazino]-benzoic acid, bpy = 2,2‧-bipyridine, Phen = 1,10 phenanthroline) are synthesized and characterized using spectroscopic techniques (FT-IR, 1H NMR, 13C NMR, electronic absorption and emission) and elemental analysis data. The assembly of the complexes involving intramolecular H-bonding is displayed using corresponding crystal structure. Binding of the complexes separately with Calf Thymus DNA is monitored using UV-vis spectral titrations. The displacement of ethidium bromide (EB) bound to DNA by the complexes, in phosphate buffer solution (pH ∼ 7.2) is monitored using fluorescence spectral titrations. Nuclease activity of the complexes follow the order 4 > 3 > 1 > 2. The gel electrophoretic mobility assay measurement in presence of minor groove binder 4‧,6-diamidino-2-phenylindole (DAPI), suggests that complexes preferably bind with the minor groove of DNA. Topoisomerase I inhibitory activity of the complexes 3 and 4 inhibit topoisomerase I activity with IC50 values of 112 and 87 μM respectively.

  2. Mixed ligand complexes of Cu(II)/Zn(II) ions containing (m-)/(p-) carboxylato phenyl azo pentane 2,4-dione and 2,2'-bipyridine/1,10 phenanthroline: Synthesis, characterization, DNA binding, nuclease and topoisomerase I inhibitory activity.

    Science.gov (United States)

    Hasan, Md Amin; Kumari, Niraj; Singh, Kanhaiya; Singh, Kiran; Mishra, Lallan

    2016-01-05

    Metal complexes of type [Cu(L1H)2(bpy)] (1), [Zn(L1H)2(bpy)] (2), [Cu(L2H)2(bpy)] (3) and [Cu(L2H)2(Phen)] (4) (L1H2=3-[N'-(1-acetyl-2-oxo-propylidene)-hydrazino]-benzoic acid, L2H2=4-[N'-(1-acetyl-2-oxo-propylidene)-hydrazino]-benzoic acid, bpy=2,2'-bipyridine, Phen=1,10 phenanthroline) are synthesized and characterized using spectroscopic techniques (FT-IR, (1)H NMR, (13)C NMR, electronic absorption and emission) and elemental analysis data. The assembly of the complexes involving intramolecular H-bonding is displayed using corresponding crystal structure. Binding of the complexes separately with Calf Thymus DNA is monitored using UV-vis spectral titrations. The displacement of ethidium bromide (EB) bound to DNA by the complexes, in phosphate buffer solution (pH∼7.2) is monitored using fluorescence spectral titrations. Nuclease activity of the complexes follow the order 4>3>1>2. The gel electrophoretic mobility assay measurement in presence of minor groove binder 4',6-diamidino-2-phenylindole (DAPI), suggests that complexes preferably bind with the minor groove of DNA. Topoisomerase I inhibitory activity of the complexes 3 and 4 inhibit topoisomerase I activity with IC50 values of 112 and 87μM respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. BES Science Network Requirements

    Energy Technology Data Exchange (ETDEWEB)

    Biocca, Alan; Carlson, Rich; Chen, Jackie; Cotter, Steve; Tierney, Brian; Dattoria, Vince; Davenport, Jim; Gaenko, Alexander; Kent, Paul; Lamm, Monica; Miller, Stephen; Mundy, Chris; Ndousse, Thomas; Pederson, Mark; Perazzo, Amedeo; Popescu, Razvan; Rouson, Damian; Sekine, Yukiko; Sumpter, Bobby; Dart, Eli; Wang, Cai-Zhuang -Z; Whitelam, Steve; Zurawski, Jason

    2011-02-01

    The Energy Sciences Network (ESnet) is the primary provider of network connectivityfor the US Department of Energy Office of Science (SC), the single largest supporter of basic research in the physical sciences in the United States. In support of the Office ofScience programs, ESnet regularly updates and refreshes its understanding of the networking requirements of the instruments, facilities, scientists, and science programs that it serves. This focus has helped ESnet to be a highly successful enabler of scientific discovery for over 20 years.

  4. BES Science Network Requirements

    International Nuclear Information System (INIS)

    Dart, Eli; Tierney, Brian; Biocca, A.; Carlson, R.; Chen, J.; Cotter, S.; Dattoria, V.; Davenport, J.; Gaenko, A.; Kent, P.; Lamm, M.; Miller, S.; Mundy, C.; Ndousse, T.; Pederson, M.; Perazzo, A.; Popescu, R.; Rouson, D.; Sekine, Y.; Sumpter, B.; Wang, C.-Z.; Whitelam, S.; Zurawski, J.

    2011-01-01

    The Energy Sciences Network (ESnet) is the primary provider of network connectivity for the US Department of Energy Office of Science (SC), the single largest supporter of basic research in the physical sciences in the United States. In support of the Office of Science programs, ESnet regularly updates and refreshes its understanding of the networking requirements of the instruments, facilities, scientists, and science programs that it serves. This focus has helped ESnet to be a highly successful enabler of scientific discovery for over 20 years.

  5. NIRVANA network requirements

    Energy Technology Data Exchange (ETDEWEB)

    Wood, B.J.

    1990-08-01

    NIRVANA is an effort to standardize electrical computer-aided design workstations at Sandia National Laboratories in Albuquerque, New Mexico. The early effect of this project will be the introduction of at least 60 new engineering workstations at Sandia National Laboratories. Albuquerque, and at Allied Signal, Kansas City Division. These workstations are expected to begin arriving in September 1990. This paper proposes a design and outlines the requirements for a network to support the NIRVANA project. The author proposes a near-term network design, describes the security profile and caveats of this design, and proposes a long-term networking strategy for NIRVANA. 6 refs., 7 figs.

  6. Finding Incorrect and Missing Quality Requirements Definitions Using Requirements Frame

    Science.gov (United States)

    Kaiya, Haruhiko; Ohnishi, Atsushi

    Defining quality requirements completely and correctly is more difficult than defining functional requirements because stakeholders do not state most of quality requirements explicitly. We thus propose a method to measure a requirements specification for identifying the amount of quality requirements in the specification. We also propose another method to recommend quality requirements to be defined in such a specification. We expect stakeholders can identify missing and unnecessary quality requirements when measured quality requirements are different from recommended ones. We use a semi-formal language called X-JRDL to represent requirements specifications because it is suitable for analyzing quality requirements. We applied our methods to a requirements specification, and found our methods contribute to defining quality requirements more completely and correctly.

  7. Section 4: Requirements Intertwining

    Science.gov (United States)

    Loucopoulos, Pericles

    Business analysts are being asked to develop increasingly complex and varied business systems that need to cater to the changing and dynamic market conditions of the new economy. This is particularly acute in today’s turbulent business environment where powerful forces such as deregulation, globalisation, mergers, advances in information and telecommunications technologies, and increasing education of people provide opportunities for organising work in ways that have never before been possible. Enterprises attempt to create wealth either by getting better at improving their products and services or by harnessing creativity and human-centred management to create innovative solutions. In these business settings, requirements become critical in bridging system solutions to organisational and societal problems. They intertwine organisational, social, cognitive, and implementation considerations and they can provide unique insights to change in systems and their business context. Such design situations often involve multiple stakeholders from different participating organisations, subcontractors, divisions, etc., who may have a diversity of expertise, come from different organisational cultures and often have competing goals. The success or failure of many projects depends, to a large extent, on understanding the contextual setting of requirements and their interaction amongst a diverse population of stakeholders.

  8. Beauty Requires Thought.

    Science.gov (United States)

    Brielmann, Aenne A; Pelli, Denis G

    2017-05-22

    The experience of beauty is a pleasure, but common sense and philosophy suggest that feeling beauty differs from sensuous pleasures such as eating or sex. Immanuel Kant [1, 2] claimed that experiencing beauty requires thought but that sensuous pleasure can be enjoyed without thought and cannot be beautiful. These venerable hypotheses persist in models of aesthetic processing [3-7] but have never been tested. Here, participants continuously rated the pleasure felt from a nominally beautiful or non-beautiful stimulus and then judged whether they had experienced beauty. The stimuli, which engage various senses, included seeing images, tasting candy, and touching a teddy bear. The observer reported the feelings that the stimulus evoked. The time course of pleasure, across stimuli, is well-fit by a model with one free parameter: pleasure amplitude. Pleasure amplitude increases linearly with the feeling of beauty. To test Kant's claim of a need for thought, we reduce cognitive capacity by adding a "two-back" task to distract the observer's thoughts. The distraction greatly reduces the beauty and pleasure experienced from stimuli that otherwise produce strong pleasure and spares that of less-pleasant stimuli. We also find that strong pleasure is always beautiful, whether produced reliably by beautiful stimuli or just occasionally by sensuous stimuli. In sum, we confirm Kant's claim that only the pleasure associated with feeling beauty requires thought and disprove his claim that sensuous pleasures cannot be beautiful. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. Finding Incorrect and Missing Quality Requirements Definitions Using Requirements Frame

    OpenAIRE

    Kaiya, Haruhiko; Ohnishi, Atsushi

    2012-01-01

    Defining quality requirements completely and correctly is more difficult than defining functional requirements because stakeholders do not state most of quality requirements explicitly. We thus propose a method to measure a requirements specification for identifying the amount of quality requirements in the specification. We also propose another method to recommend quality requirements to be defined in such a specification. We expect stakeholders can identify missing and unnecessary quality r...

  10. Display Parameters and Requirements

    Science.gov (United States)

    Bahadur, Birendra

    The following sections are included: * INTRODUCTION * HUMAN FACTORS * Anthropometry * Sensory * Cognitive * Discussions * THE HUMAN VISUAL SYSTEM - CAPABILITIES AND LIMITATIONS * Cornea * Pupil and Iris * Lens * Vitreous Humor * Retina * RODS - NIGHT VISION * CONES - DAY VISION * RODS AND CONES - TWILIGHT VISION * VISUAL PIGMENTS * MACULA * BLOOD * CHOROID COAT * Visual Signal Processing * Pathways to the Brain * Spatial Vision * Temporal Vision * Colour Vision * Colour Blindness * DICHROMATISM * Protanopia * Deuteranopia * Tritanopia * ANOMALOUS TRICHROMATISM * Protanomaly * Deuteranomaly * Tritanomaly * CONE MONOCHROMATISM * ROD MONOCHROMATISM * Using Colour Effectively * COLOUR MIXTURES AND THE CHROMATICITY DIAGRAM * Colour Matching Functions and Chromaticity Co-ordinates * CIE 1931 Colour Space * CIE PRIMARIES * CIE COLOUR MATCHING FUNCTIONS AND CHROMATICITY CO-ORDINATES * METHODS FOR DETERMINING TRISTIMULUS VALUES AND COLOUR CO-ORDINATES * Spectral Power Distribution Method * Filter Method * CIE 1931 CHROMATICITY DIAGRAM * ADDITIVE COLOUR MIXTURE * CIE 1976 Chromaticity Diagram * CIE Uniform Colour Spaces and Colour Difference Formulae * CIELUV OR L*u*v* * CIELAB OR L*a*b* * CIE COLOUR DIFFERENCE FORMULAE * Colour Temperature and CIE Standard Illuminants and source * RADIOMETRIC AND PHOTOMETRIC QUANTITIES * Photopic (Vλ and Scotopic (Vλ') Luminous Efficiency Function * Photometric and Radiometric Flux * Luminous and Radiant Intensities * Incidence: Illuminance and Irradiance * Exitance or Emittance (M) * Luminance and Radiance * ERGONOMIC REQUIREMENTS OF DISPLAYS * ELECTRO-OPTICAL PARAMETERS AND REQUIREMENTS * Contrast and Contrast Ratio * Luminance and Brightness * Colour Contrast and Chromaticity * Glare * Other Aspects of Legibility * SHAPE AND SIZE OF CHARACTERS * DEFECTS AND BLEMISHES * FLICKER AND DISTORTION * ANGLE OF VIEW * Switching Speed * Threshold and Threshold Characteristic * Measurement Techniques For Electro-optical Parameters * RADIOMETRIC

  11. SOFG: Standards requirements

    International Nuclear Information System (INIS)

    Gerganov, T.; Grigorov, S.; Kozhukharov, V.; Brashkova, N.

    2005-01-01

    It is well-known that Solid Oxide Fuel Cells will have industrial application in the nearest future. In this context, the problem of SOFC materials and SOFC systems standardization is of high level of priority. In the present study the attention is focused on the methods for physical and chemical characterization of the materials for SOFC components fabrication and about requirements on single SOFC cells tests. The status of the CEN, ISO, ASTM (ANSI, ASSN) and JIS class of standards has been verified. Standards regarding the test methods for physical-chemical characterization of vitreous materials (as sealing SOFC component), ceramic materials (as electrodes and electrolyte components, including alternative materials used) and metallic materials (interconnect components) are subject of overview. It is established that electrical, mechanical, surface and interfacial phenomena, chemical durability and thermal corrosion behaviour are the key areas for standardization of the materials for SOFC components

  12. SDC detector foundation requirements

    International Nuclear Information System (INIS)

    Western, J.L.; Butalla, M.W.

    1992-01-01

    The Solenoidal Detector Collaboration (SDC) Detector weighs approximately 32,000 metric tons, and its ability to perform to design specifications is directly related to its internal alignment. The limits of the misalignment tolerance envelope in combination with the detector weight impose a set of tolerance limits of performance directly upon the foundation structure. The foundation must accommodate different detector loading conditions during installation, operation, maintenance, and future enhancements. The foundation must also respond to the loading conditions within a restrictive set of displacement limitations in order to maintain the detector's critical alignment, thereby guaranteeing its operational integrity. This paper will present the results of this study, which has been issued to the Architect Engineer/Construction Manager as user requirements of design. The total structural system performance of the combination of both the detector and its foundation will be discussed

  13. Equipment Operational Requirements

    Energy Technology Data Exchange (ETDEWEB)

    Greenwalt, B; Henderer, B; Hibbard, W; Mercer, M

    2009-06-11

    The Iraq Department of Border Enforcement is rich in personnel, but poor in equipment. An effective border control system must include detection, discrimination, decision, tracking and interdiction, capture, identification, and disposition. An equipment solution that addresses only a part of this will not succeed, likewise equipment by itself is not the answer without considering the personnel and how they would employ the equipment. The solution should take advantage of the existing in-place system and address all of the critical functions. The solutions are envisioned as being implemented in a phased manner, where Solution 1 is followed by Solution 2 and eventually by Solution 3. This allows adequate time for training and gaining operational experience for successively more complex equipment. Detailed descriptions of the components follow the solution descriptions. Solution 1 - This solution is based on changes to CONOPs, and does not have a technology component. It consists of observers at the forts and annexes, forward patrols along the swamp edge, in depth patrols approximately 10 kilometers inland from the swamp, and checkpoints on major roads. Solution 2 - This solution adds a ground sensor array to the Solution 1 system. Solution 3 - This solution is based around installing a radar/video camera system on each fort. It employs the CONOPS from Solution 1, but uses minimal ground sensors deployed only in areas with poor radar/video camera coverage (such as canals and streams shielded by vegetation), or by roads covered by radar but outside the range of the radar associated cameras. This document provides broad operational requirements for major equipment components along with sufficient operational details to allow the technical community to identify potential hardware candidates. Continuing analysis will develop quantities required and more detailed tactics, techniques, and procedures.

  14. DNA sequences required for regulated expresson of the β-globin genes in murine erythroleukaemia cells.

    NARCIS (Netherlands)

    S. Wright; E. de Boer (Ernie); A. Rosenthal; R.A. Flavell (Richard); F.G. Grosveld (Frank)

    1984-01-01

    textabstractWe have introduced into murine erythroleukaemia (MEL) cells a series of human globin gene cosmids and two sets of hybrid genes constructed from the human beta-globin gene and the human gamma-globin or murine H-2Kbm1 genes. S1-nuclease analysis of the mRNA products from these genes before

  15. ASCR Science Network Requirements

    Energy Technology Data Exchange (ETDEWEB)

    Dart, Eli; Tierney, Brian

    2009-08-24

    The Energy Sciences Network (ESnet) is the primary provider of network connectivity for the US Department of Energy Office of Science, the single largest supporter of basic research in the physical sciences in the United States. In support of the Office of Science programs, ESnet regularly updates and refreshes its understanding of the networking requirements of the instruments, facilities, scientists, and science programs that it serves. This focus has helped ESnet to be a highly successful enabler of scientific discovery for over 20 years. In April 2009 ESnet and the Office of Advanced Scientific Computing Research (ASCR), of the DOE Office of Science, organized a workshop to characterize the networking requirements of the programs funded by ASCR. The ASCR facilities anticipate significant increases in wide area bandwidth utilization, driven largely by the increased capabilities of computational resources and the wide scope of collaboration that is a hallmark of modern science. Many scientists move data sets between facilities for analysis, and in some cases (for example the Earth System Grid and the Open Science Grid), data distribution is an essential component of the use of ASCR facilities by scientists. Due to the projected growth in wide area data transfer needs, the ASCR supercomputer centers all expect to deploy and use 100 Gigabit per second networking technology for wide area connectivity as soon as that deployment is financially feasible. In addition to the network connectivity that ESnet provides, the ESnet Collaboration Services (ECS) are critical to several science communities. ESnet identity and trust services, such as the DOEGrids certificate authority, are widely used both by the supercomputer centers and by collaborations such as Open Science Grid (OSG) and the Earth System Grid (ESG). Ease of use is a key determinant of the scientific utility of network-based services. Therefore, a key enabling aspect for scientists beneficial use of high

  16. Requirements of quality standards

    International Nuclear Information System (INIS)

    Mueller, J.

    1977-01-01

    The lecture traces the development of nuclear standards, codes, and Federal regulations on quality assurance (QA) for nuclear power plants and associated facilities. The technical evolution of the last twelve years, especially in the area of nuclear technology, led to different activities and regulatory initiatives, and the present result is: several nations have their own homemade standards. The lecture discusses the former and especially current activities in standard development, and gives a description of the requirements of QA-standards used in USA and Europe, especially Western Germany. Furthermore the lecture attempts to give a comparison and an evaluation of the international quality standards from the author's viewpoint. Finally the lecture presents an outlook for the future international implications of QA-standards. There is an urgent need within the nuclear industry for simplification and standardization of QA-standards. The relationship between the various standards, and the applicability of the standards need clarification and a better transparancy. To point out these problems is the purpose of the lecture. (orig.) [de

  17. Internationalization of regulatory requirements.

    Science.gov (United States)

    Juillet, Y

    2003-02-01

    The aim of harmonisation of medicines regulatory requirements is to allow the patient quicker access to new drugs and to avoid animal and human duplications. Harmonisation in the European Union (EU) is now completed, and has led to the submission of one dossier in one language study leading to European marketing authorizations, thanks in particular to efficacy guidelines published at the European level. With the benefit of the European experience since 1989, more than 40 guidelines have been harmonised amongst the EU, Japan and the USA through the International Conference on Harmonisation (ICH). ICH is a unique process gathering regulators and industry experts from the three regions. Its activity is built on expertise and trust. The Common Technical Document (CTD), an agreed common format for application in the three regions, is a logical follow-up to the ICH first phase harmonising the content of the dossier. The CTD final implementation in July 2003 will have considerable influence on the review process and on the exchange of information in the three regions.

  18. THE EQUALITY PRINCIPLE REQUIREMENTS

    Directory of Open Access Journals (Sweden)

    CLAUDIA ANDRIŢOI

    2013-05-01

    Full Text Available The problem premises and the objectives followed: the idea of inserting the equality principle between the freedom and the justice principles is manifested in positive law in two stages, as a general idea of all judicial norms and as requirement of the owner of a subjective right of the applicants of an objective law. Equality in face of the law and of public authorities can not involve the idea of standardization, of uniformity, of enlisting of all citizens under the mark of the same judicial regime, regardless of their natural or socio-professional situation. Through the Beijing Platform and the position documents of the European Commission we have defined the integrative approach of equality as representing an active and visible integration of the gender perspective in all sectors and at all levels. The research methods used are: the conceptualist method, the logical method and the intuitive method necessary as means of reasoning in order to argue our demonstration. We have to underline the fact that the system analysis of the research methods of the judicial phenomenon doesn’t agree with “value ranking”, because one value cannot be generalized in rapport to another. At the same time, we must fight against a methodological extremism. The final purpose of this study is represented by the reaching of the perfecting/excellence stage by all individuals through the promotion of equality and freedom. This supposes the fact that the existence of a non-discrimination favourable frame (fairness represents a means and a condition of self-determination, and the state of perfection/excellency is a result of this self-determination, the condition necessary for the obtaining of this nondiscrimination frame for all of us and in conditions of freedom for all individuals, represents the same condition that promotes the state of perfection/excellency. In conclusion we may state the fact that the equality principle represents a true catalyst of the

  19. Requirements Reasoning for Distributed Requirements Analysis using Semantic Wiki

    NARCIS (Netherlands)

    Liang, Peng; Avgeriou, Paris; Clerc, Viktor

    2009-01-01

    In large-scale collaborative software projects, thousands of requirements with complex interdependencies and different granularity spreading in different levels are elicited, documented, and evolved during the project lifecycle. Non-technical stakeholders involved in requirements engineering

  20. Requirement Assurance: A Verification Process

    Science.gov (United States)

    Alexander, Michael G.

    2011-01-01

    Requirement Assurance is an act of requirement verification which assures the stakeholder or customer that a product requirement has produced its "as realized product" and has been verified with conclusive evidence. Product requirement verification answers the question, "did the product meet the stated specification, performance, or design documentation?". In order to ensure the system was built correctly, the practicing system engineer must verify each product requirement using verification methods of inspection, analysis, demonstration, or test. The products of these methods are the "verification artifacts" or "closure artifacts" which are the objective evidence needed to prove the product requirements meet the verification success criteria. Institutional direction is given to the System Engineer in NPR 7123.1A NASA Systems Engineering Processes and Requirements with regards to the requirement verification process. In response, the verification methodology offered in this report meets both the institutional process and requirement verification best practices.

  1. Waste management system requirements document

    International Nuclear Information System (INIS)

    1991-02-01

    This volume defines the top level requirements for the Mined Geologic Disposal System (MGDS). It is designed to be used in conjunction with Volume 1 of the WMSR, General System Requirements. It provides a functional description expanding the requirements allocated to the MGDS in Volume 1 and elaborates on each requirement by providing associated performance criteria as appropriate. Volumes 1 and 4 of the WMSR provide a minimum set of requirements that must be satisfied by the final MGDS design. This document sets forth specific requirements that must be fulfilled. It is not the intent or purpose of this top level document to describe how each requirement is to be satisfied in the final MGDS design. Each subsequent level of the technical document hierarchy must provide further guidance and definition as to how each of these requirements is to be implemented in the design. It is expected that each subsequent level of requirements will be significantly more detailed. Section 2 of this volume provides a functional description of the MGDS. Each function is addressed in terms of requirements, and performance criteria. Section 3 provides a list of controlling documents. Each document cited in a requirement of Chapter 2 is included in this list and is incorporated into this document as a requirement on the final system. The WMSR addresses only federal requirements (i.e., laws, regulations and DOE orders). State and local requirements are not addressed. However, it will be specifically noted at the potentially affected WMSR requirements that there could be additional or more stringent regulations imposed by a state or local requirements or administering agency over the cited federal requirements

  2. Imperfect Requirements in Software Development

    NARCIS (Netherlands)

    Noppen, J.A.R.; van den Broek, P.M.; Aksit, Mehmet; Sawyer, Pete; Paech, Barbara; Heymans, Patrick

    2007-01-01

    Requirement Specifications are very difficult to define. Due to lack of information and differences in interpretation, software engineers are faced with the necessity to redesign and iterate. This imperfection in software requirement specifications is commonly addressed by incremental design. In

  3. Requirement Development Process and Tools

    Science.gov (United States)

    Bayt, Robert

    2017-01-01

    Requirements capture the system-level capabilities in a set of complete, necessary, clear, attainable, traceable, and verifiable statements of need. Requirements should not be unduly restrictive, but should set limits that eliminate items outside the boundaries drawn, encourage competition (or alternatives), and capture source and reason of requirement. If it is not needed by the customer, it is not a requirement. They establish the verification methods that will lead to product acceptance. These must be reproducible assessment methods.

  4. Waste Management System Requirement document

    International Nuclear Information System (INIS)

    1990-04-01

    This volume defines the top level technical requirements for the Monitored Retrievable Storage (MRS) facility. It is designed to be used in conjunction with Volume 1, General System Requirements. Volume 3 provides a functional description expanding the requirements allocated to the MRS facility in Volume 1 and, when appropriate, elaborates on requirements by providing associated performance criteria. Volumes 1 and 3 together convey a minimum set of requirements that must be satisfied by the final MRS facility design without unduly constraining individual design efforts. The requirements are derived from the Nuclear Waste Policy Act of 1982 (NWPA), the Nuclear Waste Policy Amendments Act of 1987 (NWPAA), the Environmental Protection Agency's (EPA) Environmental Standards for the Management and Disposal of Spent Nuclear Fuel (40 CFR 191), NRC Licensing Requirements for the Independent Storage of Spent Nuclear and High-Level Radioactive Waste (10 CFR 72), and other federal statutory and regulatory requirements, and major program policy decisions. This document sets forth specific requirements that will be fulfilled. Each subsequent level of the technical document hierarchy will be significantly more detailed and provide further guidance and definition as to how each of these requirements will be implemented in the design. Requirements appearing in Volume 3 are traceable into the MRS Design Requirements Document. Section 2 of this volume provides a functional breakdown for the MRS facility. 1 tab

  5. Physician Requirements-1990. For Cardiology.

    Science.gov (United States)

    Tracy, Octavious; Birchette-Pierce, Cheryl

    Professional requirements for physicians specializing in cardiology were estimated to assist policymakers in developing guidelines for graduate medical education. The determination of physician requirements was based on an adjusted needs rather than a demand or utilization model. For each illness, manpower requirements were modified by the…

  6. Capital Requirements and Banks' Leniency

    DEFF Research Database (Denmark)

    Dietrich, J. Kimball; Wihlborg, Clas

    2003-01-01

    We investigate the effect of changes in capital regulation on the strictness(leniency) of loan terms using a simple model of bank capital requirements andasset quality examinations. Banks offer different levels of `leniency' in the senseof willingness to offer automatic extensions of loans...... rates. As capital requirements increase thedifference between initial capital levels and between interest rates of strict andlenient banks decrease. Thus, higher capital requirements in recessions tend toreduce the interest rate premium paid for leniency. If a recession is interpreted asan increase...... in the required return, the interest rate premium paid for leniency isincreased in recession at a given level of required capital....

  7. Tool-based requirement traceability between requirement and design artifacts

    CERN Document Server

    Turban, Bernhard

    2013-01-01

    Processes for developing safety-critical systems impose special demands on ensuring requirements traceability. Achieving valuable traceability information, however, is especially difficult concerning the transition from requirements to design. Bernhard Turban analyzes systems and software engineering theories cross-cutting the issue (embedded systems development, systems engineering, software engineering, requirements engineering and management, design theory and processes for safety-critical systems). As a solution, the author proposes a new tool approach to support designers in their thinkin

  8. Piv site-specific invertase requires a DEDD motif analogous to the catalytic center of the RuvC Holliday junction resolvases.

    Science.gov (United States)

    Buchner, John M; Robertson, Anne E; Poynter, David J; Denniston, Shelby S; Karls, Anna C

    2005-05-01

    Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertion elements from the IS110/IS492 family and shows no similarity to the site-specific recombinases of the tyrosine- or serine-recombinase families. Piv tertiary structure is predicted to include the RNase H-like fold that typically encompasses the catalytic site of the recombinases or nucleases of the retroviral integrase superfamily, including transposases and RuvC-like Holliday junction resolvases. Analogous to the DDE and DEDD catalytic motifs of transposases and RuvC, respectively, four Piv acidic residues D9, E59, D101, and D104 appear to be positioned appropriately within the RNase H fold to coordinate two divalent metal cations. This suggests mechanistic similarity between site-specific inversion mediated by Piv and transposition or endonucleolytic reactions catalyzed by enzymes of the retroviral integrase superfamily. The role of the DEDD motif in Piv catalytic activity was addressed using Piv variants that are substituted individually or multiply at these acidic residues and assaying for in vivo inversion, intermolecular recombination, and DNA binding activities. The results indicate that all four residues of the DEDD motif are required for Piv catalytic activity. The DEDD residues are not essential for inv recombination site recognition and binding, but this acidic tetrad does appear to contribute to the stability of Piv-inv interactions. On the basis of these results, a working model for Piv-mediated inversion that includes resolution of a Holliday junction is presented.

  9. National Ignition Facility site requirements

    International Nuclear Information System (INIS)

    1996-07-01

    The Site Requirements (SR) provide bases for identification of candidate host sites for the National Ignition Facility (NIF) and for the generation of data regarding potential actual locations for the facilities. The SR supplements the NIF Functional Requirements (FR) with information needed for preparation of responses to queries for input to HQ DOE site evaluation. The queries are to include both documents and explicit requirements for the potential host site responses. The Sr includes information extracted from the NIF FR (for convenience), data based on design approaches, and needs for physical and organization infrastructure for a fully operational NIF. The FR and SR describe requirements that may require new construction or may be met by use or modification of existing facilities. The SR do not establish requirements for NIF design or construction project planning. The SR document does not constitute an element of the NIF technical baseline

  10. UTM TCL2 Software Requirements

    Science.gov (United States)

    Smith, Irene S.; Rios, Joseph L.; McGuirk, Patrick O.; Mulfinger, Daniel G.; Venkatesan, Priya; Smith, David R.; Baskaran, Vijayakumar; Wang, Leo

    2017-01-01

    The Unmanned Aircraft Systems (UAS) Traffic Management (UTM) Technical Capability Level (TCL) 2 software implements the UTM TCL 2 software requirements described herein. These software requirements are linked to the higher level UTM TCL 2 System Requirements. Each successive TCL implements additional UTM functionality, enabling additional use cases. TCL 2 demonstrated how to enable expanded multiple operations by implementing automation for beyond visual line-of-sight, tracking operations, and operations flying over sparsely populated areas.

  11. Information requirements for enterprise systems

    OpenAIRE

    Sommerville, Ian; Lock, Russell; Storer, Tim

    2012-01-01

    In this paper, we discuss an approach to system requirements engineering, which is based on using models of the responsibilities assigned to agents in a multi-agency system of systems. The responsibility models serve as a basis for identifying the stakeholders that should be considered in establishing the requirements and provide a basis for a structured approach, described here, for information requirements elicitation. We illustrate this approach using a case study drawn from civil emergenc...

  12. Addressing the Resource Requirements Mismatch

    National Research Council Canada - National Science Library

    Braun, William

    2003-01-01

    ... on the other, appear to be developing a requirements-resource mismatch. The goals and objectives of the transformation rhetoric intuitively resonate with the military's increasingly technologic culture...

  13. Requirements for Medical Modeling Languages

    Science.gov (United States)

    van der Maas, Arnoud A.F.; Ter Hofstede, Arthur H.M.; Ten Hoopen, A. Johannes

    2001-01-01

    Objective: The development of tailor-made domain-specific modeling languages is sometimes desirable in medical informatics. Naturally, the development of such languages should be guided. The purpose of this article is to introduce a set of requirements for such languages and show their application in analyzing and comparing existing modeling languages. Design: The requirements arise from the practical experience of the authors and others in the development of modeling languages in both general informatics and medical informatics. The requirements initially emerged from the analysis of information modeling techniques. The requirements are designed to be orthogonal, i.e., one requirement can be violated without violation of the others. Results: The proposed requirements for any modeling language are that it be “formal” with regard to syntax and semantics, “conceptual,” “expressive,” “comprehensible,” “suitable,” and “executable.” The requirements are illustrated using both the medical logic modules of the Arden Syntax as a running example and selected examples from other modeling languages. Conclusion: Activity diagrams of the Unified Modeling Language, task structures for work flows, and Petri nets are discussed with regard to the list of requirements, and various tradeoffs are thus made explicit. It is concluded that this set of requirements has the potential to play a vital role in both the evaluation of existing domain-specific languages and the development of new ones. PMID:11230383

  14. Federal Environmental Requirements for Construction

    Data.gov (United States)

    Department of Veterans Affairs — This guide provides information on federal environmental requirements for construction projects. It is written primarily for owners of construction projects and for...

  15. Consensus standard requirements and guidance

    International Nuclear Information System (INIS)

    Putman, V.L.

    1995-01-01

    This report presents information from the ANS Criticality Alarm System Workshop relating to the consensus standard requirements and guidance. Topics presented include: definition; nomenclature; requirements and recommendations; purpose of criticality alarms; design criteria; signal characteristics; reliability, dependability and durability; tests; and emergency preparedness and planning

  16. Humidity requirements in WSCF Laboratories

    International Nuclear Information System (INIS)

    Evans, R.A.

    1994-01-01

    The purpose of this paper is to develop and document a position on Relative Humidity (RH) requirements in the WSCF Laboratories. A current survey of equipment vendors for Organic, Inorganic and Radiochemical laboratories indicate that 25% - 80% relative humidity may meet the environmental requirements for safe operation and protection of all the laboratory equipment

  17. Requirements Engineering for Pervasive Services

    NARCIS (Netherlands)

    Kolos, L.; Poulisse, Gert-Jan; van Eck, Pascal; Videira lopes, C.; Schaefer, S.; Clarke, S.; Elrad, T.; Jahnke, J.

    2005-01-01

    Developing pervasive mobile services for a mass market of end customers entails large up-front investments and therefore a good understanding of customer requirements is of paramount importance. This paper presents an approach for developing requirements engineering method that takes distinguishing

  18. Physician Requirements-1990. For Nephrology.

    Science.gov (United States)

    Rosenbach, Joan K.

    Professional requirements for physicians specializing in nephrology were estimated to assist policymakers in developing guidelines for graduate medical education. In estimating service requirements for nephrology, a nephrology Delphi panel reviewed reference and incidence-prevalence and utilization data for 34 conditions that are treated in the…

  19. The sp7 gene is required for maturation of osteoblast-lineage cells in medaka (Oryzias latipes) vertebral column development.

    Science.gov (United States)

    Azetsu, Yuki; Inohaya, Keiji; Takano, Yoshiro; Kinoshita, Masato; Tasaki, Mai; Kudo, Akira

    2017-11-15

    Sp7 is a zinc finger transcription factor that is essential for osteoblast differentiation in mammals. To verify the characteristic features of osteoblast-lineage cells in teleosts, we established medaka sp7 mutants using a transcription activator-like effector nuclease (TALEN) genome editing system. These mutants showed severe defects in the formation of skeletal structures. In particular, the neural and the hemal arches were not formed, although the chordal centra were formed. Analysis of the transgenic medaka revealed that sp7 mutant had normal distribution of type X collagen a1 a (col10a1a)-positive osteoblast-like cells around the centrum and at the proximal region of the vertebral arch. The sp7 mutant phenotype could be rescued by exogenous sp7 expression in col10a1a-positive cells, as well as in sp7-positive osteoblast cells. Furthermore, runx2-positive osteoblast progenitors were observed on the vertebral arches, but not on the centrum, during vertebral column development. In addition, these osteoblast progenitors differentiated into the col10a1a-positive cells. In sp7 mutant, the runx2-positive cells were normally distributed at the region of unformed vertebral arch but failed to differentiate into col10a1a-positive cells. These results indicate that osteoblast-lineage cells undergo two distinct differentiation processes during development of the vertebral arch and the centrum. Nevertheless, our results verified that sp7 gene expression in osteoblast-lineage cells is required for differentiation into mature osteoblasts to form the vertebral column and other skeletal structures. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. EVI and MDS/EVI are required for adult intestinal stem cell formation during postembryonic vertebrate development.

    Science.gov (United States)

    Okada, Morihiro; Shi, Yun-Bo

    2018-01-01

    The gene ectopic viral integration site 1 (EVI) and its variant myelodysplastic syndrome 1 (MDS)/EVI encode zinc-finger proteins that have been recognized as important oncogenes in various types of cancer. In contrast to the established role of EVI and MDS/EVI in cancer development, their potential function during vertebrate postembryonic development, especially in organ-specific adult stem cells, is unclear. Amphibian metamorphosis is strikingly similar to postembryonic development around birth in mammals, with both processes taking place when plasma thyroid hormone (T3) levels are high. Using the T3-dependent metamorphosis in Xenopus tropicalis as a model, we show here that high levels of EVI and MDS/EVI are expressed in the intestine at the climax of metamorphosis and are induced by T3. By using the transcription activator-like effector nuclease gene editing technology, we have knocked out both EVI and MDS/EVI and have shown that EVI and MDS/EVI are not essential for embryogenesis and premetamorphosis in X. tropicalis On the other hand, knocking out EVI and MDS/EVI causes severe retardation in the growth and development of the tadpoles during metamorphosis and leads to tadpole lethality at the climax of metamorphosis. Furthermore, the homozygous-knockout animals have reduced adult intestinal epithelial stem cell proliferation at the end of metamorphosis (for the few that survive through metamorphosis) or during T3-induced metamorphosis. These findings reveal a novel role of EVI and/or MDS/EVI in regulating the formation and/or proliferation of adult intestinal adult stem cells during postembryonic development in vertebrates.-Okada, M., Shi, Y.-B. EVI and MDS/EVI are required for adult intestinal stem cell formation during postembryonic vertebrate development. © FASEB.

  1. The NLC Software Requirements Methodology

    Energy Technology Data Exchange (ETDEWEB)

    Shoaee, Hamid

    2002-08-20

    We describe the software requirements and development methodology developed for the NLC control system. Given the longevity of that project, and the likely geographical distribution of the collaborating engineers, the planned requirements management process is somewhat more formal than the norm in high energy physics projects. The short term goals of the requirements process are to accurately estimate costs, to decompose the problem, and to determine likely technologies. The long term goal is to enable a smooth transition from high level functional requirements to specific subsystem and component requirements for individual programmers, and to support distributed development. The methodology covers both ends of that life cycle. It covers both the analytical and documentary tools for software engineering, and project management support. This paper introduces the methodology, which is fully described in [1].

  2. Biochemical properties of three plant nucleases with anticancer potential

    Czech Academy of Sciences Publication Activity Database

    Podzimek, Tomáš; Matoušek, Jaroslav; Lipovová, P.; Poučková, P.; Spiwok, V.; Šantrůček, J.

    2011-01-01

    Roč. 180, č. 2 (2011), s. 343-351 ISSN 0168-9452 R&D Projects: GA ČR GA521/09/1214 Institutional research plan: CEZ:AV0Z50510513 Keywords : dsRNase activity * N-glycosylation * Anti-tumor effect Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.945, year: 2011

  3. Synthesis, spectral properties and DNA binding and nuclease ...

    Indian Academy of Sciences (India)

    suggest a minor participation of 4f orbitals in bonding. 3.3 Description of the molecular structure of. [Ce(BPBH)2(NO3)3]. [Ce(BPBH)2(NO3)3] crystallized in monoclinic space group C2/c and the structure contains six neutral. Figure 1. Unit cell structure of [Ce(BPBH)2(NO3)3] with distorted icosahedrons around Ce(III) ...

  4. Crystallization of recombinant bifunctional nuclease TBN1 from tomato

    Czech Academy of Sciences Publication Activity Database

    Kovaľ, Tomáš; Lipovová, P.; Podzimek, Tomáš; Matoušek, Jaroslav; Dušková, Jarmila; Skálová, Tereza; Štěpánková, Andrea; Hašek, Jindřich; Dohnálek, Jan

    2011-01-01

    Roč. 67, č. 1 (2011), s. 124-128 ISSN 1744-3091 R&D Projects: GA ČR GA310/09/1407; GA ČR GA202/06/0757; GA ČR GA521/09/1214 Grant - others:AV ČR(CZ) AP0701 Program:Akademická prémie - Praemium Academiae Institutional research plan: CEZ:AV0Z10100521; CEZ:AV0Z40500505; CEZ:AV0Z50510513 Keywords : plant endonuclease * anti-tumor properties * crystallization * X-ray diffraction Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 0.506, year: 2011

  5. Synthesis, spectral properties and DNA binding and nuclease ...

    Indian Academy of Sciences (India)

    The central metal is 12 coordinated and the coordination polyhedron around the cerium atom can be described as a distorted icosahedron. The existence of nitrate. . . and CH. . . stacking interactions in the [Ce(BPBH)2(NO3)3] leads to a supramolecular arrangement in its network. The binding properties of these ...

  6. Synthesis, spectral properties and DNA binding and nuclease ...

    Indian Academy of Sciences (India)

    voltammetry was performed with a CH instrument 660C. Electrochemical analyzer and a conventional three elec- trode assembly, Ag/AgCl (1M KCl) as reference elec- trode, glassy carbon as working electrode and platinum as counter electrode. The E1/2 of ferrocence with respect to Ag/AgCl electrode is 0.38V. Nitrogen ...

  7. Crystal structures of two eukaryotic nucleases involved in RNA metabolism

    DEFF Research Database (Denmark)

    Jonstrup, Anette Thyssen; Midtgaard, Søren Fuglsang; Van, Lan Bich

    specific transcripts. Here, we present the crystal structure of the S. pombe Pop2p protein to 1.4 Å resolution. The high resolution structure provides a clear picture of the active site architecture. Structural alignment of single nucleotides and poly(A)-oligonucleotides from earlier co-crystal structures...... form the 3'-end of mRNA, is normally the first and also rate-limiting step in cellular mRNA degradation and therefore a key process in the control of eukaryotic mRNA turnover. Since Ccr4p is believed to be the main deadenylase the precise role of Pop2p in the complex is less clear. Nevertheless, Pop2p...

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

    Science.gov (United States)

    Freudenreich, Catherine H

    2018-01-11

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

  9. Managing System of Systems Requirements with a Requirements Screening Group

    Energy Technology Data Exchange (ETDEWEB)

    Ronald R. Barden

    2012-07-01

    Figuring out an effective and efficient way to manage not only your Requirement’s Baseline, but also the development of all your individual requirements during a Program’s/Project’s Conceptual and Development Life Cycle Stages can be both daunting and difficult. This is especially so when you are dealing with a complex and large System of Systems (SoS) Program with potentially thousands and thousands of Top Level Requirements as well as an equal number of lower level System, Subsystem and Configuration Item requirements that need to be managed. This task is made even more overwhelming when you have to add in integration with multiple requirements’ development teams (e.g., Integrated Product Development Teams (IPTs)) and/or numerous System/Subsystem Design Teams. One solution for tackling this difficult activity on a recent large System of Systems Program was to develop and make use of a Requirements Screening Group (RSG). This group is essentially a Team made up of co-chairs from the various Stakeholders with an interest in the Program of record that are enabled and accountable for Requirements Development on the Program/Project. The RSG co-chairs, often with the help of individual support team, work together as a Program Board to monitor, make decisions on, and provide guidance on all Requirements Development activities during the Conceptual and Development Life Cycle Stages of a Program/Project. In addition, the RSG can establish and maintain the Requirements Baseline, monitor and enforce requirements traceability across the entire Program, and work with other elements of the Program/Project to ensure integration and coordination.

  10. Industrial requirements in food safety.

    Science.gov (United States)

    Vincent, P M

    1990-01-01

    The principles of establishing industrial requirements in food safety are described, taking risk potentials all along the food chain into their respective account. Regulations will, in the future, lead to increased autocontrol in production. The rapid changes in food technology require constant adaptation to new problems, to keep the global quality of food at a high level. Regulatory authorities will, in the new European market, concentrate on enforcement of 'essential requirements' while industrialists will follow good manufacturing practices. Open dialogue between the latter, the former and the scientific community is highly desirable since mutual knowledge of the problem will help maintain a high level of food safety, for the benefit of everybody.

  11. Development of transportation operations requirements

    International Nuclear Information System (INIS)

    Grady, S.T.; Best, R.E.; Danese, F.L.; Peterson, R.W.; Pope, R.B.

    1990-01-01

    Transport conditions at various utility sties vary dramatically in terms of characteristics at and near the site, requirements, administrative procedures, and other factors. Continuation of design efforts for the OCRWM transportation operations system requires that the operating requirements for the transportation system -- quantity of fuel per unit time per site -- be identified so that the effect the variations have on the system can be accommodated. The approach outlined in this paper provides for an identification of specific sites, evaluation of shipment capabilities at each site, and integration of the sites into multi-site shipping campaigns to scope the logistics management problem for the transportation operations system. 1 fig., 1 tab

  12. Deaf mobile application accessibility requirements

    Science.gov (United States)

    Nathan, Shelena Soosay; Hussain, Azham; Hashim, Nor Laily

    2016-08-01

    Requirement for deaf mobile applications need to be analysed to ensure the disabilities need are instilled into the mobile applications developed for them. Universal design is understandable to comply every user needs, however specific disability is argued by the authors to have different need and requirements. These differences are among the reasons for these applications being developed to target for a specific group of people, however they are less usable and later abandoned. This study focuses on deriving requirements that are needed by the deaf in their mobile applications that are meant specifically for them. Studies on previous literature was conducted it can be concluded that graphic, text, multimedia and sign language interpreter are among mostly required features to be included in their mobile application to ensure the applications are usable for this community.

  13. Electrocardiogram Scanner-System Requirements

    Science.gov (United States)

    1973-03-01

    An experimental and analytical study has been conducted to establish the feasibility for scanning and digitizing electrocardiogram records. The technical requirements and relative costs for two systems are discussed herein. One is designed to automat...

  14. PFP requirements development planning guide

    International Nuclear Information System (INIS)

    SINCLAIR, J.C.

    1999-01-01

    The PFP Requirements Development Planning Guide presents the strategy and process used for the identification, allocation, and maintenance of requirements within the Plutonium Finishing Plant (PFP) integrated project baseline. Future revisions to this document will be included as attachments (e.g., results of the PFP Requirements Analysis attributable to this approach). This document is intended be a Project-owned management tool. As such, this document will periodically require revisions resulting from improvements of the information, processes, and techniques as now described. Future updates may be made to this document by PFP management and final approval of the content will be accomplished in a Baseline Change Request as it impacts the Multi-Year Work Plan, or baseline information managed in the Hanford Site Systems Engineering Baseline

  15. Guest Comment: Universal Language Requirement.

    Science.gov (United States)

    Sherwood, Bruce Arne

    1979-01-01

    Explains that reading English among Scientists is almost universal, however, there are enormous problems with spoken English. Advocates the use of Esperanto as a viable alternative, and as a language requirement for graduate work. (GA)

  16. Data Requirements for Pesticide Registration

    Science.gov (United States)

    In evaluating a pesticide registration application, we assess a wide variety of potential human health and environmental effects associated with use of the product. Learn about these data requirements.

  17. Fusion technology status and requirements

    International Nuclear Information System (INIS)

    Thomassen, K.I.

    1982-01-01

    This paper summarizes the status of fusion technology and discusses the requirements to be met in order to build a demonstration fusion plant. Strategies and programmatic considerations in pursuing engineering feasibility are also outlined

  18. Grant Closeout Requirements and Reports

    Science.gov (United States)

    Requirements and reports to comply with grant closeout, including Final Federal Financial Report (FFR, SF425); Final Research Performance Progress Report (FRPPR); Interim Research Performance Progress Report (IRPPR); Final Invention Statement (FIS, HHS

  19. Requirement Tracing using Term Extraction

    OpenAIRE

    Al-Saati, Najla; Abdul-Jaleel, Raghda

    2015-01-01

    Requirements traceability is an essential step in ensuring the quality of software during the early stages of its development life cycle. Requirements tracing usually consists of document parsing, candidate link generation and evaluation and traceability analysis. This paper demonstrates the applicability of Statistical Term Extraction metrics to generate candidate links. It is applied and validated using two data sets and four types of filters two for each data set, 0.2 and 0.25 for MODIS, 0...

  20. Capturing Requirements for Autonomous Spacecraft with Autonomy Requirements Engineering

    Science.gov (United States)

    Vassev, Emil; Hinchey, Mike

    2014-08-01

    The Autonomy Requirements Engineering (ARE) approach has been developed by Lero - the Irish Software Engineering Research Center within the mandate of a joint project with ESA, the European Space Agency. The approach is intended to help engineers develop missions for unmanned exploration, often with limited or no human control. Such robotics space missions rely on the most recent advances in automation and robotic technologies where autonomy and autonomic computing principles drive the design and implementation of unmanned spacecraft [1]. To tackle the integration and promotion of autonomy in software-intensive systems, ARE combines generic autonomy requirements (GAR) with goal-oriented requirements engineering (GORE). Using this approach, software engineers can determine what autonomic features to develop for a particular system (e.g., a space mission) as well as what artifacts that process might generate (e.g., goals models, requirements specification, etc.). The inputs required by this approach are the mission goals and the domain-specific GAR reflecting specifics of the mission class (e.g., interplanetary missions).

  1. Quality Assurance Requirements and Description

    International Nuclear Information System (INIS)

    Ram Murthy

    2002-01-01

    The Quality Assurance Requirements and Description (QARD) is the principal Quality Assurance (QA) document for the Civilian Radioactive Waste Management Program (Program). It establishes the minimum requirements for the QA program [INTRODUCTION :1p2s (NOT A REQUIREMENT)]. The QARD contains regulatory requirements and program commitments necessary for the development of an effective QA program [INTRODUCTION :1p3s (NOT A REQUIREMENT)]. Implementing documents must be based on, and be consistent with the QARD. The QARD applies to the following: (1) Acceptance of spent nuclear fuel and high-level waste. (2) Transport of spent nuclear fuel and high-level waste. (3) Storage of spent nuclear fuel through receipt of storage cask certification or a facility operating license. (4) Monitored Geologic Repository, including the site characterization activities [Exploratory Studies Facility (ESF) and surface based testing], through receipt of an operating license. (5) High-level waste form development through qualification, production, and acceptance. (6) Characterization of DOE spent nuclear fuel, and conditioning through acceptance of DOE spent nuclear fuel. Section 2.0, Quality Assurance Program, defines in greater detail criteria for determining work subject to the QARD

  2. Requirement Metrics for Risk Identification

    Science.gov (United States)

    Hammer, Theodore; Huffman, Lenore; Wilson, William; Rosenberg, Linda; Hyatt, Lawrence

    1996-01-01

    The Software Assurance Technology Center (SATC) is part of the Office of Mission Assurance of the Goddard Space Flight Center (GSFC). The SATC's mission is to assist National Aeronautics and Space Administration (NASA) projects to improve the quality of software which they acquire or develop. The SATC's efforts are currently focused on the development and use of metric methodologies and tools that identify and assess risks associated with software performance and scheduled delivery. This starts at the requirements phase, where the SATC, in conjunction with software projects at GSFC and other NASA centers is working to identify tools and metric methodologies to assist project managers in identifying and mitigating risks. This paper discusses requirement metrics currently being used at NASA in a collaborative effort between the SATC and the Quality Assurance Office at GSFC to utilize the information available through the application of requirements management tools.

  3. Authorization basis requirements comparison report

    International Nuclear Information System (INIS)

    Brantley, W.M.

    1997-01-01

    The TWRS Authorization Basis (AB) consists of a set of documents identified by TWRS management with the concurrence of DOE-RL. Upon implementation of the TWRS Basis for Interim Operation (BIO) and Technical Safety Requirements (TSRs), the AB list will be revised to include the BIO and TSRs. Some documents that currently form part of the AB will be removed from the list. This SD identifies each - requirement from those documents, and recommends a disposition for each to ensure that necessary requirements are retained when the AB is revised to incorporate the BIO and TSRs. This SD also identifies documents that will remain part of the AB after the BIO and TSRs are implemented. This document does not change the AB, but provides guidance for the preparation of change documentation

  4. Authorization basis requirements comparison report

    Energy Technology Data Exchange (ETDEWEB)

    Brantley, W.M.

    1997-08-18

    The TWRS Authorization Basis (AB) consists of a set of documents identified by TWRS management with the concurrence of DOE-RL. Upon implementation of the TWRS Basis for Interim Operation (BIO) and Technical Safety Requirements (TSRs), the AB list will be revised to include the BIO and TSRs. Some documents that currently form part of the AB will be removed from the list. This SD identifies each - requirement from those documents, and recommends a disposition for each to ensure that necessary requirements are retained when the AB is revised to incorporate the BIO and TSRs. This SD also identifies documents that will remain part of the AB after the BIO and TSRs are implemented. This document does not change the AB, but provides guidance for the preparation of change documentation.

  5. Fusion Energy Sciences Network Requirements

    Energy Technology Data Exchange (ETDEWEB)

    Dart, Eli [ESNet, Berkeley, CA (United States); Tierney, Brian [ESNet, Berkeley, CA (United States)

    2012-09-26

    The Energy Sciences Network (ESnet) is the primary provider of network connectivity for the U.S. Department of Energy Office of Science, the single largest supporter of basic research in the physical sciences in the United States. In support of the Office of Science programs, ESnet regularly updates and refreshes its understanding of the networking requirements of the instruments, facilities, scientists, and science programs that it serves. This focus has helped ESnet to be a highly successful enabler of scientific discovery for over 25 years. In December 2011, ESnet and the Office of Fusion Energy Sciences (FES), of the DOE Office of Science (SC), organized a workshop to characterize the networking requirements of the programs funded by FES. The requirements identified at the workshop are summarized in the Findings section, and are described in more detail in the body of the report.

  6. Buddy Tag CONOPS and Requirements.

    Energy Technology Data Exchange (ETDEWEB)

    Brotz, Jay Kristoffer [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Deland, Sharon M. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2015-12-01

    This document defines the concept of operations (CONOPS) and the requirements for the Buddy Tag, which is conceived and designed in collaboration between Sandia National Laboratories and Princeton University under the Department of State Key VerificationAssets Fund. The CONOPS describe how the tags are used to support verification of treaty limitations and is only defined to the extent necessary to support a tag design. The requirements define the necessary functions and desired non-functional features of the Buddy Tag at a high level

  7. Overview of SSC accelerator requirements

    International Nuclear Information System (INIS)

    Dugan, G.

    1992-03-01

    This paper will present a general overview of the requirements of the Superconducting Super Collider (SSC) accelerators. Each accelerator in the injector chain will be discussed separately, followed by a discussion of the collider itself. In conclusion, the top level requirements of the overall accelerator system will be presented. For each accelerator, the primary operating parameters will be presented in tabular form. A brief narrative discussion of the principal technical features of each machine will be given. Finally, the principal technical design challenges for the machine will be noted, together with the currently planned solution to these challenges

  8. Lysine requirements of growing emus.

    Science.gov (United States)

    Mannion, P F; Kent, P B; Barram, K M; Trappett, P C; Blight, G W; Sales, J

    1999-05-01

    1. The lysine requirement of growing emus between 23 and 65 d of age was determined according to growth response variables. 2. The optimal lysine requirement of emus was found to be 0.83 and 0.90 g/MJ ME for growth rate and gain:food ratio respectively. These findings are in accordance with the recommended value of 0.80 g/MJ ME, but is lower than the recommended value for ostriches (1.02 g/MJ ME) and higher than determined values for broilers (0.75 g/MJ ME) of the same age range.

  9. An Introduction to Requirements Traceability

    NARCIS (Netherlands)

    Wieringa, Roelf J.

    This report surveys the requirements traceability literature and gives some recommendations for further research and for an approach to consultancy concerning traceability in the 2RARE project. The problem of maintaining traceability in a development project is viewed as the problem of maintaining

  10. Requirements for flexible learner monitoring

    NARCIS (Netherlands)

    Glahn, Christian; Specht, Marcus; Koper, Rob

    2007-01-01

    Glahn, C., Specht, M., & Koper, R. (2007). Requirements for flexible learner monitoring. In T. Navarette, J. Blat & R. Koper (Eds.). Proceedings of the 3rd TENCompetence Open Workshop 'Current Research on IMS Learning Design and Lifelong Competence Development Infrastructures' (pp. 89-96). June,

  11. Requirements Engineering: Solutions and Trends

    NARCIS (Netherlands)

    Ebert, C.; Wieringa, Roelf J.; Aurum, A.; Wohlin, C.

    2005-01-01

    This last chapter of the book describes solutions and trends in the discipline of RE. Starting from a wrap-up of what was presented throughout this book, it suggests a framework of requirements engineering and indicates what current solutions are available in this framework. Beyond providing a short

  12. Utilizing inheritance in requirements engineering

    Science.gov (United States)

    Kaindl, Hermann

    1994-01-01

    The scope of this paper is the utilization of inheritance for requirements specification, i.e., the tasks of analyzing and modeling the domain, as well as forming and defining requirements. Our approach and the tool supporting it are named RETH (Requirements Engineering Through Hypertext). Actually, RETH uses a combination of various technologies, including object-oriented approaches and artificial intelligence (in particular frames). We do not attempt to exclude or replace formal representations, but try to complement and provide means for gradually developing them. Among others, RETH has been applied in the CERN (Conseil Europeen pour la Rechereche Nucleaire) Cortex project. While it would be impossible to explain this project in detail here, it should be sufficient to know that it deals with a generic distributed control system. Since this project is not finished yet, it is difficult to state its size precisely. In order to give an idea, its final goal is to substitute the many existing similar control systems at CERN by this generic approach. Currently, RETH is also tested using real-world requirements for the Pastel Mission Planning System at ESOC in Darmstadt. First, we outline how hypertext is integrated into a frame system in our approach. Moreover, the usefulness of inheritance is demonstrated as performed by the tool RETH. We then summarize our experiences of utilizing inheritance in the Cortex project. Lastly, RETH will be related to existing work.

  13. Requirements for effective modelling strategies.

    NARCIS (Netherlands)

    Gaunt, J.L.; Riley, J.; Stein, A.; Penning de Vries, F.W.T.

    1997-01-01

    As the result of a recent BBSRC-funded workshop between soil scientists, modellers, statisticians and others to discuss issues relating to the derivation of complex environmental models, a set of modelling guidelines is presented and the required associated research areas are discussed.

  14. Time Elements May Require Change.

    Science.gov (United States)

    Clemons, Molly J.

    1993-01-01

    Argues that school districts may need to use a different time frame to accommodate the varied time requirements of the more flexible outcome-based education. Discusses three "alternate" scheduling methods currently in use and how they affect the teaching of journalism. (SR)

  15. Conversion of dependability deterministic requirements into probabilistic requirements

    International Nuclear Information System (INIS)

    Bourgade, E.; Le, P.

    1993-02-01

    This report concerns the on-going survey conducted jointly by the DAM/CCE and NRE/SR branches on the inclusion of dependability requirements in control and instrumentation projects. Its purpose is to enable a customer (the prime contractor) to convert into probabilistic terms dependability deterministic requirements expressed in the form ''a maximum permissible number of failures, of maximum duration d in a period t''. The customer shall select a confidence level for each previously defined undesirable event, by assigning a maximum probability of occurrence. Using the formulae we propose for two repair policies - constant rate or constant time - these probabilized requirements can then be transformed into equivalent failure rates. It is shown that the same formula can be used for both policies, providing certain realistic assumptions are confirmed, and that for a constant time repair policy, the correct result can always be obtained. The equivalent failure rates thus determined can be included in the specifications supplied to the contractors, who will then be able to proceed to their previsional justification. (author), 8 refs., 3 annexes

  16. 12 CFR 614.4935 - Escrow requirement.

    Science.gov (United States)

    2010-01-01

    ... 12 Banks and Banking 6 2010-01-01 2010-01-01 false Escrow requirement. 614.4935 Section 614.4935... Insurance Requirements § 614.4935 Escrow requirement. If a System institution requires the escrow of taxes... shall also require the escrow of all premiums and fees for any flood insurance required under § 614.4930...

  17. The European Utility Requirement Document

    International Nuclear Information System (INIS)

    Roche, I.I.

    1999-01-01

    The major European electricity producers work on a common requirement document for future LWR plants since 1992. They aim at requirements acceptable together by the owners, the public and the authorities. Thus the designers can develop standard LWR designs acceptable everywhere in Europe and the utilities can open their consultations to vendors on common bases. Such a standardisation promotes an improvement of generation costs and of safety : public and authorities acceptance should be improved as well ; significant savings are expected in development and construction costs. Since the early stages of the project, the EUR group has grown significantly. It now includes utilities from nine European countries. Utilities from two other European countries are joining the group. Specific cooperation agreements are also in progress with a few extra-European partners

  18. Documentation requirements for radiation sterilization

    DEFF Research Database (Denmark)

    Miller, A.

    1995-01-01

    Several standards are recently approved or are under development by the standard organizations ISO and CEN in the field of radiation sterilization. Particularly in Europe these standards define new requirements on some issues and on other issues they emphasize the necessary documentation for appr......Several standards are recently approved or are under development by the standard organizations ISO and CEN in the field of radiation sterilization. Particularly in Europe these standards define new requirements on some issues and on other issues they emphasize the necessary documentation...... for approval of radiation sterilized products. The impact of these standards on the radiation sterilization is discussed, with special attention given to a few special issues, mainly traceability and uncertainty of measurement results....

  19. ROS signalling - specificity is required

    DEFF Research Database (Denmark)

    Møller, Ian M; Sweetlove, Lee J

    2010-01-01

    Reactive oxygen species (ROS) production increases in plants under stress. ROS can damage cellular components, but they can also act in signal transduction to help the cell counteract the oxidative damage in the stressed compartment. H2O2 might induce a general stress response, but it does not have...... the required specificity to selectively regulate nuclear genes required for dealing with localized stress, e.g. in chloroplasts or mitochondria. Here we argue that peptides deriving from proteolytic breakdown of oxidatively damaged proteins have the requisite specificity to act as secondary ROS messengers...... and regulate source-specific genes and in this way contribute to retrograde ROS signalling during oxidative stress. Likewise, unmodified peptides deriving from the breakdown of redundant proteins could help coordinate organellar and nuclear gene expression...

  20. Grading of quality assurance requirements

    International Nuclear Information System (INIS)

    1991-01-01

    The present Manual provides guidance and illustrative examples for applying a method by which graded quality assurance requirements may be determined and adapted to the items and services of a nuclear power plant in conformance with the requirements of the IAEA Nuclear Safety Standards (NUSS) Code and Safety Guides on quality assurance. The Manual replaces the previous publication IAEA-TECDOC-303 on the same subject. Various methods of grading quality assurance are available in a number of Member States. During the development of the present Manual it was not considered practical to attempt to resolve the differences between those methods and it was preferred to identify and benefit from the good practices available in all the methods. The method presented in this Manual deals with the aspects of management, documentation, control, verification and administration which affect quality. 1 fig., 4 tabs

  1. Host City Contract operational requirements

    OpenAIRE

    2015-01-01

    The Host City Contract - Operational Requirements (the “HCC Operational Requirements”) are an important part of the Host City Contract, detailing a set of core elements for the project, which provide Olympic quality conditions for the athletes and all participants, while at the same time allowing potential host cities to responsibly match their Games concepts to their own sport, economic, social, and environmental long-term planning needs.

  2. Nutritional requirements after bariatric surgery.

    Science.gov (United States)

    Bosnic, Gordana

    2014-06-01

    This article presents an overview of postoperative nutritional requirements and goals following bariatric surgery. It summarizes current diet progression and nutrient intake guidelines geared toward optimizing weight loss and maintaining adequate nutritional status, nutrient absorption, as well as hydration. The article further emphasizes the importance of postoperative follow-up with a bariatric multidisciplinary team for appropriate postoperative care, diet management, and nutrient deficiency screenings. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Regulatory requirements for radiation protection

    International Nuclear Information System (INIS)

    Mason, E.A.; Cunningham, R.E.; Hard, J.E.; Mattson, R.J.; Smith, R.D.; Peterson, H.T. Jr.

    1977-01-01

    Regulatory requirements for radiation protection have evolved and matured over several decades. Due to the wide adoption of recommendations of the International Commission on Radiation Protection (ICRP), there exists international agreement on the principles to be followed for radiation protection. This foundation will be increasingly important due to the growing need for international agreements and standards for radiation protection and radioactive materials management. During the infancy of the commercial nuclear industry, primary reliance was placed on the protection of the individual, both in the work force and as a member of the public. With the growth of nuclear power in the 1960's and 1970's, environmental impact assessments and expert reviews of bio-effects data have focused attention on statistical risks to large population groups and the use of the collective dose commitment concept to estimate potential effects. The potential release of long-lived radionuclides from the nuclear fuel cycle requires further consideration of radionuclide accumulation in the biosphere and calls for controls conceived and implemented at the international level. The initial development efforts for addressing these concerns already have been instituted by the ICRP and the IAEA. However, formal international agreements and a unified set of international standards may be required to implement the recommendations of these groups. Further international efforts in the field of radiation protection are also called for in developing waste management practices and radioactive effluent control technology, in site selection for fuel reprocessing plants and waste dispersal facilities, and for ensuring safe transport of high-level wastes in various forms. Since the regulation of very low dose rates and doses will be involved, it will be useful to reexamine dose-effect relationships and societal goals for health protection. Improved criteria and methodologies for ''as low as readily

  4. Testing Requirements for Refractory Materials

    Science.gov (United States)

    Calle, Luz Marina; Hintze, Paul E.; Parlier, Christopher R.; Curran, Jerome P.; Kolody, Mark R.; Sampson, Jeffrey W,; Montgomery, Eliza M.

    2012-01-01

    Launch Pads 39A and 39B currently use refractory material (Fondu Fyre) in the flame trenches. This material was initially approved for the Saturn program. This material had a lifetime of 10years according to the manufacturer, and it has been used for over 40 years. As a consequence, the Fondu Fyre at Launch Complex 39 requires repair subsequent to almost every launch. A review of the literature indicates that the gunned Fondu Fyre refractory product (WA-1 G) was never tested prior to use. With the recent severe damage to the flame trenches, a new refractory material is sought to replace Fondu Fyre. In order to replace Fondu Fyre, a methodology to test and evaluate refractory products was developed. This paper outlines this methodology and discusses current testing requirements, as well as the laboratory testing that might be required. Furthermore, this report points out the necessity for subscale testing, the locations where this testing can be performed, and the parameters that will be necessary to qualify a product. The goal is to identify a more durable refractory material that has physical, chemical, and thermal properties suitable to withstand the harsh environment of the launch pads at KSC.

  5. Misplaced Cervical Screws Requiring Reoperation.

    Science.gov (United States)

    Peterson, Jeremy C; Arnold, Paul M; Smith, Zachary A; Hsu, Wellington K; Fehlings, Michael G; Hart, Robert A; Hilibrand, Alan S; Nassr, Ahmad; Rahman, Ra'Kerry K; Tannoury, Chadi A; Tannoury, Tony; Mroz, Thomas E; Currier, Bradford L; De Giacomo, Anthony F; Fogelson, Jeremy L; Jobse, Bruce C; Massicotte, Eric M; Riew, K Daniel

    2017-04-01

    A multicenter, retrospective case series. In the past several years, screw fixation of the cervical spine has become commonplace. For the most part, this is a safe, low-risk procedure. While rare, screw backout or misplaced screws can lead to morbidity and increased costs. We report our experiences with this uncommon complication. A multicenter, retrospective case series was undertaken at 23 institutions in the United States. Patients were included who underwent cervical spine surgery from January 1, 2005, to December 31, 2011, and had misplacement of screws requiring reoperation. Institutional review board approval was obtained at all participating institutions, and detailed records were sent to a central data center. A total of 12 903 patients met the inclusion criteria and were analyzed. There were 11 instances of screw backout requiring reoperation, for an incidence of 0.085%. There were 7 posterior procedures. Importantly, there were no changes in the health-related quality-of-life metrics due to this complication. There were no new neurologic deficits; a patient most often presented with pain, and misplacement was diagnosed on plain X-ray or computed tomography scan. The most common location for screw backout was C6 (36%). This study represents the largest series to tabulate the incidence of misplacement of screws following cervical spine surgery, which led to revision procedures. The data suggest this is a rare event, despite the widespread use of cervical fixation. Patients suffering this complication can require revision, but do not usually suffer neurologic sequelae. These patients have increased cost of care. Meticulous technique and thorough knowledge of the relevant anatomy are the best means of preventing this complication.

  6. Superluminal travel requires negative energies

    OpenAIRE

    Olum, Ken D.

    1998-01-01

    I investigate the relationship between faster-than-light travel and weak-energy-condition violation, i.e., negative energy densities. In a general spacetime it is difficult to define faster-than-light travel, and I give an example of a metric which appears to allow superluminal travel, but in fact is just flat space. To avoid such difficulties, I propose a definition of superluminal travel which requires that the path to be traveled reach a destination surface at an earlier time than any neig...

  7. Metabolic requirements for fetal growth.

    Science.gov (United States)

    Milley, J R; Simmons, M A

    1979-09-01

    Table 1 outlines a metabolic balance sheet for the sheep fetus. It is clear that maternal substrate concentrations as well as placental function are important in assuring the provision of adequate substrate to meet fetal metabolic and growth requirements. It is intriguing that the fetus appears to use substrates not usually regarded as important in extrauterine diets (lactate) and to use substrates for catabolic purposes normally thought to be primarily anabolic substrates (amino acids). This information emphasizes the hazards of extrapolating metabolic and nutritional patterns seen in extrauterine life in reaching conclusions concerning the fetus. It likewise emphasizes the importance of ongoing studies in maternal and fetal nutrition and metabolism.

  8. The development of safety requirements

    International Nuclear Information System (INIS)

    Jorel, M.

    2009-01-01

    This document describes the safety approach followed in France for the design of nuclear reactors. This safety approach is based on safety principles from which stem safety requirements that set limiting values for specific parameters. The improvements in computerized simulation, the use of more adequate new materials, a better knowledge of the concerned physical processes, the changes in the reactor operations (higher discharge burnups for instance) have to be taken into account for the definition of safety criteria and the setting of limiting values. The developments of the safety criteria linked to the risks of cladding failure and loss of primary coolant are presented. (A.C.)

  9. Physical requirements in Olympic sailing

    DEFF Research Database (Denmark)

    Bojsen-Møller, J; Larsson, B; Aagaard, Per

    2015-01-01

    Abstract Physical fitness and muscular strength are important performance parameters in Olympic sailing although their relative importance changes between classes. The Olympic format consists of eight yacht types combined into 10 so-called events with total 15 sailors (male and female) in a compl......Abstract Physical fitness and muscular strength are important performance parameters in Olympic sailing although their relative importance changes between classes. The Olympic format consists of eight yacht types combined into 10 so-called events with total 15 sailors (male and female....... Another group of studies has investigated boardsailing and provided evidence to show that windsurfing requires very high aerobic and anaerobic capacity. Although data exist on other types of sailors, the information is limited, and moreover the profile of the Olympic events has changed markedly over...... the last few years to involve more agile, fast and spectacular yachts. The change of events in Olympic sailing has likely added to physical requirements; however, data on sailors in the modern-type yachts are scarce. The present paper describes the recent developments in Olympic sailing with respect...

  10. Quality requirements for EHR archetypes.

    Science.gov (United States)

    Kalra, Dipak; Tapuria, Archana; Austin, Tony; De Moor, Georges

    2012-01-01

    The realisation of semantic interoperability, in which any EHR data may be communicated between heterogeneous systems and fully understood by computers as well as people on receipt, is a challenging goal. Despite the use of standardised generic models for the EHR and standard terminology systems, too much optionality and variability exists in how particular clinical entries may be represented. Clinical archetypes provide a means of defining how generic models should be shaped and bound to terminology for specific kinds of clinical data. However, these will only contribute to semantic interoperability if libraries of archetypes can be built up consistently. This requires the establishment of design principles, editorial and governance policies, and further research to develop ways for archetype authors to structure clinical data and to use terminology consistently. Drawing on several years of work within communities of practice developing archetypes and implementing systems from them, this paper presents quality requirements for the development of archetypes. Clinical engagement on a wide scale is also needed to help grow libraries of good quality archetypes that can be certified. Vendor and eHealth programme engagement is needed to validate such archetypes and achieve safe, meaningful exchange of EHR data between systems.

  11. 12 CFR 760.5 - Escrow requirement.

    Science.gov (United States)

    2010-01-01

    ... 12 Banks and Banking 6 2010-01-01 2010-01-01 false Escrow requirement. 760.5 Section 760.5 Banks... HAVING SPECIAL FLOOD HAZARDS § 760.5 Escrow requirement. If a credit union requires the escrow of taxes... shall also require the escrow of all premiums and fees for any flood insurance required under § 760.3...

  12. 12 CFR 339.5 - Escrow requirement.

    Science.gov (United States)

    2010-01-01

    ... 12 Banks and Banking 4 2010-01-01 2010-01-01 false Escrow requirement. 339.5 Section 339.5 Banks... IN AREAS HAVING SPECIAL FLOOD HAZARDS § 339.5 Escrow requirement. If a bank requires the escrow of... bank shall also require the escrow of all premiums and fees for any flood insurance required under...

  13. 12 CFR 572.5 - Escrow requirement.

    Science.gov (United States)

    2010-01-01

    ... 12 Banks and Banking 5 2010-01-01 2010-01-01 false Escrow requirement. 572.5 Section 572.5 Banks... FLOOD HAZARDS § 572.5 Escrow requirement. If a savings association requires the escrow of taxes... association shall also require the escrow of all premiums and fees for any flood insurance required under...

  14. Nuclear Energy, Long Term Requirements

    International Nuclear Information System (INIS)

    Knapp, V.

    2006-01-01

    There are serious warnings about depletion of oil and gas and even more serious warnings about dangers of climate change caused by emission of carbon dioxide. Should developed countries be called to replace CO2 emitting energy sources as soon as possible, and the time available may not be longer then few decades, can nuclear energy answer the call and what are the requirements? Assuming optimistic contribution of renewable energy sources, can nuclear energy expand to several times present level in order to replace large part of fossil fuels use? Paper considers intermediate and long-term requirements. Future of nuclear power depends on satisfactory answers on several questions. First group of questions are those important for near and intermediate future. They deal with economics and safety of nuclear power stations in the first place. On the same time scale a generally accepted concept for radioactive waste disposal is also required. All these issues are in the focus of present research and development. Safer and more economical reactors are targets of international efforts in Generation IV and INPRO projects, but aiming further ahead these innovative projects are also addressing issues such as waste reduction and proliferation resistance. However, even assuming successful technical development of these projects, and there is no reason to doubt it, long term and large-scale nuclear power use is thereby not yet secured. If nuclear power is to play an essential role in the long-term future energy production and in reduction of CO2 emission, than several additional questions must be replied. These questions will deal with long-term nuclear fuel sufficiency, with necessary contribution of nuclear power in sectors of transport and industrial processes and with nuclear proliferation safety. This last issue is more political then technical, thus sometimes neglected by nuclear engineers, yet it will have essential role for the long-term prospects of nuclear power. The

  15. Requirements and Markets for Nanoelectronics

    Science.gov (United States)

    Hoefflinger, Bernd

    The semiconductor market grew 2010 by 70Bio. against 2009, more than in the previous 9 years taken together, and the semiconductor industry launched the biggest investment program in its history with 100Bio. over a 2-year period. This was the overture to a decade with great potential and great challenges. We look at the market segments and the required electronic functions, and we highlight four product and service areas: Approaching 6 Billion mobile-phone subscribers Access to education for any child One Carebot (personal robot) per family Efficient and safe personal mobility. At the level of over four billion active mobile phones 2010, it is clear that mobile electronic companions have become the drivers of nanoelectronic innovations with growth only limited by the creation and support of new, attractive features and services. Energy, bandwidth, size and weight requirements of these consumer products provide the largest pressure for System-on-Chip (SoC) architectures. Other exemplary new products are selected for their significance, some for their lengthy path into the market. Health care is such an example: The non-invasive glucose sensor and the portable ECG recorder" with automatic, neuroprocessor-driven event detection in the size of a quarter would serve hundreds of millions of people. Nanoelectronics for self-guided health is an area of public policy in view of the cost of "a posteriori" medical care. Access to information and education for any child/student will be provided by 1 tablets where service contracts and the spin-offs from surfing and cloud-computing will generate the revenue. Personal robots, coined by the ageing Japanese nation as the key product after the PC and ridiculed by others, will arrive as carebots for education, entertainment, rehabilitation, and home-service, accepted as a large-scale need by 2020 in most developed countries including China. Accident prevention systems on rail and road already would make millions of units per year

  16. The Anti-Apoptotic Properties of APEX1 in the Endothelium Require the First 20 Amino Acids and Converge on Thioredoxin-1.

    Science.gov (United States)

    Dyballa-Rukes, Nadine; Jakobs, Philipp; Eckers, Anna; Ale-Agha, Niloofar; Serbulea, Vlad; Aufenvenne, Karin; Zschauer, Tim-Christian; Rabanter, Lothar L; Jakob, Sascha; von Ameln, Florian; Eckermann, Olaf; Leitinger, Norbert; Goy, Christine; Altschmied, Joachim; Haendeler, Judith

    2017-04-20

    The APEX nuclease (multifunctional DNA repair enzyme) 1 (APEX1) has a disordered N-terminus, a redox, and a DNA repair domain. APEX1 has anti-apoptotic properties, which have been linked to both domains depending on cell type and experimental conditions. As protection against apoptosis is a hallmark of vessel integrity, we wanted to elucidate whether APEX1 acts anti-apoptotic in primary human endothelial cells and, if so, what the underlying mechanisms are. APEX1 inhibits apoptosis in endothelial cells by reducing Cathepsin D (CatD) cleavage, potentially by binding to the unprocessed form. Diminished CatD activation results in increased Thioredoxin-1 protein levels leading to reduced Caspase 3 activation. Consequently, apoptosis rates are decreased. This depends on the first twenty amino acids in APEX1, because APEX1 (21-318) induces CatD activity, decreases Thioredoxin-1 protein levels, and, thus, increases Caspase 3 activity and apoptosis. Along the same lines, APEX1 (1-20) inhibits Caspase 3 cleavage and apoptosis. Furthermore, re-expression of Thioredoxin-1 via lentiviral transduction rescues endothelial cells from APEX1 (21-318)-induced apoptosis. In an in vivo model of restenosis, which is characterized by oxidative stress, endothelial activation, and smooth muscle cell proliferation, Thioredoxin-1 protein levels are reduced in the endothelium of the carotids. APEX1 acts anti-apoptotic in endothelial cells. This anti-apoptotic effect depends on the first 20 amino acids of APEX1. As proper function of the endothelium during life span is a hallmark for individual health span, a detailed characterization of the functions of the APEX1N-terminus is required to understand all its cellular properties. Antioxid. Redox Signal. 26, 616-629.

  17. SGA Requirements in Coming Years

    Science.gov (United States)

    Lindsay, Stephen J.

    Refineries report various physical and chemical properties of Smelting Grade Alumina, SGA, on Certificate of Analysis data sheets. Without strong understanding of customer needs this data may fall short of meeting the true needs of smelters and downstream customers. For example, improving excess fluoride control with no specification or target for the variability of Na2O content may be a challenge. Downstream customers for aluminum conductor products require excellent conductivity, but this does not always translate itself into alumina shipping limits for Cr2O3, MnO or V2O5. The author presents these and other examples for consideration during the joint sessions between Alumina & Bauxite and Aluminum Reduction Technology.

  18. [Regulatory requirements for topical preparations].

    Science.gov (United States)

    Wohlrab, J; Klauck, D; Savtcheva, E

    2014-03-01

    Professional use of topical treatment in dermatological practice requires not only knowledge about the pharmacological properties, efficacy, safety and pharmaceutical quality of a preparation, but also about its regulatory classification. The latter essentially determines the physician's prescription practice and therapeutic freedom. The regulatory framework with which one is confronted unfortunately lacks transparency. It regulates not only the prescribability and reimbursability of proprietary medicinal products and extemporaneous preparations, but also the obligation to give information as well as the details of liability of both the prescriber (physician) and the manufacturer (pharmaceutical company or pharmacist). The prescriber needs to be aware of to what extent the pharmacist has the possibility and even obligation to change the prescribed preparation. In some cases this can directly affect the therapeutic concept of the physician and even impair the effectiveness and safety of the chosen therapy.

  19. Chilling requirement of Ribes cultivars

    Directory of Open Access Journals (Sweden)

    Hamlyn eJones

    2015-01-01

    Full Text Available It is usually thought that adequate winter chill is required for the full flowering of many temperate woody species. This paper investigates the sensitivity of blackcurrant bud burst and flowering to natural weather fluctuations in a temperate maritime climate, and compares a range of chill models that have been proposed for assessing the accumulation of winter chill. Bud break for four contrasting cultivars are compared in an exceptionally cold and in a mild winter in Eastern Scotland. The results confirm the importance of chilling at temperatures lower than 0ºC and demonstrate that no single chilling function applies equally to all blackcurrant cultivars. There is a pressing need for further model development to take into account the relationship between chilling temperatures and warming temperatures occurring both during and after the chill accumulation period.

  20. Programmable data communications controller requirements

    Science.gov (United States)

    1977-01-01

    The design requirements for a Programmable Data Communications Controller (PDCC) that reduces the difficulties in attaching data terminal equipment to a computer are presented. The PDCC is an interface between the computer I/O channel and the bit serial communication lines. Each communication line is supported by a communication port that handles all line control functions and performs most terminal control functions. The port is fabricated on a printed circuit board that plugs into a card chassis, mating with a connector that is joined to all other card stations by a data bus. Ports are individually programmable; each includes a microprocessor, a programmable read-only memory for instruction storage, and a random access memory for data storage.

  1. Requirements engineering for digital health

    CERN Document Server

    Thümmler, Christoph; Gavras, Anastasius

    2015-01-01

    Healthcare and well-being have captured the attention of established software companies, start-ups, and investors. Software is starting to play a central role for addressing the problems of the aging society and the escalating cost of healthcare services. Enablers of such digital health are a growing number of sensors for sensing the human body and communication infrastructure for remote meetings, data sharing, and messaging. The challenge that lies in front of us is how to effectively make use of these capabilities, for example to empower patients and to free the scarce resources of medical personnel. Requirements engineering is the process by which the capabilities of a software product are aligned with stakeholder needs and a shared understanding between the stakeholders and development team established. This book provides guide for what to look for and do when inquiring and specifying software that targets healthcare and well-being, helping readers avoid the pitfalls of the highly regulated and sensible h...

  2. The EURS (European utilities requirements)

    International Nuclear Information System (INIS)

    Berbey, P.

    2000-01-01

    The major European electricity producers have worked on a common requirement document for future LWR plants since 1992 to get specifications acceptable together by the owners, the public and the authorities. Thus the designers can develop standard LWR designs that could be acceptable everywhere in Europe and the utilities can open their consultations to vendors on common bases. Public and authority's acceptance should be improved as well. Significant saving are expected in development and construction costs. Since the release of the last versions of the EUR texts in 1996, a lot of work has been carried out: reviews by the regulators and other external organisations, comparisons, assessment of compliance of designs vs. EUR and clarification works on the controversial topics that deserved changes or clarification. At the beginning of 1999 enough material was available to start a complete revision of the EUR document

  3. Requirements for an energy policy

    International Nuclear Information System (INIS)

    Conant, M.A.

    1987-01-01

    The central issue facing the US today lies in the rise of oil imports. No supergiant (5 billion barrels) oil discoveries have been made in the US. Production from existing fields is declining. The 1985-86 oil price collapse from $26 to less than $15 per barrel had a disastrous effect on the budgets of smaller oil companies which do most of the exploring, and on the service industry. Budgets for overseas exploration has been generally sustained. Oil prices are not expected to sustain domestic exploration. Gulf oil sources will, in the next five years, supply some 75 percent of all oil in international trade. Without an energy policy, involvement in Middle East oil will grow exponentially, as will the needs of others for Gulf oil. The natural gas situation is different, with a spare producing capacity of one trillion cubic feet this year, which could double next year. Natural gas deregulation has created an unbelievable mess in the requirements of producers/suppliers and purchasers to have dependable business arrangements. Coal is plentiful and will be until the end of time. Public opposition to emission problems and the greenhouse effect are an obstacle to greater use of coal. The nuclear option may be dead, with no new orders since 1978. Statistics are provided on proven world reserves of conventional crude oil, recoverable heavy oils, tar sands, and shale oil; which indicates for the long term an ability to transform the Geopolitics of oil away from the middle east. Energy options require energy R ampersand D, use of Alaskan gas, conservation and efficiency in energy use, strategic reserves, close energy relations with allies, and a government-industry link which insures meeting the US oil needs from the Western Hemisphere

  4. Reversed field pinch ignition requirements

    International Nuclear Information System (INIS)

    Werley, K.A.

    1991-01-01

    Plasma models are described and used to calculated numerically the transport confinement (nτ E ) requirements and steady state operation points for both the reversed field pinch (RFP) and the tokamak. The models are used to examine the CIT tokamak ignition conditions and the RFP experimental and ignition conditions. Physics differences between RFPs and tokamaks and their consequences for a D-T ignition machine are discussed. Compared with a tokamak, the ignition RFP has many physics advantages, including Ohmic heating to ignition (no need for auxiliary heating systems), higher beta, lower ignition current, less sensitivity of ignition requirements to impurity effects, no hard disruptions (associated with beta or density limits) and successful operation with high radiation fractions (f RAD ∼ 0.95). These physics advantages, coupled with important engineering advantages associated with lower external magnetic field, larger aspect ratios and smaller plasma cross-sections, translate to significant cost reductions for both ignition and reactor applications. The primary drawback of the RFP is the uncertainty that the present scaling will extrapolate to reactor regimes. Devices that are under construction should go a long way toward resolving this scaling uncertainty. The 4 MA ZTH is expected to extend the nτ E transport scaling data by three orders of magnitude above the results of ZT-40M, and, if the present scaling holds, ZTH is expected to achieve a D-T equivalent scientific energy breakeven, Q = 1. A base case RFP ignition point is identified with a plasma current of 8.1 MA and no auxiliary heating. (author). 19 refs, 11 figs, 3 tabs

  5. Physical System Requirements: Transport Waste

    International Nuclear Information System (INIS)

    1992-04-01

    The Nuclear Waste Policy Act (NWPA) of 1982 assigned to the Department of Energy (DOE) the responsibility for managing the disposal of spent nuclear fuel and high-level radioactive waste and established the Office of Civilian Radioactive Waste Management (OCRWM) for that purpose. The Secretary of Energy, in his November 1989 report to Congress (DOE/RW-0247), announced three new initiatives for the conduct of the Civilian Radioactive Waste Management (CRWM) program. One of these initiatives was to establish improved management structure and procedures. In response, OCRWM performed a management study and the Director subsequently issued the Management Systems Improvement Strategy (MSIS) on August 10, 1990, calling for a rigorous implementation of systems engineering principles with a special emphasis on functional analysis. The functional analysis approach establishes a framework for integrating the program management efforts with the technical requirements analysis into a single, unified, and consistent program. This approach recognizes that just as the facilities and equipment comprising the physical waste management system must perform certain functions, so must certain programmatic and management functions be performed within the program in order to successfully bring the physical system into being. The objective of this document is to establish the essential functions, requirements, interfaces, and system architecture for the Transport Waste mission. Based upon the Nuclear Waste Policy Act, the mission of the Waste Transportation System is to transport SNF and/or HLW from the purchaser's/producer's facilities to, and between, NWMS facilities in a manner that protects the health and safety of the public and of workers and the quality of the environment makes effective use of financial and other resources, and to the fullest extent possible uses the private sector

  6. Meeting US and European supplier control requirements.

    Science.gov (United States)

    Donawa, Maria

    2009-01-01

    Medical device manufacturers operating under European quality system requirements are sometimes surprised to learn that their supplier control procedures do not fully meet United States (US) requirements. This article discusses important differences between US and European requirements for controlling suppliers.

  7. Design requirement on HYPER blanket fuel assembly

    International Nuclear Information System (INIS)

    Hwang, Woan; Lee, B. O.; Nam, C.; Ryu, W. S.; Lee, B. S.; Park, W. S.

    2000-07-01

    This document describes design requirements which are needed for designing the blanket assembly of the HYPER as design guidance. The blanket assembly of the HYPER consists of blanket fuel rods, mounting rail, spacer, upper nozzle with handling socket, bottom nozzle with mounting rail and skeleton structure. The blanket fuel rod consists of top end plug, bottom end plug with key way, blanket fuel slug, and cladding. In the assembly, the rods are in a triangular pitch array. This report contains functional requirements, performance and operational requirements, interfacing systems requirements, core restraint and interface requirements, design limits and strength requirements, system configuration and essential feature requirements, seismic requirements, structural requirements, environmental requirements, reliability and safety requirements, standard and codes, QA programs, and other requirements for the blanket fuel assembly of the HYPER

  8. Rapid quality assurance with requirements smells

    OpenAIRE

    Femmer, Henning; Méndez Fernández, Daniel; Wagner, Stefan; Eder, Sebastian

    2016-01-01

    Context: Bad requirements quality can cause expensive consequences during the software development lifecycle, especially if iterations are long and feedback comes late. Objectives: We aim at a light-weight static requirements analysis approach that allows for rapid checks immediately when requirements are written down. Method: We transfer the concept of code smells to Requirements Engineering as Requirements Smells. To evaluate the benefits and limitations, we define Requirements Smells, real...

  9. Cold vacuum drying facility design requirements

    International Nuclear Information System (INIS)

    IRWIN, J.J.

    1999-01-01

    This document provides the detailed design requirements for the Spent Nuclear Fuel Project Cold Vacuum Drying Facility. Process, safety, and quality assurance requirements and interfaces are specified

  10. Cold vacuum drying facility design requirements

    Energy Technology Data Exchange (ETDEWEB)

    IRWIN, J.J.

    1999-07-01

    This document provides the detailed design requirements for the Spent Nuclear Fuel Project Cold Vacuum Drying Facility. Process, safety, and quality assurance requirements and interfaces are specified.

  11. Menorrhagia in adolescents requiring hospitalization.

    Science.gov (United States)

    Smith, Y R; Quint, E H; Hertzberg, R B

    1998-02-01

    This study was undertaken to assess the causes and treatments of menorrhagia in adolescents hospitalized for this menstrual disorder. A retrospective chart review was performed of all adolescents adolescents with 46 admissions for menorrhagia were identified. The average age of menarche was 12.9 years and the average age at admission was 15.9 years. Nineteen adolescents had significant medical diseases. For the 46 admissions, causes of menorrhagia were anovulation (21), hematologic disease (15), chemotherapy-related (5), and infections (5). Transfusions of blood products were performed in 28 of the admissions. Treatments included oral contraceptive pills or progestins (30), intravenous conjugated estrogens (8), antibiotics (4), immune gammaglobulin (3), DDAVP (3), and prednisone (1). Twelve surgical procedures were performed, including eight dilatation and curettages (D&Cs), three laparoscopies, and one hysterectomy. Sixty-one percent of admissions for adolescent menorrhagia were in adolescents with significant medical problems. The patients with menorrhagia who required admission had severe anemia and were transfused in 63% of cases. The predominant causes for these admissions included anovulation in 46%, hematologic disease in 33%, chemotherapy in 11%, and infection in 11%. Hormonal regulation or suppression of menses should be considered in adolescents with significant medical disease.

  12. Global Land Transport Infrastructure Requirements

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2013-06-01

    Over the next four decades, global passenger and freight travel is expected to double over 2010 levels. In order to accommodate this growth, it is expected that the world will need to add nearly 25 million paved road lane-kilometres and 335 000 rail track kilometres. In addition, it is expected that between 45 000 square kilometres and 77 000 square kilometres of new parking spaces will be added to accommodate vehicle stock growth. These land transport infrastructure additions, when combined with operations, maintenance and repairs, are expected to cost as much as USD 45 trillion by 2050. This publication reports on the International Energy Agency’s (IEA) analysis of infrastructure requirements to support projected road and rail travel through 2050, using the IEA Mobility Model. It considers land transport infrastructure additions to support travel growth to 2050. It also considers potential savings if countries pursue “avoid and shift” policies: in this scenario, cumulative global land transport infrastructure spending could decrease as much as USD 20 trillion by 2050 over baseline projections.

  13. Plutonium storage: Requirements and challenges

    International Nuclear Information System (INIS)

    Cunningham, P.T.; Haschke, J.M.; Martz, J.C.

    1993-01-01

    The retirement of large numbers of nuclear weapons will necessitate management of unprecedented quantities of excess plutonium. In addition, surplus material and residues from previous weapon production activities comprise a substantial quantity of concentrated plutonium that exists in a variety of chemical forms. Storage of plutonium for an indefinite period will be necessary until a decision regarding ultimate disposition is made. Selection of the most suitable storage option(s) for this interim period is complicated by technical issues, nuclear proliferation concerns, contingency planning, political factors, and uncertainty regarding the length of the interim period. Options for excess plutonium include storage as intact weapon components and storage as extracted nuclear material. Specific advantages for storage of excess material in a variety of chemical forms have been presented. In this paper, technical issues associated with various storage options are examined with emphasis on relevant physical and chemical properties of candidate materials. Technology and facility requirements for preparing and certifying storage forms are considered and recommendations, based on our assessment of options, are presented

  14. General lighting requirements for photosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Geiger, D.R. [Univ. of Dayton, OH (United States)

    1994-12-31

    A review of the general lighting requirements for photosynthesis reveals that four aspects of light are important: irradiance, quality, timing and duration. These properties of light affect photosynthesis by providing the energy that drives carbon assimilation as well as by exerting control over physiology, structure and morphology of plants. Irradiance, expressed as energy flux, W m{sup -2}, or photon irradiance, {mu}mol m{sup -2} s{sup -1}, determines the rate at which energy is being delivered to the photosynthetic reaction centers. Spectral quality, the wavelength composition of light, is important because photons differ in their probability of being absorbed by the light harvesting complex and hence their ability to drive carbon assimilation. Also the various light receptors for light-mediated regulation of plant form and physiology have characteristic absorption spectra and hence photons differ in their effectiveness for eliciting responses. Duration is important because both carbon assimilation and regulation are affected by the total energy or integrated irradiance delivered during a given period. Many processes associated with photosynthesis are time-dependent, increasing or decreasing with duration. Timing is important because the effectiveness of light in the regulation of plant processes varies with the phase of the diumal cycle as determined by the plant`s time-measuring mechanisms.

  15. Physical system requirements: Overall system

    International Nuclear Information System (INIS)

    1992-01-01

    The Nuclear Waste Policy Act (NWPA) of 1982 assigned to the Department of Energy (DOE) the responsibility for managing the disposal of spent nuclear fuel and high-level radioactive waste and established the Office of Civilian Radioactive Waste Management (OCRWM) for that purpose. The Secretary of Energy, in his November 1989 report to Congress (DOE/RW-0247), announced three new initiatives for conduct of the Civilian Radioactive Waste Management (CRWM) program. One of these initiatives was to establish improved management structure and procedures. In response, OCRWM performed a management study and the Direct subsequently issued the Management Systems Improvement Strategy (MSIS) on August 10, 1990, calling for a rigorous implementation of systems engineering principles with a special emphasis on functional analysis. This approach establishes a framework for integrating the program management efforts with the technical requirements analysis into a single, unified, and consistent program. The functional analysis approach recognizes that just the facilities and equipment comprising the physical waste management system must perform certain functions, so must certain programmatic and management functions be performed within the program in order to successfully bring the physical system into being

  16. 12 CFR 22.5 - Escrow requirement.

    Science.gov (United States)

    2010-01-01

    ... 12 Banks and Banking 1 2010-01-01 2010-01-01 false Escrow requirement. 22.5 Section 22.5 Banks and... HAZARDS § 22.5 Escrow requirement. If a bank requires the escrow of taxes, insurance premiums, fees, or..., increased, extended, or renewed on or after October 1, 1996, the bank shall also require the escrow of all...

  17. 17 CFR 41.45 - Required margin.

    Science.gov (United States)

    2010-04-01

    ... 17 Commodity and Securities Exchanges 1 2010-04-01 2010-04-01 false Required margin. 41.45 Section... PRODUCTS Customer Accounts and Margin Requirements § 41.45 Required margin. (a) Applicability. Each security futures intermediary shall determine the required margin for the security futures and related...

  18. 21 CFR 1304.11 - Inventory requirements.

    Science.gov (United States)

    2010-04-01

    ... the inventory of the registered location to which they are subject to control or to which the person... 21 Food and Drugs 9 2010-04-01 2010-04-01 false Inventory requirements. 1304.11 Section 1304.11... REGISTRANTS Inventory Requirements § 1304.11 Inventory requirements. (a) General requirements. Each inventory...

  19. 10 CFR 61.59 - Institutional requirements.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Institutional requirements. 61.59 Section 61.59 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSING REQUIREMENTS FOR LAND DISPOSAL OF RADIOACTIVE WASTE Technical Requirements for Land Disposal Facilities § 61.59 Institutional requirements. (a) Land ownership...

  20. 20 CFR 655.152 - Advertising requirements.

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 3 2010-04-01 2010-04-01 false Advertising requirements. 655.152 Section 655... Employment in the United States (H-2A Workers) Post-Acceptance Requirements § 655.152 Advertising requirements. All advertising conducted to satisfy the required recruitment activities under § 655.151 must...

  1. Definition and validation of requirements management measures

    OpenAIRE

    Loconsole, Annabella

    2007-01-01

    The quality of software systems depends on early activities in the software development process, of which the management of requirements is one. When requirements are not managed well, a project can fail or become more costly than intended, and the quality of the software developed can decrease. Among the requirements management practices, it is particularly important to quantify and predict requirements volatility, i.e., how much the requirements are likely to change over time. Software meas...

  2. Virtual Observatories: Requirements for Utility

    Science.gov (United States)

    Paxton, L. J.

    2008-12-01

    The principal act that separates science from engineering is that of discovery. Virtual Observatories are a development with great potential for advancing our ability to do science by enabling us to do research effectively and to do research across disciplines. Access to data is one of the factors that enables discovery. A well-designed VO should enable discovery as well as providing for a uniform means by which data are accessed: thus, enabling discovery is the key challenge of a VO in fact it is and should be the principle that distinguishes a VO from a traditional archive. As the number of satellites in the Heliophysics Great observatory starts to decline due to the slower launch cadence and the reduction in funding for extended missions, it becomes more imperative that the community have the means to fully utilize and access the available resources. With the proliferation of low-cost computing and community-based models, cross-disciplinary studies become the new frontier. Many, if not the great majority of research papers are, at this time, confined to a particular discipline. Some of this "stove piping" may be due to the difficulty in accessing products from outside one's own discipline. One would hope and expect that VOs would address this. Two of the principal challenges associated with the vitality of the VOs, aside from the provision of the funds required to maintain the VOs, is 1) the limitation on the availability of data from non-NASA sources and 2) the need for some level of continued support for expertise on the data accessed through the VOs. The first issue is one of culture - some organizations support the view that the data belong to the PI whereas in Heliophysics "data rights" are curtailed. The second issue is to be addressed by the concept of the Resident Archive. This talk will provide an overview of the issues and challenges associated with VOs, Resident Archives, data rights, space missions, and instruments and their associated ground data

  3. Closed Loop Requirements and Analysis Management

    Science.gov (United States)

    Lamoreaux, Michael; Verhoef, Brett

    2015-01-01

    Effective systems engineering involves the use of analysis in the derivation of requirements and verification of designs against those requirements. The initial development of requirements often depends on analysis for the technical definition of specific aspects of a product. Following the allocation of system-level requirements to a product's components, the closure of those requirements often involves analytical approaches to verify that the requirement criteria have been satisfied. Meanwhile, changes that occur in between these two processes need to be managed in order to achieve a closed-loop requirement derivation/verification process. Herein are presented concepts for employing emerging Team center capabilities to jointly manage requirements and analysis data such that analytical techniques are utilized to effectively derive and allocate requirements, analyses are consulted and updated during the change evaluation processes, and analyses are leveraged during the design verification process. Recommendations on concept validation case studies are also discussed.

  4. Personal belief exemptions from school vaccination requirements.

    Science.gov (United States)

    Diekema, Douglas S

    2014-01-01

    Despite the impact vaccination has had on the control and prevention of many infectious diseases, some parents choose not to vaccinate their children. Although there is no federal law requiring vaccination of children in the United States, all states require evidence of vaccination against at least some diseases as a condition of school entry. Which vaccines are required; how many doses are required; whether entry requirements apply to child care, kindergarten, or middle school; and whether exemptions from vaccine requirements will be allowed all differ by state. All but two states allow some kind of personal belief exemption from school vaccination requirements. This article reviews the history of school vaccination requirements and exemptions, the legal status of state vaccination laws and exemptions, the impact of school vaccination requirements and personal belief exemptions on vaccination rates and disease incidence, and strategies for maintaining adequate vaccination rates in states that allow personal belief exemptions.

  5. 7 CFR 1416.7 - Insurance requirements.

    Science.gov (United States)

    2010-01-01

    ... requirements. For the Citrus, Fruit and Vegetable, Tropical Fruit and Nursery Disaster Programs: (a) Payment... producers required to purchase a citrus policy may purchase a fruit or tree policy; or (2) NAP coverage. (c...

  6. Higher speed freight truck design : performance requirements.

    Science.gov (United States)

    2013-10-01

    This proposed requirements document combines a set of requirements for high-speed freight car truck design and performance : from the generally accepted standards in the U.S. Code of Federal Regulation (CFR), the Association of American Railroads : (...

  7. Insulin requirements in type 1 diabetic pregnancy

    DEFF Research Database (Denmark)

    Callesen, Nicoline; Ringholm, Lene; Stage, Edna

    2012-01-01

    To evaluate the insulin requirements in women with type 1 diabetes during twin pregnancy compared with singleton pregnancy.......To evaluate the insulin requirements in women with type 1 diabetes during twin pregnancy compared with singleton pregnancy....

  8. Requirements Engineering for Software Integrity and Safety

    Science.gov (United States)

    Leveson, Nancy G.

    2002-01-01

    Requirements flaws are the most common cause of errors and software-related accidents in operational software. Most aerospace firms list requirements as one of their most important outstanding software development problems and all of the recent, NASA spacecraft losses related to software (including the highly publicized Mars Program failures) can be traced to requirements flaws. In light of these facts, it is surprising that relatively little research is devoted to requirements in contrast with other software engineering topics. The research proposed built on our previous work. including both criteria for determining whether a requirements specification is acceptably complete and a new approach to structuring system specifications called Intent Specifications. This grant was to fund basic research on how these ideas could be extended to leverage innovative approaches to the problems of (1) reducing the impact of changing requirements, (2) finding requirements specification flaws early through formal and informal analysis, and (3) avoiding common flaws entirely through appropriate requirements specification language design.

  9. Ecological flow requirements for South African rivers

    CSIR Research Space (South Africa)

    Ferrar, AA

    1989-01-01

    Full Text Available This document contains the proceedings of a workshop which was convened to debate the ecological flow requirements of South African rivers. Topics which are discussed include the influence of weirs and impoundments, the quantity requirements...

  10. Security Requirements for Post-Transition Cuba

    National Research Council Canada - National Science Library

    Crowther, Glenn A

    2007-01-01

    .... With that change, Cuba's security requirements will change as well. This monograph analyzes security requirements that the new Cuba will face and proposes what missions and structure the Cuban security forces might have after a...

  11. Business System Planning Project System Requirements Specification

    Energy Technology Data Exchange (ETDEWEB)

    NELSON, R.E.

    2000-09-08

    The purpose of the Business Systems Planning Project System Requirements Specification (SRS) is to provide the outline and contents of the requirements for the CH2M HILL Hanford Group, Inc. (CHG) integrated business and technical information systems. The SRS will translate proposed objectives into the statement of the functions that are to be performed and data and information flows that they require. The requirements gathering methodology will use (1) facilitated group requirement sessions; (2) individual interviews; (3) surveys; and (4) document reviews. The requirements will be verified and validated through coordination of the technical requirement team and CHG Managers. The SRS document used the content and format specified in Lockheed Martin Services, Inc. Organization Standard Software Practices in conjunction with the Institute of Electrical and Electronics Engineers Standard 8340-1984 for Systems Requirements Documents.

  12. 8 CFR 1216.2 - Notification requirements.

    Science.gov (United States)

    2010-01-01

    ... second time of the requirement that the alien and the petitioning spouse or alien entrepreneur must file... does not relieve the alien and the petitioning spouse, or alien entrepreneur of the requirement to file...

  13. Rapid quality assurance with Requirements Smells

    OpenAIRE

    Femmer, H.; Fernández, D. Méndez; Wagner, S.; Eder, S.

    2016-01-01

    Bad requirements quality can cause expensive consequences during the software development lifecycle, especially if iterations are long and feedback comes late. %-- the faster a problem is found, the cheaper it is to fix. This makes explicit the need of a lightweight detection mechanism of requirements quality violations. We aim at a light-weight static requirements analysis approach that allows for rapid checks immediately when requirements are written down. We transfer the concept of code sm...

  14. Meta-requirements that Model Change

    OpenAIRE

    Gouri Prakash

    2010-01-01

    One of the common problems encountered in software engineering is addressing and responding to the changing nature of requirements. While several approaches have been devised to address this issue, ranging from instilling resistance to changing requirements in order to mitigate impact to project schedules, to developing an agile mindset towards requirements, the approach discussed in this paper is one of conceptualizing the delta in requirement and modeling it, in order t...

  15. Cloud computing security requirements: a systematic review

    OpenAIRE

    Iankoulova, Iliana; Daneva, Maia; Rolland, C; Castro, J.; Pastor, O

    2012-01-01

    Many publications have dealt with various types of security requirements in cloud computing but not all types have been explored in sufficient depth. It is also hard to understand which types of requirements have been under-researched and which are most investigated. This paper's goal is to provide a comprehensive and structured overview of cloud computing security requirements and solutions. We carried out a systematic review and identified security requirements from previous publications th...

  16. Dosimeter characteristics and service performance requirements

    International Nuclear Information System (INIS)

    Ambrosi, P.; Bartlett, D.T.

    1999-01-01

    The requirements for personal dosimeters and dosimetry services given by ICRP 26, ICRP 35, ICRP 60 and ICRP 75 are summarised and compared with the requirements given in relevant international standards. Most standards could be made more relevant to actual workplace conditions. In some standards, the required tests of energy and angular dependence of the response are not sufficient, or requirements on overall uncertainty are lacking. (author)

  17. 40 CFR 141.80 - General requirements.

    Science.gov (United States)

    2010-07-01

    ... service line replacement, and public education. These requirements are triggered, in some cases, by lead... requirements. (1) All water systems shall install and operate optimal corrosion control treatment as defined in... specified by the State under § 141.83. (f) Lead service line replacement requirements. Any system exceeding...

  18. 7 CFR 1948.96 - Audit requirements.

    Science.gov (United States)

    2010-01-01

    ... Program § 1948.96 Audit requirements. (a) Audit requirements for Site Development and Acquisition Grants will be made in accordance with FmHA Instruction 1942-G. (b) Audits for planning grants made in... 7 Agriculture 13 2010-01-01 2009-01-01 true Audit requirements. 1948.96 Section 1948.96...

  19. Understand the Design Requirement in Companies

    DEFF Research Database (Denmark)

    Li, Xuemeng; Ahmed-Kristensen, Saeema

    2015-01-01

    requirements can lead to inappropriate products (Hall, et al., 2002). Understanding the nature of design requirements and the sources, from where they can or should be generated, is critical to before developing methods and processes to support this process. Requirement Engineering research, originated from...

  20. Capturing security requirements for software systems

    Science.gov (United States)

    El-Hadary, Hassan; El-Kassas, Sherif

    2014-01-01

    Security is often an afterthought during software development. Realizing security early, especially in the requirement phase, is important so that security problems can be tackled early enough before going further in the process and avoid rework. A more effective approach for security requirement engineering is needed to provide a more systematic way for eliciting adequate security requirements. This paper proposes a methodology for security requirement elicitation based on problem frames. The methodology aims at early integration of security with software development. The main goal of the methodology is to assist developers elicit adequate security requirements in a more systematic way during the requirement engineering process. A security catalog, based on the problem frames, is constructed in order to help identifying security requirements with the aid of previous security knowledge. Abuse frames are used to model threats while security problem frames are used to model security requirements. We have made use of evaluation criteria to evaluate the resulting security requirements concentrating on conflicts identification among requirements. We have shown that more complete security requirements can be elicited by such methodology in addition to the assistance offered to developers to elicit security requirements in a more systematic way. PMID:25685514

  1. 24 CFR 241.1069 - Escrow requirements.

    Science.gov (United States)

    2010-04-01

    ... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Escrow requirements. 241.1069...-Eligibility Requirements § 241.1069 Escrow requirements. (a) An equity loan provided in connection with a plan... behalf of the borrower, 10 percent of the loan amount in an escrow account, controlled by the...

  2. 12 CFR 16.31 - Escrow requirement.

    Science.gov (United States)

    2010-01-01

    ... 12 Banks and Banking 1 2010-01-01 2010-01-01 false Escrow requirement. 16.31 Section 16.31 Banks... RULES § 16.31 Escrow requirement. The OCC may require that any funds received in connection with an offer or sale of securities be held in an independent escrow account at an unrelated insured depository...

  3. 38 CFR 36.4704 - Escrow requirement.

    Science.gov (United States)

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false Escrow requirement. 36...) LOAN GUARANTY Sale of Loans, Guarantee of Payment, and Flood Insurance § 36.4704 Escrow requirement. If the Secretary requires the escrow of taxes, insurance premiums, fees, or any other charges for a loan...

  4. 28 CFR 80.12 - Accounting requirements.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Accounting requirements. 80.12 Section 80... PROCEDURE § 80.12 Accounting requirements. Neither the submission of a request for an FCPA Opinion, its... comply with the accounting requirements of 15 U.S.C. 78m(b)(2) and (3). ...

  5. Understanding the Requirements for Open Source Software

    Science.gov (United States)

    2009-06-17

    Elicitation in the Post-Methodology Era’, Requirements Engineering, 5, 67-73, (2000). 3. Bollinger , T.: Use of Free and Open-Source Software (FOSS) in the U.S...Paris, France , December, (2003). 46. Robinson, W.: A Requirements Monitoring Framework for Enterprise Systems, Requirements Engineering, 11(1), 17

  6. 49 CFR 383.111 - Required knowledge.

    Science.gov (United States)

    2010-10-01

    ... importance of proper visual search, and proper visual search methods. (6) Communication. The principles and procedures for proper communications and the hazards of failure to signal properly. (7) Speed management. The... STANDARDS; REQUIREMENTS AND PENALTIES Required Knowledge and Skills § 383.111 Required knowledge. All...

  7. 7 CFR 1770.13 - Accounting requirements.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 12 2010-01-01 2010-01-01 false Accounting requirements. 1770.13 Section 1770.13... AGRICULTURE (CONTINUED) ACCOUNTING REQUIREMENTS FOR RUS TELECOMMUNICATIONS BORROWERS Uniform System of Accounts § 1770.13 Accounting requirements. (a) Each borrower shall maintain its books of accounts on the...

  8. 22 CFR 126.13 - Required information.

    Science.gov (United States)

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Required information. 126.13 Section 126.13... PROVISIONS § 126.13 Required information. (a) All applications for licenses (DSP-5, DSP-61, DSP-73, and DSP... are multiple consignors, consignees or freight forwarders, and all the required information cannot be...

  9. 50 CFR 660.15 - Equipment requirements.

    Science.gov (United States)

    2010-10-01

    ... operational requirements for scales used to weigh catch at sea, scales used to weigh catch at IFQ first... software requirements. (i) A personal computer with Pentium 75-MHz or higher. Random Access Memory (RAM... required under this subsection are fully operational and functional whenever the Pacific whiting primary...

  10. 46 CFR 298.13 - Financial requirements.

    Science.gov (United States)

    2010-10-01

    ... the reporting requirements imposed by § 298.42. (2) “Working Capital” means the excess, if any, of... Working Capital requirements, as set forth in paragraph (f) of this section. (1) The various financial..., minimum requirements at Closing usually are as follows: (i) Working Capital. The Company's Working Capital...

  11. 10 CFR 4.21 - General requirements.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 1 2010-01-01 2010-01-01 false General requirements. 4.21 Section 4.21 Energy NUCLEAR... and Title IV of the Energy Reorganization Act of 1974 Assurances Required § 4.21 General requirements... purpose involving the provision of similar services or benefits. In the case of personal property the...

  12. Variations in land requirements for meat production

    NARCIS (Netherlands)

    Elferink, E. V.; Nonhebel, S.

    2007-01-01

    Production of meat requires substantial amounts of feed grains which in turn require vast amounts of land. Future population growth and increase in consumption will raise the demand for meat and with it the land required for meat production. This paper analyses the various factors that affect land

  13. 40 CFR 35.6450 - General requirements.

    Science.gov (United States)

    2010-07-01

    ... STATE AND LOCAL ASSISTANCE Cooperative Agreements and Superfund State Contracts for Superfund Response Actions Copyright Requirements Under A Cooperative Agreement § 35.6450 General requirements. The recipient... comply with the requirements regarding contract copyright provisions described in § 35.6595(b)(2). Use of...

  14. 75 FR 12803 - Fingerprint Submission Requirements Rule

    Science.gov (United States)

    2010-03-17

    ... NATIONAL CRIME PREVENTION AND PRIVACY COMPACT COUNCIL Fingerprint Submission Requirements Rule... Fingerprint Submission Requirements Rule, title 28 Code of Federal Regulations (CFR), part 901. FOR FURTHER... the Fingerprint Submission Requirements Rule (28 CFR, part 901) when health or safety of vulnerable...

  15. 29 CFR 96.12 - Audit requirements.

    Science.gov (United States)

    2010-07-01

    .... (b) The audit requirements contained in 29 CFR part 99 shall be followed for audits of all fiscal... 29 Labor 1 2010-07-01 2010-07-01 true Audit requirements. 96.12 Section 96.12 Labor Office of the Secretary of Labor AUDIT REQUIREMENTS FOR GRANTS, CONTRACTS, AND OTHER AGREEMENTS Audits of States, Local...

  16. 23 CFR 1313.4 - General requirements.

    Science.gov (United States)

    2010-04-01

    ... ALCOHOL-IMPAIRED DRIVING PREVENTION PROGRAMS § 1313.4 General requirements. (a) Qualification requirements... enforcement of alcohol-impaired driving prevention programs in § 1313.6 and other associated costs permitted... certification that it has an alcohol-impaired driving prevention program that meets the requirements of 23 U.S.C...

  17. 10 CFR 61.40 - General requirement.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false General requirement. 61.40 Section 61.40 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) LICENSING REQUIREMENTS FOR LAND DISPOSAL OF RADIOACTIVE WASTE Performance Objectives § 61.40 General requirement. Land disposal facilities must be sited, designed, operated, closed...

  18. 40 CFR 63.181 - Recordkeeping requirements.

    Science.gov (United States)

    2010-07-01

    ... monitoring, quality improvement) for each type of equipment. All records and information required by this... service is not required. Equipment subject to the provisions of this subpart may be identified on a plant... information shall be recorded for each dual mechanical seal system: (i) Design criteria required in §§ 63.163...

  19. 40 CFR 97.6 - Standard requirements.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 20 2010-07-01 2010-07-01 false Standard requirements. 97.6 Section 97... General Provisions § 97.6 Standard requirements. (a) Permit requirements. (1) The NOX authorized account... a complete NOX Budget permit application under § 97.22 in accordance with the deadlines specified in...

  20. 76 FR 50881 - Required Scale Tests

    Science.gov (United States)

    2011-08-17

    ..., Packers and Stockyards Administration 9 CFR Part 201 RIN 0580-AB10 Required Scale Tests AGENCY: Grain... January 20, 2011, and on April 4, 2011, concerning required scale tests. Those documents defined ``limited...), concerning required scale tests. Those documents incorrectly defined limited seasonal basis in Sec. 201.72(a...

  1. From document to database: modernizing requirements management

    International Nuclear Information System (INIS)

    Giajnorio, J.; Hamilton, S.

    2007-01-01

    The creation, communication, and management of design requirements are central to the successful completion of any large engineering project, both technically and commercially. Design requirements in the Canadian nuclear industry are typically numbered lists in multiple documents created using word processing software. As an alternative, GE Nuclear Products implemented a central requirements management database for a major project at Bruce Power. The database configured the off-the-shelf software product, Telelogic Doors, to GE's requirements structure. This paper describes the advantages realized by this scheme. Examples include traceability from customer requirements through to test procedures, concurrent engineering, and automated change history. (author)

  2. Legal requirements governing proxy voting in Denmark

    DEFF Research Database (Denmark)

    Werlauff, Erik

    2008-01-01

    The requirements in Danish company law concerning proxy voting in companies whose shares have been accepted for listing on a regulated market have been successively tightened in recent years, and corporate governance principles have also led to the introduction of several requirements concerning...... proxy holders. A thorough knowledge of these requirements is important not only for the listed companies but also for their advisers and investors in Denmark and abroad. This article considers these requirements as well as the additional requirements which will derive from Directive 2007...

  3. TRACER - TRACING AND CONTROL OF ENGINEERING REQUIREMENTS

    Science.gov (United States)

    Turner, P. R.

    1994-01-01

    TRACER (Tracing and Control of Engineering Requirements) is a database/word processing system created to document and maintain the order of both requirements and descriptive material associated with an engineering project. A set of hierarchical documents are normally generated for a project whereby the requirements of the higher level documents levy requirements on the same level or lower level documents. Traditionally, the requirements are handled almost entirely by manual paper methods. The problem with a typical paper system, however, is that requirements written and changed continuously in different areas lead to misunderstandings and noncompliance. The purpose of TRACER is to automate the capture, tracing, reviewing, and managing of requirements for an engineering project. The engineering project still requires communications, negotiations, interactions, and iterations among people and organizations, but TRACER promotes succinct and precise identification and treatment of real requirements separate from the descriptive prose in a document. TRACER permits the documentation of an engineering project's requirements and progress in a logical, controllable, traceable manner. TRACER's attributes include the presentation of current requirements and status from any linked computer terminal and the ability to differentiate headers and descriptive material from the requirements. Related requirements can be linked and traced. The program also enables portions of documents to be printed, individual approval and release of requirements, and the tracing of requirements down into the equipment specification. Requirement "links" can be made "pending" and invisible to others until the pending link is made "binding". Individuals affected by linked requirements can be notified of significant changes with acknowledgement of the changes required. An unlimited number of documents can be created for a project and an ASCII import feature permits existing documents to be incorporated

  4. Managing Legal Texts in Requirements Engineering

    Science.gov (United States)

    Otto, Paul N.; Antón, Annie I.

    Laws and regulations are playing an increasingly important role in requirements engineering and systems development. Monitoring systems for requirements and policy compliance has been recognized in the requirements engineering community as a key area for research. Similarly, legal compliance is critical in systems development, especially given that non-compliance can result in both financial and criminal penalties. Working with legal texts can be very challenging, however, because they contain numerous ambiguities, cross-references, domain-specific definitions, and acronyms, and are frequently amended via new statutes, regulations, and case law. Requirements engineers and compliance auditors must be able to identify relevant legal texts, extract requirements and other key concepts, and monitor compliance. This chapter surveys research efforts over the past 50 years in handling legal texts for systems development. This survey can aid requirements engineers and auditors to better specify, test, and monitor systems for compliance.

  5. Requirements Analysis in the Value Methodology

    Energy Technology Data Exchange (ETDEWEB)

    Conner, Alison Marie

    2001-05-01

    The Value Methodology (VM) study brings together a multidisciplinary team of people who own the problem and have the expertise to identify and solve it. With the varied backgrounds and experiences the team brings to the study, come different perspectives on the problem and the requirements of the project. A requirements analysis step can be added to the Information and Function Analysis Phases of a VM study to validate whether the functions being performed are required, either regulatory or customer prescribed. This paper will provide insight to the level of rigor applied to a requirements analysis step and give some examples of tools and techniques utilized to ease the management of the requirements and functions those requirements support for highly complex problems.

  6. Waste Acceptance System Requirements document (WASRD)

    International Nuclear Information System (INIS)

    1993-01-01

    This Waste Acceptance System Requirements document (WA-SRD) describes the functions to be performed and the technical requirements for a Waste Acceptance System for accepting spent nuclear fuel (SNF) and high-level radioactive waste (HLW) into the Civilian Radioactive Waste Management System (CRWMS). This revision of the WA-SRD addresses the requirements for the acceptance of HLW. This revision has been developed as a top priority document to permit DOE's Office of Environmental Restoration and Waste Management (EM) to commence waste qualification runs at the Savannah River Site's (SRS) Defense Waste Processing Facility (DWPF) in a timely manner. Additionally, this revision of the WA-SRD includes the requirements from the Physical System Requirements -- Accept Waste document for the acceptance of SNF. A subsequent revision will fully address requirements relative to the acceptance of SNF

  7. Virtual phosphorus ore requirement of Japanese economy.

    Science.gov (United States)

    Matsubae, Kazuyo; Kajiyama, Jun; Hiraki, Takehito; Nagasaka, Tetsuya

    2011-08-01

    Phosphorus is indispensable for agricultural production. Hence, the consumption of imported food indirectly implies the import of phosphorus resources. The global consumption of agricultural products depends on a small number of ore-producing countries. For sustainable management of phosphorus resources, the global supply and demand network should be clarified. In this study, we propose the virtual phosphorus ore requirement as a new indicator of the direct and indirect phosphorus requirements for our society. The virtual phosphorus ore requirement indicates the direct and indirect demands for phosphorus ore transformed into agricultural products and fertilizer. In this study, the virtual phosphorus ore requirement was evaluated for the Japanese economy in 2005. Importantly, the results show that our society requires twice as much phosphorus ore as the domestic demand for fertilizer production. The phosphorus contained in "eaten" agricultural products was only 12% of virtual phosphorus ore requirement. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. Mined Geologic Disposal System Requirements Document

    International Nuclear Information System (INIS)

    1993-01-01

    This Mined Geologic Disposal System Requirements document (MGDS-RD) describes the functions to be performed by, and the requirements for, a Mined Geologic Disposal System (MGDS) for the permanent disposal of spent nuclear fuel (SNF) and commercial and defense high level radioactive waste (HLW) in support of the Civilian Radioactive Waste Management System (CRWMS). The development and control of the MGDS-RD is quality-affecting work and is subject to the Department of Energy (DOE) Office of Civilian Radioactive Waste Management (OCRWM) Quality Assurance Requirements Document (QARD). As part of the technical requirements baseline, it is also subject to Baseline Management Plan controls. The MGDS-RD and the other program-level requirements documents have been prepared and managed in accordance with the Technical Document Preparation Plan (TDPP) for the Preparation of System Requirements Documents

  9. SOFTWARE REQUIREMENTS SPECIFICATION SINAPRA BERBASIS SISTEM INFORMASI

    Directory of Open Access Journals (Sweden)

    Nur Hadi Waryanto

    2015-07-01

    Full Text Available Sistem Informasi Sarana dan Prasarana (SINAPRA merupakan bagian dari beberapa sistem informasi yang dipakai oleh Univeristas Negeri Yogyakarta. SINAPRA merupakan salah satu sistem yang akan dikembangkan dalam Sistem Informasi Terpadu (SIPADU. Software Requirements Specification SINAPRA merupakan acuan teknis developer dalam mengembangkan sistem untuk tahap selanjutnya. Software Requirements Specification SINAPRA dikembangkan menggunakan model WSU-TC CptS 322  dengan berbasis sistem informasi terpadu UNY Kata Kunci : Software Requirements Specification, WSU-TC CptS 322, SINAPRA

  10. Identify and Manage the Software Requirements Volatility

    OpenAIRE

    Khloud Abd Elwahab; Mahmoud Abd EL Latif; Sherif Kholeif

    2016-01-01

    Management of software requirements volatility through development of life cycle is a very important stage. It helps the team to control significant impact all over the project (cost, time and effort), and also it keeps the project on track, to finally satisfy the user which is the main success criteria for the software project. In this research paper, we have analysed the root causes of requirements volatility through a proposed framework presenting the requirements volatility causes and how...

  11. Training Requirements and Information Management System

    Energy Technology Data Exchange (ETDEWEB)

    Cillan, T.F.; Hodgson, M.A.

    1992-05-01

    This is the software user's guide for the Training Requirements and Information Management System. This guide defines and describes the software operating procedures as they apply to the end user of the software program. This guide is intended as a reference tool for the user who already has an indepth knowledge of the Training Requirements and Information Management System functions and data reporting requirement.

  12. Requirements engineering for human activity systems

    CSIR Research Space (South Africa)

    Erasmus, J

    2014-10-01

    Full Text Available Guide for System Life Cycle Processes. Version 3.2. Edited by C. Haskins. San Diego, California: International Council on Systems Engineering, 2010. Jordan, J. Scott, ed. Systems Theories and A Priori Aspects of Perception. Amsterdam: Elsevier... to ensure that such requirements are properly developed and managed, by applying well defined rules and techniques (Hull, Jackson and Dick 2011). For example, a requirement should not be unrealistic and each requirement must be accompanied by criteria...

  13. Towards a framework for improving requirement traceability

    Directory of Open Access Journals (Sweden)

    Marco Antonio Toranzo Céspedes

    2012-01-01

    Full Text Available Work regarding requirement traceability focuses on programming aspects instead of identifying, analysing and modelling all traceable data in a software project. This paper describes the development of a framework for improving software requirement traceability. The framework consisted of classifying traced information, defining and using relationship types regarding traced information, a set of guidelines for developing a requirements traceability model for a software project and using my management tool (MyMT tool to support developing a requirement traceability model. A university library management system was used to illustrate applying the framework.

  14. Safety of Research Reactors. Safety Requirements

    International Nuclear Information System (INIS)

    2010-01-01

    The main objective of this Safety Requirements publication is to provide a basis for safety and a basis for safety assessment for all stages in the lifetime of a research reactor. Another objective is to establish requirements on aspects relating to regulatory control, the management of safety, site evaluation, design, operation and decommissioning. Technical and administrative requirements for the safety of research reactors are established in accordance with these objectives. This Safety Requirements publication is intended for use by organizations engaged in the site evaluation, design, manufacturing, construction, operation and decommissioning of research reactors as well as by regulatory bodies

  15. 36 CFR 254.3 - Requirements.

    Science.gov (United States)

    2010-07-01

    ...; consolidation of split estates; expansion of communities; accommodation of existing or planned land use... required to exchange any Federal lands. Land exchanges are discretionary, voluntary real estate...

  16. EMS certification requirements for flight nurses.

    Science.gov (United States)

    Frakes, Michael A; Lord, Wendy R

    2004-01-01

    Emergency medical technician (EMT) or paramedic (EMTP) certification requirements for flight nurses (FNs) providing on-scene patient care vary. We surveyed those requirements and evaluated the relationships between flight team composition or program location and FN EMS certification. Telephone survey of all 184 rotor-wing programs responding with a nurse to scenes The overall EMS training requirement for FNs was: none-57.6%, EMT-21.7%, EMTP-14.7%, local credential (not EMT or EMTP)-6.0%. Second team members were EMTP, RN, physician, or respiratory therapist (RRT). Overall, team configuration related significantly to FN EMS certification (P =.01). FN/EMTP and FN/RRT teams were individually significant (P EMTP teams tending not to require certification and all FN/RRT teams tending toward a certification requirement. Neither FN/FN nor FN/physician pairings related significantly with FN EMS certification requirements. Regional patterns emerged to both crew configuration and FN EMS certification requirements. Most flight programs do not require FN EMT/EMTP certification. Team configuration and geography are related to those requirements.

  17. SE Requirements Development Tool User Guide

    Energy Technology Data Exchange (ETDEWEB)

    Benson, Faith Ann [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2016-05-13

    The LANL Systems Engineering Requirements Development Tool (SERDT) is a data collection tool created in InfoPath for use with the Los Alamos National Laboratory’s (LANL) SharePoint sites. Projects can fail if a clear definition of the final product requirements is not performed. For projects to be successful requirements must be defined early in the project and those requirements must be tracked during execution of the project to ensure the goals of the project are met. Therefore, the focus of this tool is requirements definition. The content of this form is based on International Council on Systems Engineering (INCOSE) and Department of Defense (DoD) process standards and allows for single or collaborative input. The “Scoping” section is where project information is entered by the project team prior to requirements development, and includes definitions and examples to assist the user in completing the forms. The data entered will be used to define the requirements and once the form is filled out, a “Requirements List” is automatically generated and a Word document is created and saved to a SharePoint document library. SharePoint also includes the ability to download the requirements data defined in the InfoPath from into an Excel spreadsheet. This User Guide will assist you in navigating through the data entry process.

  18. Hydrogen tomorrow: Demands and technology requirements

    Science.gov (United States)

    1975-01-01

    National needs for hydrogen are projected and the technologies of production, handling, and utilization are evaluated. Research and technology activities required to meet the projected needs are determined.

  19. Defense Logistics Agency Family Housing Requirements

    National Research Council Canada - National Science Library

    Granetto, Paul

    1999-01-01

    The Director, Defense Logistics Agency (Installation Support Group) requested the audit to review the process the Defense Logistics Agency installations use to determine family housing requirements...

  20. Nursing staff requirements for neonatal intensive care.

    OpenAIRE

    Williams, S; Whelan, A; Weindling, A M; Cooke, R W

    1993-01-01

    A study to estimate the number of nursing staff required for neonatal nursing was undertaken. Certain nursing tasks, such as transporting any infant, caring for the dying infant, and looking after the very unstable infant required continuous attention by one nurse (5.5 whole time equivalent (wte) nurses for each cot). The stable ventilated infant required 10.5 nursing hours each day-that is, 2.4 wte/cot. Infants with intravenous infusions, but not ventilated, required only slightly less nursi...

  1. 7 CFR 352.8 - Marking requirements.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 5 2010-01-01 2010-01-01 false Marking requirements. 352.8 Section 352.8 Agriculture Regulations of the Department of Agriculture (Continued) ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE PLANT QUARANTINE SAFEGUARD REGULATIONS § 352.8 Marking requirements. Prohibited and...

  2. 40 CFR 264.1065 - Reporting requirements.

    Science.gov (United States)

    2010-07-01

    ... Air Emission Standards for Equipment Leaks § 264.1065 Reporting requirements. (a) A semiannual report...). (iii) The equipment identification number of each compressor for which a leak was not repaired as... monitoring required by § 264.1060 and was not corrected within 24 hours, the duration and cause of each...

  3. 40 CFR 63.982 - Requirements.

    Science.gov (United States)

    2010-07-01

    ... Standards for Closed Vent Systems, Control Devices, Recovery Devices and Routing to a Fuel Gas System or a Process § 63.982 Requirements. (a) General compliance requirements for storage vessels, process vents... subpart apply to emissions being routed to a fuel gas system or process. (e) Final recovery devices...

  4. Requirements engineering: foundation for software quality

    NARCIS (Netherlands)

    Daneva, Maia; Pastor, Oscar

    2016-01-01

    Welcome to the proceedings of the 22nd edition of REFSQ: the International Working Conference on Requirements Engineering – Foundation for Software Quality! Requirements engineering (RE) has been recognized as a critical factor that impacts the quality of software, systems, and services. Since the

  5. Regulatory capital requirements and bail in mechanisms

    NARCIS (Netherlands)

    Joosen, B.P.M.; Haentjens, M.; Wessels, B.

    2015-01-01

    With the introduction of the Capital Requirements Regulation (CRR) in the European Union, the qualitative requirements for bank regulatory capital have changed. These changes aim at implementing in Europe the Basel III principles for better bank capital that is able to absorb losses of banks,

  6. 24 CFR 290.11 - Notification requirements.

    Science.gov (United States)

    2010-04-01

    ... URBAN DEVELOPMENT HUD-OWNED PROPERTIES DISPOSITION OF MULTIFAMILY PROJECTS AND SALE OF HUD-HELD MULTIFAMILY MORTGAGES Disposition of Multifamily Projects § 290.11 Notification requirements. (a) In general. HUD may combine two or more of the required notifications, as appropriate, to simplify the disposition...

  7. Quality assurance management policies and requirements

    International Nuclear Information System (INIS)

    1985-10-01

    The purpose of this document is to: set forth overall, integrated quality assurance management policies and requirements for the entire Civilian Radioactive Waste Management Program; define management responsibilities for assuring quality; and provide a general framework for the development of more detailed quality assurance management policies and requirements by program, project, and contractor organizations

  8. 12 CFR 614.4255 - Independence requirements.

    Science.gov (United States)

    2010-01-01

    ... Collateral Evaluation Requirements § 614.4255 Independence requirements. (a) Prohibitions. For all personal and intangible property, and for all real property exempted under § 614.4260(c) of this subpart, no... indirect interest, financial or otherwise, in the loan or subject property; (2) As a director, vote on or...

  9. 12 CFR 615.5050 - Collateral requirements.

    Science.gov (United States)

    2010-01-01

    ... 12 Banks and Banking 6 2010-01-01 2010-01-01 false Collateral requirements. 615.5050 Section 615... POLICIES AND OPERATIONS, AND FUNDING OPERATIONS Collateral § 615.5050 Collateral requirements. (a) Each... property acquired in connection with loans made under the Act, obligations of the United States or any...

  10. 12 CFR 614.4260 - Evaluation requirements.

    Science.gov (United States)

    2010-01-01

    ... on real property has been taken as collateral in an abundance of caution, and the application, when... Collateral Evaluation Requirements § 614.4260 Evaluation requirements. (a) Valuation. Valuations of personal and intangible property, as well as real property exempted under paragraph (c) of this section, shall...

  11. 42 CFR 433.74 - Reporting requirements.

    Science.gov (United States)

    2010-10-01

    ... Financial Participation § 433.74 Reporting requirements. (a) Beginning with the first quarter of Federal fiscal year 1993, each State must submit to CMS quarterly summary information on the source and use of... 42 Public Health 4 2010-10-01 2010-10-01 false Reporting requirements. 433.74 Section 433.74...

  12. 29 CFR 4010.3 - Filing requirement.

    Science.gov (United States)

    2010-07-01

    ... Relating to Labor (Continued) PENSION BENEFIT GUARANTY CORPORATION CERTAIN REPORTING AND DISCLOSURE REQUIREMENTS ANNUAL FINANCIAL AND ACTUARIAL INFORMATION REPORTING § 4010.3 Filing requirement. (a) General....10, all information specified in § 4010.6(a) with respect to all members of a controlled group and...

  13. 40 CFR 59.105 - Reporting requirements.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 5 2010-07-01 2010-07-01 false Reporting requirements. 59.105 Section... Volatile Organic Compound Emission Standards for Automobile Refinish Coatings § 59.105 Reporting requirements. (a) Each regulated entity must submit an initial report no later than January 11, 1999 or within...

  14. 42 CFR 54.7 - Nondiscrimination requirement.

    Science.gov (United States)

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false Nondiscrimination requirement. 54.7 Section 54.7 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS CHARITABLE CHOICE... PROJECTS FOR ASSISTANCE IN TRANSITION FROM HOMELESSNESS GRANTS § 54.7 Nondiscrimination requirement. A...

  15. 42 CFR 441.208 - Recordkeeping requirements.

    Science.gov (United States)

    2010-10-01

    ... 42 Public Health 4 2010-10-01 2010-10-01 false Recordkeeping requirements. 441.208 Section 441.208 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES... Abortions § 441.208 Recordkeeping requirements. Medicaid agencies must maintain copies of the certifications...

  16. 46 CFR 108.213 - Heating requirements.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Heating requirements. 108.213 Section 108.213 Shipping... EQUIPMENT Construction and Arrangement Accommodation Spaces § 108.213 Heating requirements. (a) Each accommodation space must be heated by a heating system that can maintain at least 20°C. (68°F.). (b) Radiators...

  17. 38 CFR 21.3102 - Required counseling.

    Science.gov (United States)

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false Required counseling. 21.... Chapter 35 Counseling § 21.3102 Required counseling. (a) Child. The VA counseling psychologist will provide counseling and assist in preparing the educational plan only if the eligible child or his or her...

  18. 46 CFR 184.100 - General requirement.

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false General requirement. 184.100 Section 184.100 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) VESSEL CONTROL AND MISCELLANEOUS SYSTEMS AND EQUIPMENT General Provisions § 184.100 General requirement...

  19. 40 CFR 761.40 - Marking requirements.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Marking requirements. 761.40 Section 761.40 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL... PROHIBITIONS Marking of PCBs and PCB Items § 761.40 Marking requirements. (a) Each of the following items in...

  20. 11 CFR 200.2 - Procedural requirements.

    Science.gov (United States)

    2010-01-01

    ... 11 Federal Elections 1 2010-01-01 2010-01-01 false Procedural requirements. 200.2 Section 200.2 Federal Elections FEDERAL ELECTION COMMISSION ADMINISTRATIVE REGULATIONS PETITIONS FOR RULEMAKING § 200.2 Procedural requirements. (a) Any interested person may file with the Commission a written petition for the...