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Sample records for large-scale transposon mutagenesis

  1. Transposon mutagenesis of Bacteroides fragilis using a mariner transposon vector.

    PubMed

    Veeranagouda, Yaligara; Husain, Fasahath; Wexler, Hannah M

    2013-08-01

    The mariner transposon vector pYV07 was tested for use in the mutagenesis of Bacteroides fragilis 638R. The transposon vector efficiently generated mutants in B. fragilis 638R. The transposon disrupted genes were scattered throughout the genome of B. fragilis 638R. This method serves as a powerful tool to study B. fragilis. PMID:23664906

  2. Genome-Wide Transposon Mutagenesis in Saccharomyces cerevisiae and Candida albicans

    PubMed Central

    Xu, Tao; Bharucha, Nikë; Kumar, Anuj

    2016-01-01

    Transposon mutagenesis is an effective method for generating large sets of random mutations in target DNA, with applicability toward numerous types of genetic screens in prokaryotes, single-celled eukaryotes, and metazoans alike. Relative to methods of random mutagenesis by chemical/UV treatment, transposon insertions can be easily identified in mutants with phenotypes of interest. The construction of transposon insertion mutants is also less labor-intensive on a genome-wide scale than methods for targeted gene replacement, although transposon insertions are not precisely targeted to a specific residue, and thus coverage of the target DNA can be problematic. The collective advantages of transposon mutagenesis have been well demonstrated in studies of the budding yeast Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans, as transposon mutagenesis has been used extensively for phenotypic screens in both yeasts. Consequently, we present here protocols for the generation and utilization of transposon-insertion DNA libraries in S. cerevisiae and C. albicans. Specifically, we present methods for the large-scale introduction of transposon insertion alleles in a desired strain of S. cerevisiae. Methods are also presented for transposon mutagenesis of C. albicans, encompassing both the construction of the plasmid-based transposon-mutagenized DNA library and its introduction into a desired strain of Candida. In total, these methods provide the necessary information to implement transposon mutagenesis in yeast, enabling the construction of large sets of identifiable gene disruption mutations, with particular utility for phenotypic screening in nonstandard genetic backgrounds. PMID:21815095

  3. Construction of a large-scale Burkholderia cenocepacia J2315 transposon mutant library

    NASA Astrophysics Data System (ADS)

    Wong, Yee-Chin; Pain, Arnab; Nathan, Sheila

    2014-09-01

    Burkholderia cenocepacia, a pathogenic member of the Burkholderia cepacia complex (Bcc), has emerged as a significant threat towards cystic fibrosis patients, where infection often leads to the fatal clinical manifestation known as cepacia syndrome. Many studies have investigated the pathogenicity of B. cenocepacia as well as its ability to become highly resistant towards many of the antibiotics currently in use. In addition, studies have also been undertaken to understand the pathogen's capacity to adapt and survive in a broad range of environments. Transposon based mutagenesis has been widely used in creating insertional knock-out mutants and coupled with recent advances in sequencing technology, robust tools to study gene function in a genome-wide manner have been developed based on the assembly of saturated transposon mutant libraries. In this study, we describe the construction of a large-scale library of B. cenocepacia transposon mutants. To create transposon mutants of B. cenocepacia strain J2315, electrocompetent bacteria were electrotransformed with the EZ-Tn5 transposome. Tetracyline resistant colonies were harvested off selective agar and pooled. Mutants were generated in multiple batches with each batch consisting of ˜20,000 to 40,000 mutants. Transposon insertion was validated by PCR amplification of the transposon region. In conclusion, a saturated B. cenocepacia J2315 transposon mutant library with an estimated total number of 500,000 mutants was successfully constructed. This mutant library can now be further exploited as a genetic tool to assess the function of every gene in the genome, facilitating the discovery of genes important for bacterial survival and adaptation, as well as virulence.

  4. Genome-Wide Synthetic Genetic Screening by Transposon Mutagenesis in Candida albicans

    PubMed Central

    Horton, Brooke N.; Kumar, Anuj

    2016-01-01

    Transposon-based mutagenesis is an effective method for genetic screening on a genome-wide scale, with particular applicability in organisms possessing compact genomes where transforming DNA tends to integrate by homologous recombination. Methods for transposon mutagenesis have been applied with great success in the budding yeast Saccharomyces cerevisiae and in the related pathogenic yeast Candida albicans. In C. albicans, we have implemented transposon mutagenesis to generate heterozygous mutations for the analysis of complex haploinsufficiency, a type of synthetic genetic interaction wherein a pair of non-complementing heterozygous mutations results in a stronger phenotype then either individual mutation in isolation. Genes exhibiting complex haploinsufficiency typically function within a regulatory pathway, in parallel pathways, or in parallel branches within a single pathway. Here, we present protocols to implement transposon mutagenesis for complex haploinsufficiency screening in C. albicans, indicating methods for transposon construction, mutagenesis, phenotypic screening, and identification of insertion sites in strains of interest. In total, the approach is a useful means to implement large-scale synthetic genetic screening in the diploid C. albicans. PMID:25636616

  5. Transposon mutagenesis of Pseudomonas aeruginosa exoprotease genes.

    PubMed Central

    Stapleton, M J; Jagger, K S; Warren, R L

    1984-01-01

    Transposon Tn5 was used to generate protease-deficient insertion mutants of Pseudomonas aeruginosa. The presence of Tn5 in the chromosome of P. aeruginosa was demonstrated by transduction and DNA-DNA hybridization. The altered protease production and kanamycin resistance were cotransduced into a wild-type P. aeruginosa strain. A radiolabeled probe of Tn5 DNA hybridized to specific BamHI fragments isolated from the insertion mutants. Two independently isolated Tn5 insertion mutants had reduced protease production, partially impaired elastase activity, and no immunologically reactive alkaline protease. Images PMID:6317657

  6. Genome-wide transposon mutagenesis in pathogenic Leptospira species.

    PubMed

    Murray, Gerald L; Morel, Viviane; Cerqueira, Gustavo M; Croda, Julio; Srikram, Amporn; Henry, Rebekah; Ko, Albert I; Dellagostin, Odir A; Bulach, Dieter M; Sermswan, Rasana W; Adler, Ben; Picardeau, Mathieu

    2009-02-01

    Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans. PMID:19047402

  7. Sleeping Beauty transposon insertional mutagenesis based mouse models for cancer gene discovery

    PubMed Central

    Moriarity, Branden S; Largaespada, David A

    2016-01-01

    Large-scale genomic efforts to study human cancer, such as the cancer gene atlas (TCGA), have identified numerous cancer drivers in a wide variety of tumor types. However, there are limitations to this approach, the mutations and expression or copy number changes that are identified are not always clearly functionally relevant, and only annotated genes and genetic elements are thoroughly queried. The use of complimentary, nonbiased, functional approaches to identify drivers of cancer development and progression is ideal to maximize the rate at which cancer discoveries are achieved. One such approach that has been successful is the use of the Sleeping Beauty (SB) transposon-based mutagenesis system in mice. This system uses a conditionally expressed transposase and mutagenic transposon allele to target mutagenesis to somatic cells of a given tissue in mice to cause random mutations leading to tumor development. Analysis of tumors for transposon common insertion sites (CIS) identifies candidate cancer genes specific to that tumor type. While similar screens have been performed in mice with the PiggyBac (PB) transposon and viral approaches, we limit extensive discussion to SB. Here we discuss the basic structure of these screens, screens that have been performed, methods used to identify CIS. PMID:26051241

  8. The Hermes Transposon of Musca domestica Is an Efficient Tool for the Mutagenesis of Schizosaccharomyces pombe

    PubMed Central

    Evertts, Adam G.; Plymire, Christopher; Craig, Nancy L.; Levin, Henry L.

    2007-01-01

    Currently, no transposon-based method for the mutagenesis of Schizosaccharomyces pombe exists. We have developed such a system based on the introduction of the hermes transposon from the housefly into S. pombe. This system efficiently disrupts open reading frames and allows the insertion sites to be readily identified. PMID:17947404

  9. The hermes transposon of Musca domestica is an efficient tool for the mutagenesis of Schizosaccharomyces pombe.

    PubMed

    Evertts, Adam G; Plymire, Christopher; Craig, Nancy L; Levin, Henry L

    2007-12-01

    Currently, no transposon-based method for the mutagenesis of Schizosaccharomyces pombe exists. We have developed such a system based on the introduction of the hermes transposon from the housefly into S. pombe. This system efficiently disrupts open reading frames and allows the insertion sites to be readily identified. PMID:17947404

  10. Random insertion and gene disruption via transposon mutagenesis of Ureaplasma parvum using a mini-transposon plasmid.

    PubMed

    Aboklaish, Ali F; Dordet-Frisoni, Emilie; Citti, Christine; Toleman, Mark A; Glass, John I; Spiller, O Brad

    2014-11-01

    While transposon mutagenesis has been successfully used for Mycoplasma spp. to disrupt and determine non-essential genes, previous attempts with Ureaplasma spp. have been unsuccessful. Using a polyethylene glycol-transformation enhancing protocol, we were able to transform three separate serovars of Ureaplasma parvum with a Tn4001-based mini-transposon plasmid containing a gentamicin resistance selection marker. Despite the large degree of homology between Ureaplasma parvum and Ureaplasma urealyticum, all attempts to transform the latter in parallel failed, with the exception of a single clinical U. urealyticum isolate. PCR probing and sequencing were used to confirm transposon insertion into the bacterial genome and identify disrupted genes. Transformation of prototype serovar 3 consistently resulted in transfer only of sequence between the mini-transposon inverted repeats, but some strains showed additional sequence transfer. Transposon insertion occurred randomly in the genome resulting in unique disruption of genes UU047, UU390, UU440, UU450, UU520, UU526, UU582 for single clones from a panel of screened clones. An intergenic insertion between genes UU187 and UU188 was also characterised. Two phenotypic alterations were observed in the mutated strains: Disruption of a DEAD-box RNA helicase (UU582) altered growth kinetics, while the U. urealyticum strain lost resistance to serum attack coincident with disruption of gene UUR10_137 and loss of expression of a 41 kDa protein. Transposon mutagenesis was used successfully to insert single copies of a mini-transposon into the genome and disrupt genes leading to phenotypic changes in Ureaplasma parvum strains. This method can now be used to deliver exogenous genes for expression and determine essential genes for Ureaplasma parvum replication in culture and experimental models. PMID:25444567

  11. PiggyBac transposon mutagenesis: a tool for cancer gene discovery in mice.

    PubMed

    Rad, Roland; Rad, Lena; Wang, Wei; Cadinanos, Juan; Vassiliou, George; Rice, Stephen; Campos, Lia S; Yusa, Kosuke; Banerjee, Ruby; Li, Meng Amy; de la Rosa, Jorge; Strong, Alexander; Lu, Dong; Ellis, Peter; Conte, Nathalie; Yang, Fang Tang; Liu, Pentao; Bradley, Allan

    2010-11-19

    Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here, we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon-transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed that PiggyBac is capable of genome-wide mutagenesis. The PiggyBac screen uncovered many cancer genes not identified in previous retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-related transcription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty have different integration preferences. To maximize the utility of the tool, we engineered 21 mouse lines to be compatible with both transposon systems in constitutive, tissue- or temporal-specific mutagenesis. Mice with different transposon types, copy numbers, and chromosomal locations support wide applicability. PMID:20947725

  12. Transposon mutagenesis as an approach to improved understanding of Borrelia pathogenesis and biology

    PubMed Central

    Lin, Tao; Troy, Erin B.; Hu, Linden T.; Gao, Lihui; Norris, Steven J.

    2014-01-01

    Transposon insertion provides a method for near-random mutation of bacterial genomes, and has been utilized extensively for the study of bacterial pathogenesis and biology. This approach is particularly useful for organisms that are relatively refractory to genetic manipulation, including Lyme disease Borrelia. In this review, progress to date in the application of transposon mutagenesis to the study of Borrelia burgdorferi is reported. An effective Himar1-based transposon vector has been developed and used to acquire a sequence-defined library of nearly 4500 mutants in the infectious, moderately transformable B. burgdorferi B31 derivative 5A18NP1. Analysis of these transposon mutants using signature-tagged mutagenesis (STM) and Tn-seq approaches has begun to yield valuable information regarding the genes important in the pathogenesis and biology of this organism. PMID:24904839

  13. Insertional Mutagenesis by a Hybrid PiggyBac and Sleeping Beauty Transposon in the Rat

    PubMed Central

    Furushima, Kenryo; Jang, Chuan-Wei; Chen, Diane W.; Xiao, Ningna; Overbeek, Paul A.; Behringer, Richard R.

    2012-01-01

    A hybrid piggyBac/Sleeping Beauty transposon-based insertional mutagenesis system that can be mobilized by simple breeding was established in the rat. These transposons were engineered to include gene trap sequences and a tyrosinase (Tyr) pigmentation reporter to rescue the albinism of the genetic background used in the mutagenesis strategy. Single-copy transposon insertions were transposed into the rat genome by co-injection of plasmids carrying the transposon and RNA encoding piggyBac transposase into zygotes. The levels of transgenic Tyr expression were influenced by chromosomal context, leading to transgenic rats with different pigmentation that enabled visual genotyping. Transgenic rats designed to ubiquitously express either piggyBac or Sleeping Beauty transposase were generated by standard zygote injection also on an albino background. Bigenic rats carrying single-copy transposons at known loci and transposase transgenes exhibited coat color mosaicism, indicating somatic transposition. PiggyBac or Sleeping Beauty transposase bigenic rats bred with wild-type albino rats yielded offspring with pigmentation distinct from the initial transposon insertions as a consequence of germline transposition to new loci. The germline transposition frequency for Sleeping Beauty and piggyBac was ∼10% or about one new insertion per litter. Approximately 50% of the insertions occurred in introns. Chimeric transcripts containing endogenous and gene trap sequences were identified in Gabrb1 mutant rats. This mutagenesis system based on simple crosses and visual genotyping can be used to generate a collection of single-gene mutations in the rat. PMID:23023007

  14. Sleeping Beauty transposon mutagenesis identifies genes that cooperate with mutant Smad4 in gastric cancer development.

    PubMed

    Takeda, Haruna; Rust, Alistair G; Ward, Jerrold M; Yew, Christopher Chin Kuan; Jenkins, Nancy A; Copeland, Neal G

    2016-04-01

    Mutations in SMAD4 predispose to the development of gastrointestinal cancer, which is the third leading cause of cancer-related deaths. To identify genes driving gastric cancer (GC) development, we performed a Sleeping Beauty (SB) transposon mutagenesis screen in the stomach of Smad4(+/-) mutant mice. This screen identified 59 candidate GC trunk drivers and a much larger number of candidate GC progression genes. Strikingly, 22 SB-identified trunk drivers are known or candidate cancer genes, whereas four SB-identified trunk drivers, including PTEN, SMAD4, RNF43, and NF1, are known human GC trunk drivers. Similar to human GC, pathway analyses identified WNT, TGF-β, and PI3K-PTEN signaling, ubiquitin-mediated proteolysis, adherens junctions, and RNA degradation in addition to genes involved in chromatin modification and organization as highly deregulated pathways in GC. Comparative oncogenomic filtering of the complete list of SB-identified genes showed that they are highly enriched for genes mutated in human GC and identified many candidate human GC genes. Finally, by comparing our complete list of SB-identified genes against the list of mutated genes identified in five large-scale human GC sequencing studies, we identified LDL receptor-related protein 1B (LRP1B) as a previously unidentified human candidate GC tumor suppressor gene. In LRP1B, 129 mutations were found in 462 human GC samples sequenced, and LRP1B is one of the top 10 most deleted genes identified in a panel of 3,312 human cancers. SB mutagenesis has, thus, helped to catalog the cooperative molecular mechanisms driving SMAD4-induced GC growth and discover genes with potential clinical importance in human GC. PMID:27006499

  15. Excavating the Genome: Large Scale Mutagenesis Screening for the Discovery of New Mouse Models

    PubMed Central

    Sundberg, John P.; Dadras, Soheil S.; Silva, Kathleen A.; Kennedy, Victoria E.; Murray, Stephen A.; Denegre, James; Schofield, Paul N.; King, Lloyd E.; Wiles, Michael; Pratt, C. Herbert

    2016-01-01

    Technology now exists for rapid screening of mutated laboratory mice to identify phenotypes associated with specific genetic mutations. Large repositories exist for spontaneous mutants and those induced by chemical mutagenesis, many of which have never been studied or comprehensively evaluated. To supplement these resources, a variety of techniques have been consolidated in an international effort to create mutations in all known protein coding genes in the mouse. With targeted embryonic stem cell lines now available for almost all protein coding genes and more recently CRISPR/Cas9 technology, large-scale efforts are underway to create novel mutant mouse strains and to characterize their phenotypes. However, accurate diagnosis of skin, hair, and nail diseases still relies on careful gross and histological analysis. While not automated to the level of the physiological phenotyping, histopathology provides the most direct and accurate diagnosis and correlation with human diseases. As a result of these efforts, many new mouse dermatological disease models are being developed. PMID:26551941

  16. A Plasmid-Transposon Hybrid Mutagenesis System Effective in a Broad Range of Enterobacteria

    PubMed Central

    Monson, Rita; Smith, Debra S.; Matilla, Miguel A.; Roberts, Kevin; Richardson, Elizabeth; Drew, Alison; Williamson, Neil; Ramsay, Josh; Welch, Martin; Salmond, George P. C.

    2015-01-01

    Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii, and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways. PMID:26733980

  17. Evaluating Risks of Insertional Mutagenesis by DNA Transposons in Gene Therapy

    PubMed Central

    Hackett, Perry B.; Largaespada, David A.; Switzer, Kirsten C.; Cooper, Laurence J.N.

    2013-01-01

    Investigational therapy can be successfully undertaken using viral- and non-viral-mediated ex vivo gene transfer. Indeed, recent clinical trials have established the potential for genetically modified T cells to improve and restore health. Recently the Sleeping Beauty (SB) transposon/transposase system has been applied in clinical trials to stably insert a chimeric antigen receptor (CAR) to redirect T-cell specificity. We discuss the context in which the SB system can be harnessed for gene therapy and describe the human application of SB-modified CAR+ T cells. We have focused on theoretical issues relating to insertional mutagenesis in the context of human genomes that are naturally subjected to remobilization of transposons and the experimental evidence over the last decade of employing SB transposons for defining genes that induce cancer. These findings are put into the context of the use of SB transposons in the treatment of human disease. PMID:23313630

  18. Development of an inducible transposon system for efficient random mutagenesis in Clostridium acetobutylicum

    PubMed Central

    Zhang, Ying; Xu, Shu; Chai, Changsheng; Yang, Sheng; Jiang, Weihong; Minton, Nigel P.; Gu, Yang

    2016-01-01

    Clostridium acetobutylicum is an industrially important Gram-positive organism, which is capable of producing economically important chemicals in the ABE (Acetone, Butanol and Ethanol) fermentation process. Renewed interests in the ABE process necessitate the availability of additional genetics tools to facilitate the derivation of a greater understanding of the underlying metabolic and regulatory control processes in operation through forward genetic strategies. In this study, a xylose inducible, mariner-based, transposon system was developed and shown to allow high-efficient random mutagenesis in the model strain ATCC 824. Of the thiamphenicol resistant colonies obtained, 91.9% were shown to be due to successful transposition of the catP-based mini-transposon element. Phenotypic screening of 200 transposon clones revealed a sporulation-defective clone with an insertion in spo0A, thereby demonstrating that this inducible transposon system can be used for forward genetic studies in C. acetobutylicum. PMID:27001972

  19. Development of an inducible transposon system for efficient random mutagenesis in Clostridium acetobutylicum.

    PubMed

    Zhang, Ying; Xu, Shu; Chai, Changsheng; Yang, Sheng; Jiang, Weihong; Minton, Nigel P; Gu, Yang

    2016-04-01

    Clostridium acetobutylicum is an industrially important Gram-positive organism, which is capable of producing economically important chemicals in the ABE (Acetone, Butanol and Ethanol) fermentation process. Renewed interests in the ABE process necessitate the availability of additional genetics tools to facilitate the derivation of a greater understanding of the underlying metabolic and regulatory control processes in operation through forward genetic strategies. In this study, a xylose inducible, mariner-based, transposon system was developed and shown to allow high-efficient random mutagenesis in the model strain ATCC 824. Of the thiamphenicol resistant colonies obtained, 91.9% were shown to be due to successful transposition of the catP-based mini-transposon element. Phenotypic screening of 200 transposon clones revealed a sporulation-defective clone with an insertion in spo0A, thereby demonstrating that this inducible transposon system can be used for forward genetic studies in C. acetobutylicum. PMID:27001972

  20. Isolation of Magnetospirillum magneticum AMB-1 mutants defective in bacterial magnetic particle synthesis by transposon mutagenesis.

    PubMed

    Wahyudi, A T; Takeyama, H; Matsunaga, T

    2001-01-01

    Nonmagnetic mutants of Magnetospirillum magneticum AMB-1 were recovered following mini-Tn5 transposon mutagenesis. Transconjugants with kanamycin resistance were obtained at a frequency of 2.7 x 10(-7) per recipient. Of 3327 transconjugants, 62 were defective for bacterial magnetic particle (BMP) synthesis. The frequency of independent transposition events for nonmagnetic mutants was about 1.4% in transconjugants. Further analysis of DNA sequences flanking transposon by inverted polymerase chain reaction allowed isolation of at least 10 genes or DNA sequences involved in BMP synthesis in M. magneticum AMB-1. PMID:11963843

  1. Excavating the Genome: Large-Scale Mutagenesis Screening for the Discovery of New Mouse Models.

    PubMed

    Sundberg, John P; Dadras, Soheil S; Silva, Kathleen A; Kennedy, Victoria E; Murray, Stephen A; Denegre, James M; Schofield, Paul N; King, Lloyd E; Wiles, Michael V; Pratt, C Herbert

    2015-11-01

    Technology now exists for rapid screening of mutated laboratory mice to identify phenotypes associated with specific genetic mutations. Large repositories exist for spontaneous mutants and those induced by chemical mutagenesis, many of which have never been fully studied or comprehensively evaluated. To supplement these resources, a variety of techniques have been consolidated in an international effort to create mutations in all known protein coding genes in the mouse. With targeted embryonic stem cell lines now available for almost all protein coding genes and more recently CRISPR/Cas9 technology, large-scale efforts are underway to create further novel mutant mouse strains and to characterize their phenotypes. However, accurate diagnosis of skin, hair, and nail diseases still relies on careful gross and histological analysis, and while not automated to the level of the physiological phenotyping, histopathology still provides the most direct and accurate diagnosis and correlation with human diseases. As a result of these efforts, many new mouse dermatological disease models are being characterized and developed. PMID:26551941

  2. An inducible tool for random mutagenesis in Aspergillus niger based on the transposon Vader.

    PubMed

    Paun, Linda; Nitsche, Benjamin; Homan, Tim; Ram, Arthur F; Kempken, Frank

    2016-07-01

    The ascomycete Aspergillus niger is widely used in the biotechnology, for instance in producing most of the world's citric acid. It is also known as a major food and feed contaminant. While generation of gene knockouts for functional genomics has become feasible in ku70 mutants, analyzing gene functions or metabolic pathways remains a laborious task. An unbiased transposon-based mutagenesis approach may aid this process of analyzing gene functions by providing mutant libraries in a short time. The Vader transposon is a non-autonomous DNA-transposon, which is activated by the homologous tan1-transposase. However, in the most commonly used lab strain of A. niger (N400 strain and derivatives), we found that the transposase, encoded by the tan1 gene, is mutated and inactive. To establish a Vader transposon-based mutagenesis system in the N400 background, we expressed the functional transposase of A. niger strain CBS 513.88 under the control of an inducible promoter based on the Tet-on system, which is activated in the presence of the antibiotic doxycycline (DOX). Increasing amounts of doxycycline lead to higher Vader excision frequencies, whereas little to none activity of Vader was observed without addition of doxycycline. Hence, this system appears to be suitable for producing stable mutants in the A. niger N400 background. PMID:27003267

  3. Transposon Mutagenesis of the Extremely Thermophilic Bacterium Thermus thermophilus HB27

    PubMed Central

    Carr, Jennifer F.; Gregory, Steven T.; Dahlberg, Albert E.

    2014-01-01

    Thermus thermophilus is an extremely thermophilic bacterium that grows between 50 °C and 80 °C and is an excellent model organism not only for understanding life at high temperature but also for its biotechnological and industrial applications. Multiple molecular capabilities are available including targeted gene inactivation and the use of shuttle plasmids that replicate in T. thermophilus and Escherichia coli; however the ability to disrupt gene function randomly by transposon insertion has not been developed. Here we report a detailed method of transposon mutagenesis of T. thermophilus HB27 based on the EZ-Tn5 system from Epicentre Biotechnologies. We were able to generate insertion mutations throughout the chromosome by in vitro transposition and transformation with mutagenized genomic DNA. We also report that an additional step, one that fills in single stranded gaps in donor DNA generated by the transposition reaction, was essential for successful mutagenesis. We anticipate that our method of transposon mutagenesis will enable further genetic development of T. thermophilus and may also be valuable for similar endeavors with other under-developed organisms. PMID:24948436

  4. Steady-state transposon mutagenesis in inbred maize.

    PubMed

    McCarty, Donald R; Settles, Andrew Mark; Suzuki, Masaharu; Tan, Bao Cai; Latshaw, Susan; Porch, Tim; Robin, Kevin; Baier, John; Avigne, Wayne; Lai, Jinsheng; Messing, Joachim; Koch, Karen E; Hannah, L Curtis

    2005-10-01

    We implement a novel strategy for harnessing the power of high-copy transposons for functional analysis of the maize genome, and report behavioral features of the Mutator system in a uniform inbred background. The unique UniformMu population and database facilitate high-throughput molecular analysis of Mu-tagged mutants and gene knockouts. Key features of the population include: (i) high mutation frequencies (7% independent seed mutations) and moderation of copy number (approximately 57 total Mu elements; 1-2 MuDR copies per plant) were maintained by continuous back-crossing into a phenotypically uniform inbred background; (ii) a bz1-mum9 marker enabled selection of stable lines (loss of MuDR), inhibiting further transpositions in lines selected for molecular analysis; (iii) build-up of mutation load was prevented by screening Mu-active parents to exclude plants carrying pre-existing seed mutations. To create a database of genomic sequences flanking Mu insertions, selected mutant lines were analyzed by sequencing of MuTAIL PCR clone libraries. These sequences were annotated and clustered to facilitate bioinformatic subtraction of ancestral elements and identification of insertions unique to mutant lines. New insertions targeted low-copy, gene-rich sequences, and in silico mapping revealed a random distribution of insertions over the genome. Our results indicate that Mu populations differ markedly in the occurrence of Mu insertion hotspots and the frequency of suppressible mutations. We suggest that controlled MuDR copy number in UniformMu lines is a key determinant of these differences. The public database (http://uniformmu.org; http://endosperm.info) includes pedigree and phenotypic data for over 2000 independent seed mutants selected from a population of 31 548 F2 lines and integrated with analyses of 34 255 MuTAIL sequences. PMID:16167895

  5. Transposon mutagenesis of Campylobacter jejuni identifies a bipartite energy taxis system required for motility.

    PubMed

    Hendrixson, D R; Akerley, B J; DiRita, V J

    2001-04-01

    Campylobacter jejuni constitutes the leading cause of bacterial gastroenteritis in the United States and a major cause of diarrhoea worldwide. Little is known about virulence mechanisms in this organism because of the scarcity of suitable genetic tools. We have developed an efficient system of in vitro transposon mutagenesis using a mariner-based transposon and purified mariner transposase. Through in vitro transposition of C. jejuni chromosomal DNA followed by natural transformation of the transposed DNA, large random transposon mutant libraries consisting of approximately 16 000 individual mutants were generated. The first genetic screen of C. jejuni using a transposon-generated mutant library identified 28 mutants defective for flagellar motility, one of the few known virulence determinants of this pathogen. We developed a second genetic system, which allows for the construction of defined chromosomal deletions in C. jejuni, and demonstrated the requirement of sigma28 and sigma54 for motility. In addition, we show that sigma28 is involved in the transcription of flaA and that sigma54 is required for transcription of three other flagellar genes, flaB and flgDE. We also identified two previously uncharacterized genes required for motility encoding proteins that we call CetA and CetB, which mediate energy taxis responses. Through our analysis of the Cet proteins, we propose a unique mechanism for sensing energy levels and mediating energy taxis in C. jejuni. PMID:11298288

  6. Random Transposon Mutagenesis for Cell-Envelope Resistant to Phage Infection.

    PubMed

    Reyes-Cortés, Ruth; Arguijo-Hernández, Emma S; Carballo-Ontiveros, Marco A; Martínez-Peñafiel, Eva; Kameyama, Luis

    2016-01-01

    In order to identify host components involved in the infective process of bacteriophages, we developed a wide-range strategy to obtain cell envelope mutants, using Escherichia coli W3110 and its specific phage mEp213. The strategy consisted in four steps: (1) random mutagenesis using transposon miniTn10Km(r); (2) selection of phage-resistant mutants by replica-plating; (3) electroporation of the phage-resistant mutants with mEp213 genome, followed by selection of those allowing phage development; and (4) sequencing of the transposon-disrupted genes. This strategy allowed us to distinguish the host factors related to phage development or multiplication within the cell, from those involved in phage infection at the level of the cell envelope. PMID:27311665

  7. A Transposon-Based Tool for Transformation and Mutagenesis in Trypanosomatid Protozoa

    PubMed Central

    Damasceno, Jeziel D.; Beverley, Stephen M.; Tosi, Luiz R.O.

    2014-01-01

    The ability of transposable elements to mobilize across genomes and affect the expression of genes makes them exceptional tools for genetic manipulation methodologies. Several transposon-based systems have been modified and incorporated into shuttle mutagenesis approaches in a variety of organisms. We have found that the Mos1 element, a DNA transposon from Drosophila mauritiana, is suitable and readily adaptable to a variety of strategies to the study of trypanosomatid parasitic protozoa. Trypanosomatids are the causative agents of a wide range of neglected diseases in underdeveloped regions of the globe. In this chapter we describe the basic elements and the available protocols for the in vitro use of Mos1 derivatives in the protozoan parasite Leishmania. PMID:25388118

  8. Transposon mutagenesis identifies genes driving hepatocellular carcinoma in a chronic hepatitis B mouse model

    PubMed Central

    Bard-Chapeau, Emilie A.; Nguyen, Anh-Tuan; Rust, Alistair G.; Sayadi, Ahmed; Lee, Philip; Chua, Belinda Q; New, Lee-Sun; de Jong, Johann; Ward, Jerrold M.; Chin, Christopher KY.; Chew, Valerie; Toh, Han Chong; Abastado, Jean-Pierre; Benoukraf, Touati; Soong, Richie; Bard, Frederic A.; Dupuy, Adam J.; Johnson, Randy L.; Radda, George K.; Chan, Eric CY.; Wessels, Lodewyk FA.; Adams, David J.

    2014-01-01

    The most common risk factor for developing hepatocellular carcinoma (HCC) is chronic infection with hepatitis B virus (HBV). To better understand the evolutionary forces driving HCC we performed a near saturating transposon mutagenesis screen in a mouse HBV model of HCC. This screen identified 21 candidate early stage drivers, and a bewildering number (2860) of candidate later stage drivers, that were enriched for genes mutated, deregulated, or that function in signaling pathways important for human HCC, with a striking 1199 genes linked to cellular metabolic processes. Our study provides a comprehensive overview of the genetic landscape of HCC. PMID:24316982

  9. Development of an Efficient In Vivo System (Pjunc-TpaseIS1223) for Random Transposon Mutagenesis of Lactobacillus casei

    PubMed Central

    Licandro-Seraut, Hélène; Brinster, Sophie; van de Guchte, Maarten; Scornec, Hélène; Maguin, Emmanuelle; Sansonetti, Philippe

    2012-01-01

    The random transposon mutagenesis system Pjunc-TpaseIS1223 is composed of plasmids pVI129, expressing IS1223 transposase, and pVI110, a suicide transposon plasmid carrying the Pjunc sequence, the substrate of the IS1223 transposase. This system is particularly efficient in Lactobacillus casei, as more than 10,000 stable, random mutants were routinely obtained via electroporation. PMID:22610425

  10. Sleeping Beauty Transposon Mutagenesis as a Tool for Gene Discovery in the NOD Mouse Model of Type 1 Diabetes

    PubMed Central

    Elso, Colleen M.; Chu, Edward P. F.; Alsayb, May A.; Mackin, Leanne; Ivory, Sean T.; Ashton, Michelle P.; Bröer, Stefan; Silveira, Pablo A.; Brodnicki, Thomas C.

    2015-01-01

    A number of different strategies have been used to identify genes for which genetic variation contributes to type 1 diabetes (T1D) pathogenesis. Genetic studies in humans have identified >40 loci that affect the risk for developing T1D, but the underlying causative alleles are often difficult to pinpoint or have subtle biological effects. A complementary strategy to identifying “natural” alleles in the human population is to engineer “artificial” alleles within inbred mouse strains and determine their effect on T1D incidence. We describe the use of the Sleeping Beauty (SB) transposon mutagenesis system in the nonobese diabetic (NOD) mouse strain, which harbors a genetic background predisposed to developing T1D. Mutagenesis in this system is random, but a green fluorescent protein (GFP)-polyA gene trap within the SB transposon enables early detection of mice harboring transposon-disrupted genes. The SB transposon also acts as a molecular tag to, without additional breeding, efficiently identify mutated genes and prioritize mutant mice for further characterization. We show here that the SB transposon is functional in NOD mice and can produce a null allele in a novel candidate gene that increases diabetes incidence. We propose that SB transposon mutagenesis could be used as a complementary strategy to traditional methods to help identify genes that, when disrupted, affect T1D pathogenesis. PMID:26438296

  11. Sleeping Beauty Transposon Mutagenesis as a Tool for Gene Discovery in the NOD Mouse Model of Type 1 Diabetes.

    PubMed

    Elso, Colleen M; Chu, Edward P F; Alsayb, May A; Mackin, Leanne; Ivory, Sean T; Ashton, Michelle P; Bröer, Stefan; Silveira, Pablo A; Brodnicki, Thomas C

    2015-12-01

    A number of different strategies have been used to identify genes for which genetic variation contributes to type 1 diabetes (T1D) pathogenesis. Genetic studies in humans have identified >40 loci that affect the risk for developing T1D, but the underlying causative alleles are often difficult to pinpoint or have subtle biological effects. A complementary strategy to identifying "natural" alleles in the human population is to engineer "artificial" alleles within inbred mouse strains and determine their effect on T1D incidence. We describe the use of the Sleeping Beauty (SB) transposon mutagenesis system in the nonobese diabetic (NOD) mouse strain, which harbors a genetic background predisposed to developing T1D. Mutagenesis in this system is random, but a green fluorescent protein (GFP)-polyA gene trap within the SB transposon enables early detection of mice harboring transposon-disrupted genes. The SB transposon also acts as a molecular tag to, without additional breeding, efficiently identify mutated genes and prioritize mutant mice for further characterization. We show here that the SB transposon is functional in NOD mice and can produce a null allele in a novel candidate gene that increases diabetes incidence. We propose that SB transposon mutagenesis could be used as a complementary strategy to traditional methods to help identify genes that, when disrupted, affect T1D pathogenesis. PMID:26438296

  12. Large-scale mutagenesis of the mouse to understand the genetic bases of nervous system structure and function

    PubMed Central

    Goldowitz, Dan; Frankel, Wayne N.; Takahashi, Joseph S.; Holtz-Vitaterna, Martha; Bult, Carol; Kibbe, Warren A.; Snoddy, Jay; Li, Yanxia; Pretel, Stephanie; Yates, Jeana; Swanson, Douglas J.

    2013-01-01

    N-ethyl-N-nitrosourea (ENU) mutagenesis is presented as a powerful approach to developing models for human disease. The efforts of three NIH Mutagenesis Centers established for the detection of neuroscience-related phenotypes are described. Each center has developed an extensive panel of phenotype screens that assess nervous system structure and function. In particular, these screens focus on complex behavioral traits from drug and alcohol responses to circadian rhythms to epilepsy. Each of these centers has developed a bioinformatics infrastructure to track the extensive number of transactions that are inherent in these large-scale projects. Over 100 new mouse mutant lines have been defined through the efforts of these three mutagenesis centers and are presented to the research community via the centralized Web presence of the Neuromice.org consortium (http://www.neuromice.org). This community resource provides visitors with the ability to search for specific mutant phenotypes, to view the genetic and phenotypic details of mutant mouse lines, and to order these mice for use in their own research program. PMID:15582151

  13. Implementation of a large-scale ENU mutagenesis program: towards increasing the mouse mutant resource.

    PubMed

    Nolan, P M; Peters, J; Vizor, L; Strivens, M; Washbourne, R; Hough, T; Wells, C; Glenister, P; Thornton, C; Martin, J; Fisher, E; Rogers, D; Hagan, J; Reavill, C; Gray, I; Wood, J; Spurr, N; Browne, M; Rastan, S; Hunter, J; Brown, S D

    2000-07-01

    Systematic approaches to mouse mutagenesis will be vital for future studies of gene function. We have begun a major ENU mutagenesis program incorporating a large genome-wide screen for dominant mutations. Progeny of ENU-mutagenized mice are screened for visible defects at birth and weaning, and at 5 weeks of age by using a systematic and semi-quantitative screening protocol-SHIRPA. Following this, mice are screened for abnormal locomotor activity and for deficits in prepulse inhibition of the acoustic startle response. Moreover, in the primary screen, blood is collected from mice and subjected to a comprehensive clinical biochemical analysis. Subsequently, secondary and tertiary screens of increasing complexity can be used on animals demonstrating deficits in the primary screen. Frozen sperm is archived from all the male mice passing through the screen. In addition, tail tips are stored for DNA. Overall, the program will provide an extensive new resource of mutant and phenotype data to the mouse and human genetics communities at large. The challenge now is to employ the expanding mouse mutant resource to improve the mutant map of the mouse. An improved mutant map of the mouse will be an important asset in exploiting the growing gene map of the mouse and assisting with the identification of genes underlying novel mutations-with consequent benefits for the analysis of gene function and the identification of novel pathways. PMID:10886012

  14. Transposon mutagenesis identifies genes and cellular processes driving epithelial-mesenchymal transition in hepatocellular carcinoma.

    PubMed

    Kodama, Takahiro; Newberg, Justin Y; Kodama, Michiko; Rangel, Roberto; Yoshihara, Kosuke; Tien, Jean C; Parsons, Pamela H; Wu, Hao; Finegold, Milton J; Copeland, Neal G; Jenkins, Nancy A

    2016-06-14

    Epithelial-mesenchymal transition (EMT) is thought to contribute to metastasis and chemoresistance in patients with hepatocellular carcinoma (HCC), leading to their poor prognosis. The genes driving EMT in HCC are not yet fully understood, however. Here, we show that mobilization of Sleeping Beauty (SB) transposons in immortalized mouse hepatoblasts induces mesenchymal liver tumors on transplantation to nude mice. These tumors show significant down-regulation of epithelial markers, along with up-regulation of mesenchymal markers and EMT-related transcription factors (EMT-TFs). Sequencing of transposon insertion sites from tumors identified 233 candidate cancer genes (CCGs) that were enriched for genes and cellular processes driving EMT. Subsequent trunk driver analysis identified 23 CCGs that are predicted to function early in tumorigenesis and whose mutation or alteration in patients with HCC is correlated with poor patient survival. Validation of the top trunk drivers identified in the screen, including MET (MET proto-oncogene, receptor tyrosine kinase), GRB2-associated binding protein 1 (GAB1), HECT, UBA, and WWE domain containing 1 (HUWE1), lysine-specific demethylase 6A (KDM6A), and protein-tyrosine phosphatase, nonreceptor-type 12 (PTPN12), showed that deregulation of these genes activates an EMT program in human HCC cells that enhances tumor cell migration. Finally, deregulation of these genes in human HCC was found to confer sorafenib resistance through apoptotic tolerance and reduced proliferation, consistent with recent studies showing that EMT contributes to the chemoresistance of tumor cells. Our unique cell-based transposon mutagenesis screen appears to be an excellent resource for discovering genes involved in EMT in human HCC and potentially for identifying new drug targets. PMID:27247392

  15. Transposon mutagenesis identifies genetic drivers of Braf(V600E) melanoma.

    PubMed

    Mann, Michael B; Black, Michael A; Jones, Devin J; Ward, Jerrold M; Yew, Christopher Chin Kuan; Newberg, Justin Y; Dupuy, Adam J; Rust, Alistair G; Bosenberg, Marcus W; McMahon, Martin; Print, Cristin G; Copeland, Neal G; Jenkins, Nancy A

    2015-05-01

    Although nearly half of human melanomas harbor oncogenic BRAF(V600E) mutations, the genetic events that cooperate with these mutations to drive melanogenesis are still largely unknown. Here we show that Sleeping Beauty (SB) transposon-mediated mutagenesis drives melanoma progression in Braf(V600E) mutant mice and identify 1,232 recurrently mutated candidate cancer genes (CCGs) from 70 SB-driven melanomas. CCGs are enriched in Wnt, PI3K, MAPK and netrin signaling pathway components and are more highly connected to one another than predicted by chance, indicating that SB targets cooperative genetic networks in melanoma. Human orthologs of >500 CCGs are enriched for mutations in human melanoma or showed statistically significant clinical associations between RNA abundance and survival of patients with metastatic melanoma. We also functionally validate CEP350 as a new tumor-suppressor gene in human melanoma. SB mutagenesis has thus helped to catalog the cooperative molecular mechanisms driving BRAF(V600E) melanoma and discover new genes with potential clinical importance in human melanoma. PMID:25848750

  16. Transposon mutagenesis identifies genetic drivers of BrafV600E melanoma

    PubMed Central

    Mann, Michael B; Black, Michael A; Jones, Devin J; Ward, Jerrold M; Yew, Christopher Chin Kuan; Newberg, Justin Y; Dupuy, Adam J; Rust, Alistair G; Bosenberg, Marcus W; McMahon, Martin; Print, Cristin G; Copeland, Neal G; Jenkins, Nancy A

    2016-01-01

    Although nearly half of human melanomas harbor oncogenic BRAFV600E mutations, the genetic events that cooperate with these mutations to drive melanogenesis are still largely unknown. Here we show that Sleeping Beauty (SB) transposon-mediated mutagenesis drives melanoma progression in BrafV600E mutant mice and identify 1,232 recurrently mutated candidate cancer genes (CCGs) from 70 SB-driven melanomas. CCGs are enriched in Wnt, PI3K, MAPK and netrin signaling pathway components and are more highly connected to one another than predicted by chance, indicating that SB targets cooperative genetic networks in melanoma. Human orthologs of >500 CCGs are enriched for mutations in human melanoma or showed statistically significant clinical associations between RNA abundance and survival of patients with metastatic melanoma. We also functionally validate CEP350 as a new tumor-suppressor gene in human melanoma. SB mutagenesis has thus helped to catalog the cooperative molecular mechanisms driving BRAFV600E melanoma and discover new genes with potential clinical importance in human melanoma. PMID:25848750

  17. In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis.

    PubMed Central

    McAdam, R A; Weisbrod, T R; Martin, J; Scuderi, J D; Brown, A M; Cirillo, J D; Bloom, B R; Jacobs, W R

    1995-01-01

    Insertional mutagenesis in Mycobacterium bovis BCG, a member of the slow-growing M. tuberculosis complex, was accomplished with transposons engineered from the Mycobacterium smegmatis insertion element IS1096. Transposons were created by placing a kanamycin resistance gene in several different positions in IS1096, and the resulting transposons were electroporated into BCG on nonreplicating plasmids. These analyses demonstrated that only one of the two open reading frames was necessary for transposition. A library of insertions was generated. Southern analysis of 23 kanamycin-resistant clones revealed that the transposons had inserted directly, with no evidence of cointegrate formation, into different restriction fragments in each clone. Sequence analysis of nine of the clones revealed junctional direct 8-bp repeats with only a slight similarity in target sites. These results suggest that IS1096-derived transposons transposed into the BCG genome in a relatively random fashion. Three auxotrophs, two for leucine and one for methionine, were isolated from the library of transposon insertions in BCG. They were characterized by sequencing and found to be homologous to the leuD gene of Escherichia coli and a sulfate-binding protein of cyanobacteria, respectively. When inoculated intravenously into C57BL/6 mice, the leucine auxotrophs, in contrast to the parent BCG strain or the methionine auxotroph, showed an inability to grow in vivo and were cleared within 7 weeks from the lungs and spleen. PMID:7868221

  18. piggyBac transposon somatic mutagenesis with an activated reporter and tracker (PB-SMART) for genetic screens in mice.

    PubMed

    Landrette, Sean F; Cornett, Jonathan C; Ni, Thomas K; Bosenberg, Marcus W; Xu, Tian

    2011-01-01

    Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB) transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease. PMID:22039523

  19. A facile and efficient transposon mutagenesis method for generation of multi-codon deletions in protein sequences.

    PubMed

    Liu, Shu-Su; Wei, Xuan; Ji, Qun; Xin, Xiu; Jiang, Biao; Liu, Jia

    2016-06-10

    Substitutions, insertions and deletions are all important mutation events in natural and laboratory protein evolution. However, protein engineering using insertions and deletions (indels) is hindered by the lack of a convenient mutagenesis method. Here, we describe a general transposon mutagenesis method that allows for removal of up to five consecutive in-frame codons from a random position of a target protein. This method, referred to as codon deletion mutagenesis (CDM), relies on an engineered Mu transposon that carries asymmetric terminal sequences flanking the MuA transposase recognition sites. CDM requires minimal DNA manipulations, and can generate multi-codon deletions with high efficiency (>90%). As a proof of principle, we constructed five libraries of green fluorescent protein (GFP) containing one to five random codon deletions, respectively. Several variants with multi-codon deletions remained fluorescent, none of which could be easily identified using traditional mutagenesis method. CDM provides a facile and efficient approach to sampling a protein sequence with multi-codon deletions. It will not only facilitate our understanding of the effects of amino acid deletions on protein function but also expedite protein engineering using deletion mutagenesis. PMID:27071724

  20. Novel strategies for gene trapping and insertional mutagenesis mediated by Sleeping Beauty transposon

    PubMed Central

    Song, Guili; Cui, Zongbin

    2013-01-01

    Gene and poly(A) trappings are high-throughput approaches to capture and interrupt the expression of endogenous genes within a target genome. Although a number of trapping vectors have been developed for investigation of gene functions in cells and vertebrate models, there is still room for the improvement of their efficiency and sensitivity. Recently, two novel trapping vectors mediated by Sleeping Beauty (SB) transposon have been generated by the combination of three functional cassettes that are required for finding endogenous genes, disrupting the expression of trapped genes, and inducing the excision of integrated traps from their original insertion sites and then inserting into another gene. In addition, several other strategies are utilized to improve the activities of two trapping vectors. First, activities of all components were examined in vitro before the generation of two vectors. Second, the inducible promoter from the tilapia Hsp70 gene was used to drive the expression of SB gene, which can mediate the excision of integrated transposons upon induction at 37 °C. Third, the Cre/LoxP system was introduced to delete the SB expression cassette for stabilization of gene interruption and bio-safety. Fourth, three stop codons in different reading frames were introduced downstream of a strong splice acceptor (SA) in the gene trapping vector to effectively terminate the translation of trapped endogenous genes. Fifth, the strong splicing donor (SD) and AU-rich RNA-destabilizing element exhibited no obvious insertion bias and markedly reduced SD read-through events, and the combination of an enhanced SA, a poly(A) signal and a transcript terminator in the poly(A) trapping vector efficiently disrupted the transcription of trapped genes. Thus, these two trapping vectors are alternative and effective tools for large-scale identification and disruption of endogenous genes in vertebrate cells and animals. PMID:24251071

  1. PiggyBac transposon-based polyadenylation-signal trap for genome-wide mutagenesis in mice

    PubMed Central

    Li, Limei; Liu, Peng; Sun, Liangliang; Bin Zhou; Fei, Jian

    2016-01-01

    We designed a new type of polyadenylation-signal (PAS) trap vector system in living mice, the piggyBac (PB) (PAS-trapping (EGFP)) gene trapping vector, which takes advantage of the efficient transposition ability of PB and efficient gene trap and insertional mutagenesis of PAS-trapping. The reporter gene of PB(PAS-trapping (EGFP)) is an EGFP gene with its own promoter, but lacking a poly(A) signal. Transgenic mouse lines carrying PB(PAS-trapping (EGFP)) and protamine 1 (Prm1) promoter-driven PB transposase transgenes (Prm1-PBase) were generated by microinjection. Male mice doubly positive for PB(PAS-trapping (EGFP)) and Prm1-PBase were crossed with WT females, generating offspring with various insertion mutations. We found that 44.8% (26/58) of pups were transposon-positive progenies. New transposon integrations comprised 26.9% (7/26) of the transposon-positive progenies. We found that 100% (5/5) of the EGFP fluorescence-positive mice had new trap insertions mediated by a PB transposon in transcriptional units. The direction of the EGFP gene in the vector was consistent with the direction of the endogenous gene reading frame. Furthermore, mice that were EGFP-PCR positive, but EGFP fluorescent negative, did not show successful gene trapping. Thus, the novel PB(PAS-trapping (EGFP)) system is an efficient genome-wide gene-trap mutagenesis in mice. PMID:27292714

  2. Marinomonas mediterranea MMB-1 Transposon Mutagenesis: Isolation of a Multipotent Polyphenol Oxidase Mutant

    PubMed Central

    Solano, Francisco; Lucas-Elío, Patricia; Fernández, Eva; Sanchez-Amat, Antonio

    2000-01-01

    Marinomonas mediterranea is a melanogenic marine bacterium expressing a multifunctional polyphenol oxidase (PPO) able to oxidize substrates characteristic for laccases and tyrosinases, as well as produce a classical tyrosinase. A new and quick method has been developed for screening laccase activity in culture plates to detect mutants differentially affected in this PPO activity. Transposon mutagenesis has been applied for the first time to M. mediterranea by using different minitransposons loaded in R6K-based suicide delivery vectors mobilizable by conjugation. Higher frequencies of insertions were obtained by using mini-Tn10 derivatives encoding kanamycin or gentamycin resistance. After applying this protocol, a multifunctional PPO-negative mutant was obtained. By using the antibiotic resistance cassette as a marker, flanking regions were cloned. Then the wild-type gene was amplified by PCR and was cloned and sequenced. This is the first report on cloning and sequencing of a gene encoding a prokaryotic enzyme with laccase activity. The deduced amino acid sequence shows the characteristic copper-binding sites of other blue copper proteins, including fungal laccases. In addition, it shows some extra copper-binding sites that might be related to its multipotent enzymatic capability. PMID:10850991

  3. Functional characterization of the Sindbis virus E2 glycoprotein by transposon linker-insertion mutagenesis

    SciTech Connect

    Navaratnarajah, Chanakha K.; Kuhn, Richard J. . E-mail: kuhnr@purdue.edu

    2007-06-20

    The glycoprotein envelope of alphaviruses consists of two proteins, E1 and E2. E1 is responsible for fusion and E2 is responsible for receptor binding. An atomic structure is available for E1, but one for E2 has not been reported. In this study, transposon linker-insertion mutagenesis was used to probe the function of different domains of E2. A library of mutants, containing 19 amino acid insertions in the E2 glycoprotein sequence of the prototype alphavirus, Sindbis virus (SINV), was generated. Fifty-seven independent E2 insertions were characterized, of which more than half (67%) gave rise to viable virus. The wild-type-like mutants identify regions that accommodate insertions without perturbing virus production and can be used to insert targeting moieties to direct SINV to specific receptors. The defective and lethal mutants give insight into regions of E2 important for protein stability, transport to the cell membrane, E1-E2 contacts, and receptor binding.

  4. piggyBac transposon-based insertional mutagenesis in mouse haploid embryonic stem cells.

    PubMed

    Pettitt, Stephen J; Tan, E-Pien; Yusa, Kosuke

    2015-01-01

    Forward genetic screening is a powerful non-hypothesis-driven approach to unveil the molecular mechanisms and pathways underlying phenotypes of interest. In this approach, a genome-wide mutant library is first generated and then screened for a phenotype of interest. Subsequently, genes responsible for the phenotype are identified. There have been a number of successful screens in yeasts, Caenorhabditis elegans and Drosophila. These model organisms all allow loss-of-function mutants to be generated easily on a genome-wide scale: yeasts have a haploid stage in their reproductive cycles and the latter two organisms have short generation times, allowing mutations to be systematically bred to homozygosity. However, in mammals, the diploid genome and long generation time have always hampered rapid and efficient production of homozygous mutant cells and animals. The recent discovery of several haploid mammalian cell lines promises to revolutionize recessive genetic screens in mammalian cells. In this protocol, we describe an overview of insertional mutagenesis, focusing on DNA transposons, and provide a method for an efficient generation of genome-wide mutant libraries using mouse haploid embryonic stem cells. PMID:25408399

  5. A two-component enhancer-inhibitor transposon mutagenesis system for functional analysis of the Arabidopsis genome.

    PubMed Central

    Speulman, E; Metz, P L; van Arkel, G; te Lintel Hekkert, B; Stiekema, W J; Pereira, A

    1999-01-01

    A modified Enhancer-Inhibitor transposon system was used to generate a series of mutant lines by single-seed descent such that multiple I insertions occurred per plant. The distribution of original insertions in the population was assessed by isolating transposon-flanking DNA, and a database of insertion sites was created. Approximately three-quarters of the identified insertion sites show similarity to sequences stored in public databases, which demonstrates the power of this regimen of insertional mutagenesis. To isolate insertions in specific genes, we developed three-dimensional pooling and polymerase chain reaction strategies that we then validated by identifying mutants for the regulator genes APETALA1 and SHOOT MERISTEMLESS. The system then was used to identify inserts in a class of uncharacterized genes involved in lipid biosynthesis; one such insertion conferred a fiddlehead mutant phenotype. PMID:10521517

  6. Transformation and transposon mutagenesis of Leifsonia xyli subsp. xyli, causal organism of ratoon stunting disease of sugarcane.

    PubMed

    Brumbley, Stevens M; Petrasovits, Lars A; Birch, Robert G; Taylor, Paul W J

    2002-03-01

    Conditions have been developed for genetic transformation and insertional mutagenesis in Leifsonia xyli subsp. xyli (Lxx), the causal organism of ratoon stunting disease (RSD), one of the most damaging and intractable diseases of sugarcane internationally. Transformation frequencies ranged from 1 to 10 colony forming units (CFU)/microg of plasmid DNA using Clavibacter/Escherichia coli shuttle vectors pCG188, pDM302, and pDM306 and ranged from 50 to 500 CFU/microg using cosmid cloning vectors pLAFR3 and pLAFR5-km. The transformation/transposition frequency was 0 to 70 CFU/microg of DNA, using suicide vectors pUCD623 and pSUP2021 containing transposable elements Tn4431 and Tn5, respectively. It was necessary to grow Lxx in media containing 0.1% glycine for electroporation and to amplify large plasmids in a dam-/dcm- E. coli strain and purify the DNA by anion exchange. To keep selection pressure at an optimum, the transformants were grown on nitrocellulose filters (0.2-microm pore size) on media containing the appropriate antibiotics. Transposon Tn4431 containing a promoterless lux operon from Vibrio fischeri and a tetracycline-resistance gene was introduced on the suicide vector pUCD623. All but 1% of the putative transposon mutants produce light, indicating transposition into functional Lxx genes. Southern blot analysis of these transformants indicates predominantly single transposon insertions at unique sites. The cosmid cloning vector pLAFR5-km was stably maintained in Lxx. The development of a transformation and transposon mutagenesis system opens the way for molecular analysis of pathogenicity determinants in Lxx. PMID:11952129

  7. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.

    PubMed

    Gagnon, James A; Valen, Eivind; Thyme, Summer B; Huang, Peng; Akhmetova, Laila; Ahkmetova, Laila; Pauli, Andrea; Montague, Tessa G; Zimmerman, Steven; Richter, Constance; Schier, Alexander F

    2014-01-01

    The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes. PMID:24873830

  8. Mapping of neutralizing epitopes on Renibacterium salmoninarum p57 by use of transposon mutagenesis and synthetic peptides.

    PubMed

    Wiens, Gregory D; Owen, Jennifer

    2005-06-01

    Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish. PMID:15932983

  9. Mapping of Neutralizing Epitopes on Renibacterium salmoninarum p57 by Use of Transposon Mutagenesis and Synthetic Peptides

    PubMed Central

    Wiens, Gregory D.; Owen, Jennifer

    2005-01-01

    Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish. PMID:15932983

  10. Multipurpose Transposon-Insertion Libraries in Yeast.

    PubMed

    Kumar, Anuj

    2016-01-01

    Libraries of transposon-insertion alleles constitute powerful and versatile tools for large-scale analysis of yeast gene function. Transposon-insertion libraries are constructed most simply through mutagenesis of a plasmid-based genomic DNA library; modification of the mutagenizing transposon by incorporation of yeast selectable markers, recombination sites, and an epitope tag enables the application of insertion alleles for phenotypic screening and protein localization. In particular, yeast genomic DNA libraries have been mutagenized with modified bacterial transposons carrying the URA3 marker, lox recombination sites, and sequence encoding multiple copies of the hemagglutinin (HA) epitope. Mutagenesis with these transposons has yielded a large resource of insertion alleles affecting nearly 4000 yeast genes in total. Through well-established protocols, these insertion libraries can be introduced into the desired strain backgrounds and the resulting insertional mutants can be screened or systematically analyzed. Relative to alternative methods of UV irradiation or chemical mutagenesis, transposon-insertion alleles can be easily identified by PCR-based approaches or high-throughput sequencing. Transposon-insertion libraries also provide a cost-effective alternative to targeted deletion approaches, although, in contrast to start-codon to stop-codon deletions, insertion alleles might not represent true null-mutants. For protein-localization studies, transposon-insertion alleles can provide encoded epitope tags in-frame with internal codons; in many cases, these transposon-encoded epitope tags can provide a more accurate localization for proteins in which terminal sequences are crucial for intracellular targeting. Thus, overall, transposon-insertion libraries can be used quickly and economically and have a particular utility in screening for desired phenotypes and localization patterns in nonstandard genetic backgrounds. PMID:27250950

  11. Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics

    PubMed Central

    2013-01-01

    Background The development of resistance to chemotherapies represents a significant barrier to successful cancer treatment. Resistance mechanisms are complex, can involve diverse and often unexpected cellular processes, and can vary with both the underlying genetic lesion and the origin or type of tumor. For these reasons developing experimental strategies that could be used to understand, identify and predict mechanisms of resistance in different malignant cells would be a major advance. Methods Here we describe a gain-of-function forward genetic approach for identifying mechanisms of resistance. This approach uses a modified piggyBac transposon to generate libraries of mutagenized cells, each containing transposon insertions that randomly activate nearby gene expression. Genes of interest are identified using next-gen high-throughput sequencing and barcode multiplexing is used to reduce experimental cost. Results Using this approach we successfully identify genes involved in paclitaxel resistance in a variety of cancer cell lines, including the multidrug transporter ABCB1, a previously identified major paclitaxel resistance gene. Analysis of co-occurring transposons integration sites in single cell clone allows for the identification of genes that might act cooperatively to produce drug resistance a level of information not accessible using RNAi or ORF expression screening approaches. Conclusion We have developed a powerful pipeline to systematically discover drug resistance in mammalian cells in vitro. This cost-effective approach can be readily applied to different cell lines, to identify canonical or context specific resistance mechanisms. Its ability to probe complex genetic context and non-coding genomic elements as well as cooperative resistance events makes it a good complement to RNAi or ORF expression based screens. PMID:23442791

  12. [Improvement of butanol production by Escherichia coli via Tn5 transposon mediated mutagenesis].

    PubMed

    Lin, Zhao; Dong, Hongjun; Li, Yin

    2015-12-01

    For engineering an efficient butanol-producing Escherichia coli strain, many efforts have been paid on the known genes or pathways based on current knowledge. However, many genes in the genome could also contribute to butanol production in an unexpected way. In this work, we used Tn5 transposon to construct a mutant library including 1 196 strains in a previously engineered butanol-producing E. coli strain. To screen the strains with improved titer of butanol production, we developed a high-throughput method for pyruvate detection based on dinitrophenylhydrazine reaction using 96-well microplate reader, because pyruvate is the precursor of butanol and its concentration is inversely correlated with butanol in the fermentation broth. Using this method, we successfully screened three mutants with increased butanol titer. The insertion sites of Tn5 transposon was in the ORFs of pykA, tdk, and cadC by inverse PCR and sequencing. These found genes would be efficient targets for further strain improvement. And the genome scanning strategy described here will be helpful for other microbial cell factory construction. PMID:27093834

  13. Genome-wide mining of potential virulence-associated genes in Riemerella anatipestifer using random transposon mutagenesis.

    PubMed

    Ni, Xintao; Jiang, Pan; Xing, Linlin; Ou, Changcan; Yu, Hui; Qi, Jingjing; Sun, Bingqing; Cui, Junsheng; Wang, Guijun; Hu, Qinghai

    2016-06-30

    Riemerella anatipestifer infection is a severe disease confronting the duck industry worldwide. However, little is known about the molecular basis of R. anatipestifer pathogenesis. In this study, we screened 3580 transposon Tn4351 insertion mutagenesis mutants of the highly virulent strain YZb1 in a duckling infection experiment and found 29 of them to be attenuated and 28 potential virulence-associated genes were identified. Molecular characterization of transposon insertion sites showed that of the 28 screened genes, two were predicted to encode TonB-dependent outer membrane receptor (plugs), sixteen encoded enzymes, and seven encoded hypothetical proteins. In addition, of the 28 affected genes, 19 were only found in bacteria belonging to the phylum Bacteroidetes and 10 were only found in the family Flavobacteriaceae. The median lethal dose of the mutants M11 and M29, which was affected in Riean_0060 and Riean_1537 respectively, were about 1700-fold and 210-fold higher than that of the wild-type strain YZb1, and those of the complemented strains M11(pRES-Riean_0060) and M29(pRES-Riean_1537) were decreased by 25- and 3-fold respectively compared to those of the mutants M11 and M29. Additional analysis indicated that the blood bacterial loading of ducklings infected with M11 or M29 was decreased significantly, as compared with that in ducklings infected with the wild-type strain YZb1. Thus, our results indicate that Riean_0060 and Riean_1537 were involved in R. anatipestifer pathogenesis. PMID:27259827

  14. A Mariner Transposon-Based Signature-Tagged Mutagenesis System for the Analysis of Oral Infection by Listeria monocytogenes

    PubMed Central

    Cummins, Joanne; Casey, Pat G.; Joyce, Susan A.; Gahan, Cormac G. M.

    2013-01-01

    Listeria monocytogenes is a Gram-positive foodborne pathogen and the causative agent of listerosis a disease that manifests predominately as meningitis in the non-pregnant individual or infection of the fetus and spontaneous abortion in pregnant women. Common-source outbreaks of foodborne listeriosis are associated with significant morbidity and mortality. However, relatively little is known concerning the mechanisms that govern infection via the oral route. In order to aid functional genetic analysis of the gastrointestinal phase of infection we designed a novel signature-tagged mutagenesis (STM) system based upon the invasive L. monocytogenes 4b serotype H7858 strain. To overcome the limitations of gastrointestinal infection by L. monocytogenes in the mouse model we created a H7858 strain that is genetically optimised for oral infection in mice. Furthermore our STM system was based upon a mariner transposon to favour numerous and random transposition events throughout the L. monocytogenes genome. Use of the STM bank to investigate oral infection by L. monocytogenes identified 21 insertion mutants that demonstrated significantly reduced potential for infection in our model. The sites of transposon insertion included lmOh7858_0671 (encoding an internalin homologous to Lmo0610), lmOh7858_0898 (encoding a putative surface-expressed LPXTG protein homologous to Lmo0842), lmOh7858_2579 (encoding the HupDGC hemin transport system) and lmOh7858_0399 (encoding a putative fructose specific phosphotransferase system). We propose that this represents an optimised STM system for functional genetic analysis of foodborne/oral infection by L. monocytogenes. PMID:24069416

  15. Identification of the genes involved in Riemerella anatipestifer biofilm formation by random transposon mutagenesis.

    PubMed

    Hu, Qinghai; Zhu, Yinyu; Tu, Jing; Yin, Yuncong; Wang, Xiaolan; Han, Xiangan; Ding, Chan; Zhang, Beimin; Yu, Shengqing

    2012-01-01

    Riemerella anatipestifer causes epizootics of infectious disease in poultry that result in serious economic losses to the duck industry. Our previous studies have shown that some strains of R. anatipestifer can form a biofilm, and this may explain the intriguing persistence of R. anatipestifer on duck farms post infection. In this study we used strain CH3, a strong producer of biofilm, to construct a library of random Tn4351 transposon mutants in order to investigate the genetic basis of biofilm formation by R. anatipestifer on abiotic surfaces. A total of 2,520 mutants were obtained and 39 of them showed a reduction in biofilm formation of 47%-98% using crystal violet staining. Genetic characterization of the mutants led to the identification of 33 genes. Of these, 29 genes are associated with information storage and processing, as well as basic cellular processes and metabolism; the function of the other four genes is currently unknown. In addition, a mutant strain BF19, in which biofilm formation was reduced by 98% following insertion of the Tn4351 transposon at the dihydrodipicolinate synthase (dhdps) gene, was complemented with a shuttle plasmid pCP-dhdps. The complemented mutant strain was restored to give 92.6% of the biofilm formation of the wild-type strain CH3, which indicates that the dhdp gene is associated with biofilm formation. It is inferred that such complementation applies also to other mutant strains. Furthermore, some biological characteristics of biofilm-defective mutants were investigated, indicating that the genes deleted in the mutant strains function in the biofilm formation of R. anatipestifer. Deletion of either gene will stall the biofilm formation at a specific stage thus preventing further biofilm development. In addition, the tested biofilm-defective mutants had different adherence capacity to Vero cells. This study will help us to understand the molecular mechanisms of biofilm development by R. anatipestifer and to study the

  16. A large-scale mutant panel in wheat developed using heavy-ion beam mutagenesis and its application to genetic research

    NASA Astrophysics Data System (ADS)

    Murai, Koji; Nishiura, Aiko; Kazama, Yusuke; Abe, Tomoko

    2013-11-01

    Mutation analysis is a powerful tool for studying gene function. Heavy-ion beam mutagenesis is a comparatively new approach to inducing mutations in plants and is particularly efficient because of its high linear energy transfer (LET). High LET radiation induces a higher rate of DNA double-strand breaks than other mutagenic methods. Over the last 12 years, we have constructed a large-scale mutant panel in diploid einkorn wheat (Triticum monococcum) using heavy-ion beam mutagenesis. Einkorn wheat seeds were exposed to a heavy-ion beam and then sown in the field. Selfed seeds from each spike of M1 plants were used to generate M2 lines. Every year, we obtained approximately 1000 M2 lines and eventually developed a mutant panel with 10,000 M2 lines in total. This mutant panel is being systematically screened for mutations affecting reproductive growth, and especially for flowering-time mutants. To date, we have identified several flowering-time mutants of great interest: non-flowering mutants (mvp: maintained vegetative phase), late-flowering mutants, and early-flowering mutants. These novel mutations will be of value for investigations of the genetic mechanism of flowering in wheat.

  17. Transposon mutagenesis with coat color genotyping identifies an essential role for Skor2 in sonic hedgehog signaling and cerebellum development.

    PubMed

    Wang, Baiping; Harrison, Wilbur; Overbeek, Paul A; Zheng, Hui

    2011-10-01

    Correct development of the cerebellum requires coordinated sonic hedgehog (Shh) signaling from Purkinje to granule cells. How Shh expression is regulated in Purkinje cells is poorly understood. Using a novel tyrosinase minigene-tagged Sleeping Beauty transposon-mediated mutagenesis, which allows for coat color-based genotyping, we created mice in which the Ski/Sno family transcriptional co-repressor 2 (Skor2) gene is deleted. Loss of Skor2 leads to defective Purkinje cell development, a severe reduction of granule cell proliferation and a malformed cerebellum. Skor2 is specifically expressed in Purkinje cells in the brain, where it is required for proper expression of Shh. Skor2 overexpression suppresses BMP signaling in an HDAC-dependent manner and stimulates Shh promoter activity, suggesting that Skor2 represses BMP signaling to activate Shh expression. Our study identifies an essential function for Skor2 as a novel transcriptional regulator in Purkinje cells that acts upstream of Shh during cerebellum development. PMID:21937600

  18. Transposon mutagenesis with coat color genotyping identifies an essential role for Skor2 in sonic hedgehog signaling and cerebellum development

    PubMed Central

    Wang, Baiping; Harrison, Wilbur; Overbeek, Paul A.; Zheng, Hui

    2011-01-01

    Correct development of the cerebellum requires coordinated sonic hedgehog (Shh) signaling from Purkinje to granule cells. How Shh expression is regulated in Purkinje cells is poorly understood. Using a novel tyrosinase minigene-tagged Sleeping Beauty transposon-mediated mutagenesis, which allows for coat color-based genotyping, we created mice in which the Ski/Sno family transcriptional co-repressor 2 (Skor2) gene is deleted. Loss of Skor2 leads to defective Purkinje cell development, a severe reduction of granule cell proliferation and a malformed cerebellum. Skor2 is specifically expressed in Purkinje cells in the brain, where it is required for proper expression of Shh. Skor2 overexpression suppresses BMP signaling in an HDAC-dependent manner and stimulates Shh promoter activity, suggesting that Skor2 represses BMP signaling to activate Shh expression. Our study identifies an essential function for Skor2 as a novel transcriptional regulator in Purkinje cells that acts upstream of Shh during cerebellum development. PMID:21937600

  19. Mouse Models of Cancer: Sleeping Beauty Transposons for Insertional Mutagenesis Screens and Reverse Genetic Studies

    PubMed Central

    Tschida, Barbara R.; Largaespada, David A.; Keng, Vincent W.

    2014-01-01

    The genetic complexity and heterogeneity of cancer has posed a problem in designing rationally targeted therapies effective in a large proportion of human cancer. Genomic characterization of many cancer types has provided a staggering amount of data that needs to be interpreted to further our understanding of this disease. Forward genetic screening in mice using Sleeping Beauty (SB) based insertional mutagenesis is an effective method for candidate cancer gene discovery that can aid in distinguishing driver from passenger mutations in human cancer. This system has been adapted for unbiased screens to identify drivers of multiple cancer types. These screens have already identified hundreds of candidate cancer-promoting mutations. These can be used to develop new mouse models for further study, which may prove useful for therapeutic testing. SB technology may also hold the key for rapid generation of reverse genetic mouse models of cancer, and has already been used to model glioblastoma and liver cancer. PMID:24468652

  20. Identification and Characterization of Non-Cellulose-Producing Mutants of Gluconacetobacter hansenii Generated by Tn5 Transposon Mutagenesis

    PubMed Central

    Deng, Ying; Nagachar, Nivedita; Xiao, Chaowen; Tien, Ming

    2013-01-01

    The acs operon of Gluconacetobacter is thought to encode AcsA, AcsB, AcsC, and AcsD proteins that constitute the cellulose synthase complex, required for the synthesis and secretion of crystalline cellulose microfibrils. A few other genes have been shown to be involved in this process, but their precise role is unclear. We report here the use of Tn5 transposon insertion mutagenesis to identify and characterize six non-cellulose-producing (Cel−) mutants of Gluconacetobacter hansenii ATCC 23769. The genes disrupted were acsA, acsC, ccpAx (encoding cellulose-complementing protein [the subscript “Ax” indicates genes from organisms formerly classified as Acetobacter xylinum]), dgc1 (encoding guanylate dicyclase), and crp-fnr (encoding a cyclic AMP receptor protein/fumarate nitrate reductase transcriptional regulator). Protein blot analysis revealed that (i) AcsB and AcsC were absent in the acsA mutant, (ii) the levels of AcsB and AcsC were significantly reduced in the ccpAx mutant, and (iii) the level of AcsD was not affected in any of the Cel− mutants. Promoter analysis showed that the acs operon does not include acsD, unlike the organization of the acs operon of several strains of closely related Gluconacetobacter xylinus. Complementation experiments confirmed that the gene disrupted in each Cel− mutant was responsible for the phenotype. Quantitative real-time PCR and protein blotting results suggest that the transcription of bglAx (encoding β-glucosidase and located immediately downstream from acsD) was strongly dependent on Crp/Fnr. A bglAx knockout mutant, generated via homologous recombination, produced only ∼16% of the wild-type cellulose level. Since the crp-fnr mutant did not produce any cellulose, Crp/Fnr may regulate the expression of other gene(s) involved in cellulose biosynthesis. PMID:24013627

  1. Transposon-5 mutagenesis transforms Corynebacterium matruchotii to synthesize novel hybrid fatty acids that functionally replace corynomycolic acid.

    PubMed Central

    Takayama, Kuni; Hayes, Barry; Vestling, Matha M; Massey, Randall J

    2003-01-01

    Enzymes within the biosynthetic pathway of mycolic acid (C(60)-C(90) a-alkyl,b-hydroxyl fatty acid) in Mycobacterium tuberculosis are attractive targets for developing new anti-tuberculosis drugs. We have turned to the simple model system of Corynebacterium matruchotii to study the terminal steps in the anabolic pathway of a C32 mycolic acid called corynomycolic acid. By transposon-5 mutagenesis, we transformed C. matruchotii into a mutant that is unable to synthesize corynomycolic acid. Instead, it synthesized two related series of novel fatty acids that were released by saponification from the cell wall fraction and from two chloroform/methanol-extractable glycolipids presumed to be analogues of trehalose mono- and di-corynomycolate. By chemical analyses and MS, we determined the general structure of the two series to be 2,4,6,8,10-penta-alkyl decanoic acid for the larger series (C(70)-C(77)) and 2,4,6,8-tetra-alkyl octanoic acid for the smaller series (C(52)-C(64)), both containing multiple keto groups, hydroxy groups and double bonds. The mutant was temperature-sensitive, aggregated extensively, grew very slowly relative to the wild type, and was resistant to the presence of lysozyme. We suggest that a regulatory protein that normally prevents the transfer of the condensation product back to b-ketoacyl synthase in the corynomycolate synthase system of the wild type was inactivated in the mutant. This will result in multiple Claisen-type condensation and the formation of two similar series of these complex hybrid fatty acids. A similar protein in M. tuberculosis would be an attractive target for new drug discovery. PMID:12879902

  2. ENU large-scale mutagenesis and quantitative trait linkage (QTL) analysis in mice: novel technologies for searching polygenetic determinants of craniofacial abnormalities.

    PubMed

    Nishimura, Ichiro; Drake, Thomas A; Lusis, Aldons J; Lyons, Karen M; Nadeau, Joseph H; Zernik, Joseph

    2003-01-01

    Discrepancies in size and shape of the jaws are the underlying etiology in many orthodontic and orthognathic surgery patients. Genetic factors combined with environmental interactions have been postulated to play a causal or contributory role in these craniofacial abnormalities. Along with the soon-to-be-available complete human and mouse genomic sequence data, mouse mutants have become a valuable tool in the functional mapping of genes involved in the development of human maxillofacial dysmorphologies. We review two powerful methods in such efforts: N-ethyl-N-nitrosourea (ENU) large-scale mutagenesis and quantitative trait linkage (QTL) analysis. The former aims at producing a plethora of novel variants of particular trait(s), and ultimately mapping the point mutations responsible for the appearance of these new traits. In contrast, the latter applies intensive breeding and mapping techniques to identify multiple loci (and, subsequently, genes) contributing to the phenotypic difference between the tested strains. A prerequisite for either approach to studying variations in the traits of interest is the application of effective mouse cephalometric phenotype analysis and rapid DNA mapping techniques. These approaches will produce a wealth of new data on critical genes that influence the size and shape of the human face. PMID:14530301

  3. High-quality mutant libraries of Xanthomonas oryzae pv. oryzae and X. campestris pv. campestris generated by an efficient transposon mutagenesis system.

    PubMed

    Sun, Qihong; Wu, Wei; Qian, Wei; Hu, Jun; Fang, Rongxiang; He, Chaozu

    2003-09-12

    A novel transposon mutagenesis system for the phytopathogenic bacteria Xanthomonas oryzae pv. oryzae (Xoo) and X. campestris pv. campestris (Xcc) was developed using a Tn5-based transposome. A highly efficient transformation up to 10(6) transformants per microg transposon DNA was obtained. Southern blot and thermal asymmetric interlaced polymerase chain reaction analyses of Tn5 insertion sites suggested a random mode of transposition. The transposition was stable in the transformants for 20 subcultures. Eighteen thousand and 17000 transformants for Xoo and Xcc, respectively, were generated, corresponding to 4X ORF coverage of the genomes. The libraries will facilitate the identification of pathogenicity-related genes as well as functional genomic analysis in Xoo and Xcc. PMID:13129620

  4. Identification of a proton-chloride antiporter (EriC) by Himar1 transposon mutagenesis in Lactobacillus reuteri and its role in histamine production.

    PubMed

    Hemarajata, P; Spinler, J K; Balderas, M A; Versalovic, J

    2014-03-01

    The gut microbiome may modulate intestinal immunity by luminal conversion of dietary amino acids to biologically active signals. The model probiotic organism Lactobacillus reuteri ATCC PTA 6475 is indigenous to the human microbiome, and converts the amino acid L-histidine to the biogenic amine, histamine. Histamine suppresses tumor necrosis factor (TNF) production by human myeloid cells and is a product of L-histidine decarboxylation, which is a proton-facilitated reaction. A transposon mutagenesis strategy was developed based on a single-plasmid nisin-inducible Himar1 transposase/transposon delivery system for L. reuteri. A highly conserved proton-chloride antiporter gene (eriC), a gene widely present in the gut microbiome was discovered by Himar1 transposon (Tn)-mutagenesis presented in this study. Genetic inactivation of eriC by transposon insertion and genetic recombineering resulted in reduced ability of L. reuteri to inhibit TNF production by activated human myeloid cells, diminished histamine production by the bacteria and downregulated expression of histidine decarboxylase cluster genes compared to those of WT 6475. EriC belongs to a large family of ion transporters that includes chloride channels and proton-chloride antiporters and may facilitate the availability of protons for the decarboxylation reaction, resulting in histamine production by L. reuteri. This report leverages the tools of bacterial genetics for probiotic gene discovery. The findings highlight the widely conserved nature of ion transporters in bacteria and how ion transporters are coupled with amino acid decarboxylation and contribute to microbiome-mediated immunomodulation. PMID:24488273

  5. Mutagenesis of Bordetella pertussis with transposon Tn5tac1: conditional expression of virulence-associated genes.

    PubMed Central

    Cookson, B T; Berg, D E; Goldman, W E

    1990-01-01

    The Tn5tac1 transposon contains a strong outward-facing promoter, Ptac, a lacI repressor gene, and a selectable Kanr gene. Transcription from Ptac is repressed by the lacI protein unless an inducer (isopropyl-beta-D-thiogalactopyranoside [IPTG]) is present. Thus, Tn5tac1 generates insertion mutations in Escherichia coli with conditional phenotypes because it is polar on distal gene expression when IPTG is absent and directs transcription of these genes when the inducer is present. To test the usefulness of Tn5tac1 in Bordetella pertussis, a nonenteric gram-negative bacterial pathogen, we chose the bifunctional adenylate cyclase-hemolysin determinant as an easily scored marker to monitor insertional mutagenesis. Tn5tac1 delivered to B. pertussis on conjugal suicide plasmids resulted in Kanr exconjugants at a frequency of 10(-3) per donor cell, and nonhemolytic (Hly-) mutants were found among the Kanr colonies at a frequency of about 1%. Of eight independent Kanr Hly- mutants, two were conditional and exhibited an Hly+ phenotype only in the presence of IPTG. Using a new quantitative assay for adenylate cyclase based on high-pressure liquid chromatography, we found that enzymatic activity in these two strains was specifically induced at least 500-fold in a dose-dependent fashion over the range of 0 to 125 microM IPTG. These data show that Ptac serves as a promoter, lacI is expressed and is functional, and IPTG can induce Ptac transcription in B. pertussis. Adenylate cyclase expression in whole cells, culture supernatants, and cell extracts from these strains depended upon IPTG, suggesting that the insertions do not merely alter secretion of adenylate cyclase-hemolysin. Other virulence determinants under control of the vir locus are expressed normally, implying that these Tn5tac1 insertions specifically regulate adenylate cyclase-hemolysin expression. We conclude that Tn5tac1 insertion mutations permit sensitive, exogenous control over the expression of genes of

  6. Transposon Mutagenesis Paired with Deep Sequencing of Caulobacter crescentus under Uranium Stress Reveals Genes Essential for Detoxification and Stress Tolerance

    PubMed Central

    Yung, Mimi C.; Park, Dan M.; Overton, K. Wesley; Blow, Matthew J.; Hoover, Cindi A.; Smit, John; Murray, Sean R.; Ricci, Dante P.; Christen, Beat; Bowman, Grant R.

    2015-01-01

    ABSTRACT The ubiquitous aquatic bacterium Caulobacter crescentus is highly resistant to uranium (U) and facilitates U biomineralization and thus holds promise as an agent of U bioremediation. To gain an understanding of how C. crescentus tolerates U, we employed transposon (Tn) mutagenesis paired with deep sequencing (Tn-seq) in a global screen for genomic elements required for U resistance. Of the 3,879 annotated genes in the C. crescentus genome, 37 were found to be specifically associated with fitness under U stress, 15 of which were subsequently tested through mutational analysis. Systematic deletion analysis revealed that mutants lacking outer membrane transporters (rsaFa and rsaFb), a stress-responsive transcription factor (cztR), or a ppGpp synthetase/hydrolase (spoT) exhibited a significantly lower survival rate under U stress. RsaFa and RsaFb, which are homologues of TolC in Escherichia coli, have previously been shown to mediate S-layer export. Transcriptional analysis revealed upregulation of rsaFa and rsaFb by 4- and 10-fold, respectively, in the presence of U. We additionally show that rsaFa mutants accumulated higher levels of U than the wild type, with no significant increase in oxidative stress levels. Our results suggest a function for RsaFa and RsaFb in U efflux and/or maintenance of membrane integrity during U stress. In addition, we present data implicating CztR and SpoT in resistance to U stress. Together, our findings reveal novel gene targets that are key to understanding the molecular mechanisms of U resistance in C. crescentus. IMPORTANCE Caulobacter crescentus is an aerobic bacterium that is highly resistant to uranium (U) and has great potential to be used in U bioremediation, but its mechanisms of U resistance are poorly understood. We conducted a Tn-seq screen to identify genes specifically required for U resistance in C. crescentus. The genes that we identified have previously remained elusive using other omics approaches and thus

  7. Using Yeast Transposon-Insertion Libraries for Phenotypic Screening and Protein Localization.

    PubMed

    Kumar, Anuj

    2016-01-01

    This protocol details how to use a transposon-insertion library for phenotypic screening and protein localization. The insertion library was generated by mutagenesis of a plasmid-based yeast genomic DNA library by using a multipurpose transposon; the transposon produces gene disruptions, and, by Cre-mediated recombination at lox sites incorporated within the transposon, alleles with an in-frame insertion can be truncated to a residual transposon encoding multiple copies of the hemagglutinin epitope. Insertions are generated in yeast by shuttle mutagenesis. Yeast genomic DNA containing a transposon insertion is released from the library, and the mutagenized DNA sequences are introduced into a desired strain of yeast, where the insertion alleles replace native loci by homologous recombination. The insertion mutants can be screened for phenotypes, and the site of transposon insertion can subsequently be identified in selected mutants by inverse polymerase chain reaction (PCR). In-frame insertions within genes of interest can be truncated to an epitope-tagged allele by Cre-lox recombination, and the subcellular localization of the encoded protein product can be identified by standard methods of indirect immunofluorescence. In summary, the transposon-insertion libraries represent an informative resource for large-scale mutagenesis, presenting a straightforward alternative to labor-intensive targeted approaches for the construction of deletion alleles and fluorescent protein fusions. PMID:27250939

  8. Generation of Single-Copy Transposon Insertions in Clostridium perfringens by Electroporation of Phage Mu DNA Transposition Complexes▿

    PubMed Central

    Lanckriet, A.; Timbermont, L.; Happonen, L. J.; Pajunen, M. I.; Pasmans, F.; Haesebrouck, F.; Ducatelle, R.; Savilahti, H.; Van Immerseel, F.

    2009-01-01

    Transposon mutagenesis is a tool that is widely used for the identification of genes involved in the virulence of bacteria. Until now, transposon mutagenesis in Clostridium perfringens has been restricted to the use of Tn916-based methods with laboratory reference strains. This system yields primarily multiple transposon insertions in a single genome, thus compromising its use for the identification of virulence genes. The current study describes a new protocol for transposon mutagenesis in C. perfringens, which is based on the bacteriophage Mu transposition system. The protocol was successfully used to generate a single-insertion mutant library both for a laboratory strain and for a field isolate. Thus, it can be used as a tool in large-scale screening to identify virulence genes of C. perfringens. PMID:19270116

  9. Transposon Mutagenesis Identified Chromosomal and Plasmid Genes Essential for Adaptation of the Marine Bacterium Dinoroseobacter shibae to Anaerobic Conditions

    PubMed Central

    Ebert, Matthias; Laaß, Sebastian; Burghartz, Melanie; Petersen, Jörn; Koßmehl, Sebastian; Wöhlbrand, Lars; Rabus, Ralf; Wittmann, Christoph; Jahn, Dieter

    2013-01-01

    Anaerobic growth and survival are integral parts of the life cycle of many marine bacteria. To identify genes essential for the anoxic life of Dinoroseobacter shibae, a transposon library was screened for strains impaired in anaerobic denitrifying growth. Transposon insertions in 35 chromosomal and 18 plasmid genes were detected. The essential contribution of plasmid genes to anaerobic growth was confirmed with plasmid-cured D. shibae strains. A combined transcriptome and proteome approach identified oxygen tension-regulated genes. Transposon insertion sites of a total of 1,527 mutants without an anaerobic growth phenotype were determined to identify anaerobically induced but not essential genes. A surprisingly small overlap of only three genes (napA, phaA, and the Na+/Pi antiporter gene Dshi_0543) between anaerobically essential and induced genes was found. Interestingly, transposon mutations in genes involved in dissimilatory and assimilatory nitrate reduction (napA, nasA) and corresponding cofactor biosynthesis (genomic moaB, moeB, and dsbC and plasmid-carried dsbD and ccmH) were found to cause anaerobic growth defects. In contrast, mutation of anaerobically induced genes encoding proteins required for the later denitrification steps (nirS, nirJ, nosD), dimethyl sulfoxide reduction (dmsA1), and fermentation (pdhB1, arcA, aceE, pta, acs) did not result in decreased anaerobic growth under the conditions tested. Additional essential components (ferredoxin, cccA) of the anaerobic electron transfer chain and central metabolism (pdhB) were identified. Another surprise was the importance of sodium gradient-dependent membrane processes and genomic rearrangements via viruses, transposons, and insertion sequence elements for anaerobic growth. These processes and the observed contributions of cell envelope restructuring (lysM, mipA, fadK), C4-dicarboxylate transport (dctM1, dctM3), and protease functions to anaerobic growth require further investigation to unravel the

  10. Transposon Mutagenesis of Salmonella enterica Serovar Enteritidis Identifies Genes That Contribute to Invasiveness in Human and Chicken Cells and Survival in Egg Albumen

    PubMed Central

    Zhou, Xiaohui; Kim, Hye-Young; Call, Douglas R.; Guard, Jean

    2012-01-01

    Salmonella enterica serovar Enteritidis is an important food-borne pathogen, and chickens are a primary reservoir of human infection. While most knowledge about Salmonella pathogenesis is based on research conducted on Salmonella enterica serovar Typhimurium, S. Enteritidis is known to have pathobiology specific to chickens that impacts epidemiology in humans. Therefore, more information is needed about S. Enteritidis pathobiology in comparison to that of S. Typhimurium. We used transposon mutagenesis to identify S. Enteritidis virulence genes by assay of invasiveness in human intestinal epithelial (Caco-2) cells and chicken liver (LMH) cells and survival within chicken (HD-11) macrophages as a surrogate marker for virulence. A total of 4,330 transposon insertion mutants of an invasive G1 Nalr strain were screened using Caco-2 cells. This led to the identification of attenuating mutations in a total of 33 different loci, many of which include genes previously known to contribute to enteric infection (e.g., Salmonella pathogenicity island 1 [SPI-1], SPI-4, SPI-5, CS54, fliH, fljB, csgB, spvR, and rfbMN) in S. Enteritidis and other Salmonella serovars. Several genes or genomic islands that have not been reported previously (e.g., SPI-14, ksgA, SEN0034, SEN2278, and SEN3503) or that are absent in S. Typhimurium or in most other Salmonella serovars (e.g., pegD, SEN1152, SEN1393, and SEN1966) were also identified. Most mutants with reduced Caco-2 cell invasiveness also showed significantly reduced invasiveness in chicken liver cells and impaired survival in chicken macrophages and in egg albumen. Consequently, these genes may play an important role during infection of the chicken host and also contribute to successful egg contamination by S. Enteritidis. PMID:22988017

  11. Genome-Wide Transposon Mutagenesis Indicates that Mycobacterium marinum Customizes Its Virulence Mechanisms for Survival and Replication in Different Hosts

    PubMed Central

    Weerdenburg, Eveline M.; Abdallah, Abdallah M.; Rangkuti, Farania; Abd El Ghany, Moataz; Otto, Thomas D.; Adroub, Sabir A.; Molenaar, Douwe; Ummels, Roy; ter Veen, Kars; van Stempvoort, Gunny; van der Sar, Astrid M.; Ali, Shahjahan; Langridge, Gemma C.; Thomson, Nicholas R.

    2015-01-01

    The interaction of environmental bacteria with unicellular eukaryotes is generally considered a major driving force for the evolution of intracellular pathogens, allowing them to survive and replicate in phagocytic cells of vertebrate hosts. To test this hypothesis on a genome-wide level, we determined for the intracellular pathogen Mycobacterium marinum whether it uses conserved strategies to exploit host cells from both protozoan and vertebrate origin. Using transposon-directed insertion site sequencing (TraDIS), we determined differences in genetic requirements for survival and replication in phagocytic cells of organisms from different kingdoms. In line with the general hypothesis, we identified a number of general virulence mechanisms, including the type VII protein secretion system ESX-1, biosynthesis of polyketide lipids, and utilization of sterols. However, we were also able to show that M. marinum contains an even larger set of host-specific virulence determinants, including proteins involved in the modification of surface glycolipids and, surprisingly, the auxiliary proteins of the ESX-1 system. Several of these factors were in fact counterproductive in other hosts. Therefore, M. marinum contains different sets of virulence factors that are tailored for specific hosts. Our data imply that although amoebae could function as a training ground for intracellular pathogens, they do not fully prepare pathogens for crossing species barriers. PMID:25690095

  12. DNA Repair Dependence of Somatic Mutagenesis of Transposon-Caused WHITE Alleles in DROSOPHILA MELANOGASTER after Treatment with Alkylating Agents

    PubMed Central

    Fujikawa, Kazuo; Kondo, Sohei

    1986-01-01

    DNA repair-defective alleles of the mei-9, mei-41, mus-104 and mus-101 loci of Drosophila melanogaster were introduced into stocks bearing the UZ and SZ marker sets. Males with the UZ marker set, z1 (zeste allele) and w+(TE) (genetically unstable white allele presumably caused by a transposable element), or the SZ marker set, z1 and w+R (semistable white allele caused by partial duplication of the w+ locus plus transposon insert), were exposed to EMS at the first instar. After emergence, adult males bearing red spots on lemon-yellow eyes were scored as flies with somatic reversions of w+(TE) or w +R. The relative mutabilities (relative values of reversion frequency at an equal EMS dose) of either w+(TE) or w+R in a repair-proficient strain and in mei-9, mei-41, mus-104 and mus-101 strains were 1:∼1.2:0.3:0.3:0.7, despite the fact that w+(TE) reverted two to three times as frequently as w+R under both the repair-proficient and repair-deficient genetic conditions. Similarly, after treatment with MMS, MNNG and ENNG, w+(TE) was somatically more mutable in the mei-9 strain and less mutable in the mei-41 and mus-104 strains than in the repair-proficient strain. From these results, we propose that mutagenic lesions produced in DNA by treatment with these chemicals are converted to mutant DNA sequences via the error-prone repair mechanisms dependent on the products of the genes mei-41+ (mei-41 and mus-104 being alleles of the same locus) and mus-101+, whereas they are eliminated by mei-9+-dependent excision repair. In contrast to the approximately linear responses of induced reversions of w+( TE) with ENNG in the repair-proficient, mei-9, and mei-41 strains, seemingly there were dosage insensitive ranges for induced reversion with MNNG in the repair-proficient and mei-41 strains, but not for reversion in the mei-9 strain; w+( TE) in the mus-104 strain was virtually nonmutable with MNNG and ENNG. These results suggest that O6-methylguanine (O6MeG) produced in DNA with

  13. PiggyBac Transposon-Mediated Mutagenesis in Rats Reveals a Crucial Role of Bbx in Growth and Male Fertility.

    PubMed

    Wang, Chieh-Ying; Tang, Ming-Chu; Chang, Wen-Chi; Furushima, Kenryo; Jang, Chuan-Wei; Behringer, Richard R; Chen, Chun-Ming

    2016-09-01

    Bobby sox homolog (Bbx) is an evolutionally conserved gene, but its biological function remains elusive. Here, we characterized defects of Bbx mutant rats that were created by PiggyBac-mediated insertional mutagenesis. Smaller body size and male infertility were the two major phenotypes of homozygous Bbx mutants. Bbx expression profile analysis showed that Bbx was more highly expressed in the testis and pituitary gland than in other organs. Histology and hormonal gene expression analysis of control and Bbx-null pituitary glands showed that loss of Bbx appeared to be dispensable for pituitary histogenesis and the expression of major hormones. BBX was localized in the nuclei of postmeiotic spermatids and Sertoli cells in wild-type testes, but absent in mutant testes. An increased presence of aberrant multinuclear giant cells and apoptotic cells was observed in mutant seminiferous tubules. TUNEL-positive cells costained with CREM (round spermatid marker), but not PLZF (spermatogonia marker), gammaH2Ax (meiotic spermatocyte marker), or GATA4 (Sertoli cell marker). Finally, there were drastically reduced numbers and motility of epididymal sperm from Bbx-null rats. These results suggest that loss of BBX induces apoptosis of postmeiotic spermatids and results in spermiogenesis defects and infertility. PMID:27465138

  14. Transposon Mutagenesis of the Plant-Associated Bacillus amyloliquefaciens ssp. plantarum FZB42 Revealed That the nfrA and RBAM17410 Genes Are Involved in Plant-Microbe-Interactions

    PubMed Central

    Dietel, Kristin; Beator, Barbara; Dolgova, Olga; Fan, Ben; Bleiss, Wilfrid; Ziegler, Jörg; Schmid, Michael; Hartmann, Anton; Borriss, Rainer

    2014-01-01

    Bacillus amyloliquefaciens ssp. plantarum FZB42 represents the prototype of Gram-positive plant growth promoting and biocontrol bacteria. In this study, we applied transposon mutagenesis to generate a transposon library, which was screened for genes involved in multicellular behavior and biofilm formation on roots as a prerequisite of plant growth promoting activity. Transposon insertion sites were determined by rescue-cloning followed by DNA sequencing. As in B. subtilis, the global transcriptional regulator DegU was identified as an activator of genes necessary for swarming and biofilm formation, and the DegU-mutant of FZB42 was found impaired in efficient root colonization. Direct screening of 3,000 transposon insertion mutants for plant-growth-promotion revealed the gene products of nfrA and RBAM_017140 to be essential for beneficial effects exerted by FZB42 on plants. We analyzed the performance of GFP-labeled wild-type and transposon mutants in the colonization of lettuce roots using confocal laser scanning microscopy. While the wild-type strain heavily colonized root surfaces, the nfrA mutant did not colonize lettuce roots, although it was not impaired in growth in laboratory cultures, biofilm formation and swarming motility on agar plates. The RBAM17410 gene, occurring in only a few members of the B. subtilis species complex, was directly involved in plant growth promotion. None of the mutant strains were affected in producing the plant growth hormone auxin. We hypothesize that the nfrA gene product is essential for overcoming the stress caused by plant response towards bacterial root colonization. PMID:24847778

  15. Misty somites, a maternal effect gene identified by transposon-mediated insertional mutagenesis in zebrafish that is essential for the somite boundary maintenance.

    PubMed

    Kotani, Tomoya; Kawakami, Koichi

    2008-04-15

    Somite boundary formation is crucial for segmentation of vertebrate somites and vertebrae and skeletal muscle morphogenesis. Previously, we developed a Tol2 transposon-mediated gene trap method in zebrafish. In the present study, we aimed to isolate transposon insertions that trap maternally-expressed genes. We found that homozygous female fish carrying a transposon insertion within a maternally-expressed gene misty somites (mys) produced embryos that showed obscure somite boundaries at the early segmentation stage (12-13 hpf). The somite boundaries became clear and distinct after this period and the embryos survived to adulthood. This phenotype was rescued by expression of mys cDNA in the homozygous adults, confirming that it was caused by a decreased mys activity. We analyzed a role of the mys gene by using morpholino oligonucleotides (MOs). The MO-injected embryo exhibited severer phenotypes than the insertional mutant probably because the mys gene was partially active in the insertional mutant. The MO-injected embryo also showed the obscure somite boundary phenotype. Fibronectin and phosphorylated FAK at the intersomitic regions were accumulated at the boundaries at this stage, but, unlike wild type embryos, somitic cells adjacent to the boundaries did not undergo epithelialization, suggesting that Mys is required for epithelialization of the somitic cells. Then in the MO-injected embryos, the boundaries once became clear and distinct, but, in the subsequent stages, disappeared, resulting in abnormal muscle morphogenesis. Accumulation of Fibronectin and phosphorylated FAK observed in the initial stage also disappeared. Thus, Mys is crucial for maintenance of the somite boundaries formed at the initial stage. To analyze the mys defect at the cellular level, we placed cells dissociated from the MO-injected embryo on Fibronectin-coated glasses. By this cell spreading assay, we found that the mys-deficient cells reduced the activity to form lamellipodia on

  16. Genome Sequencing and Transposon Mutagenesis of Burkholderia seminalis TC3.4.2R3 Identify Genes Contributing to Suppression of Orchid Necrosis Caused by B. gladioli.

    PubMed

    Araújo, Welington L; Creason, Allison L; Mano, Emy T; Camargo-Neves, Aline A; Minami, Sonia N; Chang, Jeff H; Loper, Joyce E

    2016-06-01

    From a screen of 36 plant-associated strains of Burkholderia spp., we identified 24 strains that suppressed leaf and pseudobulb necrosis of orchid caused by B. gladioli. To gain insights into the mechanisms of disease suppression, we generated a draft genome sequence from one suppressive strain, TC3.4.2R3. The genome is an estimated 7.67 megabases in size, with three replicons, two chromosomes, and the plasmid pC3. Using a combination of multilocus sequence analysis and phylogenomics, we identified TC3.4.2R3 as B. seminalis, a species within the Burkholderia cepacia complex that includes opportunistic human pathogens and environmental strains. We generated and screened a library of 3,840 transposon mutants of strain TC3.4.2R3 on orchid leaves to identify genes contributing to plant disease suppression. Twelve mutants deficient in suppression of leaf necrosis were selected and the transposon insertions were mapped to eight loci. One gene is in a wcb cluster that is related to synthesis of extracellular polysaccharide, a key determinant in bacterial-host interactions in other systems, and the other seven are highly conserved among Burkholderia spp. The fundamental information developed in this study will serve as a resource for future research aiming to identify mechanisms contributing to biological control. PMID:26959838

  17. Transposon Mutagenesis of Probiotic Lactobacillus casei Identifies asnH, an Asparagine Synthetase Gene Involved in Its Immune-Activating Capacity

    PubMed Central

    Ito, Masahiro; Kim, Yun-Gi; Tsuji, Hirokazu; Takahashi, Takuya; Kiwaki, Mayumi; Nomoto, Koji; Danbara, Hirofumi; Okada, Nobuhiko

    2014-01-01

    Lactobacillus casei ATCC 27139 enhances host innate immunity, and the J1 phage-resistant mutants of this strain lose the activity. A transposon insertion mutant library of L. casei ATCC 27139 was constructed, and nine J1 phage-resistant mutants out of them were obtained. Cloning and sequencing analyses identified three independent genes that were disrupted by insertion of the transposon element: asnH, encoding asparagine synthetase, and dnaJ and dnaK, encoding the molecular chaperones DnaJ and DnaK, respectively. Using an in vivo mouse model of Listeria infection, only asnH mutant showed deficiency in their ability to enhance host innate immunity, and complementation of the mutation by introduction of the wild-type asnH in the mutant strain recovered the immuno-augmenting activity. AsnH protein exhibited asparagine synthetase activity when the lysozyme-treated cell wall extracts of L. casei ATCC 27139 was added as substrate. The asnH mutants lost the thick and rigid peptidoglycan features that are characteristic to the wild-type cells, indicating that AsnH of L. casei is involved in peptidoglycan biosynthesis. These results indicate that asnH is required for the construction of the peptidoglycan composition involved in the immune-activating capacity of L. casei ATCC 27139. PMID:24416179

  18. Transposon facilitated DNA sequencing

    SciTech Connect

    Berg, D.E.; Berg, C.M.; Huang, H.V.

    1990-01-01

    The purpose of this research is to investigate and develop methods that exploit the power of bacterial transposable elements for large scale DNA sequencing: Our premise is that the use of transposons to put primer binding sites randomly in target DNAs should provide access to all portions of large DNA fragments, without the inefficiencies of methods involving random subcloning and attendant repetitive sequencing, or of sequential synthesis of many oligonucleotide primers that are used to match systematically along a DNA molecule. Two unrelated bacterial transposons, Tn5 and {gamma}{delta}, are being used because they have both proven useful for molecular analyses, and because they differ sufficiently in mechanism and specificity of transposition to merit parallel development.

  19. Large scale dynamic systems

    NASA Technical Reports Server (NTRS)

    Doolin, B. F.

    1975-01-01

    Classes of large scale dynamic systems were discussed in the context of modern control theory. Specific examples discussed were in the technical fields of aeronautics, water resources and electric power.

  20. Large Scale Computing

    NASA Astrophysics Data System (ADS)

    Capiluppi, Paolo

    2005-04-01

    Large Scale Computing is acquiring an important role in the field of data analysis and treatment for many Sciences and also for some Social activities. The present paper discusses the characteristics of Computing when it becomes "Large Scale" and the current state of the art for some particular application needing such a large distributed resources and organization. High Energy Particle Physics (HEP) Experiments are discussed in this respect; in particular the Large Hadron Collider (LHC) Experiments are analyzed. The Computing Models of LHC Experiments represent the current prototype implementation of Large Scale Computing and describe the level of maturity of the possible deployment solutions. Some of the most recent results on the measurements of the performances and functionalities of the LHC Experiments' testing are discussed.

  1. Large-Scale Disasters

    NASA Astrophysics Data System (ADS)

    Gad-El-Hak, Mohamed

    "Extreme" events - including climatic events, such as hurricanes, tornadoes, and drought - can cause massive disruption to society, including large death tolls and property damage in the billions of dollars. Events in recent years have shown the importance of being prepared and that countries need to work together to help alleviate the resulting pain and suffering. This volume presents a review of the broad research field of large-scale disasters. It establishes a common framework for predicting, controlling and managing both manmade and natural disasters. There is a particular focus on events caused by weather and climate change. Other topics include air pollution, tsunamis, disaster modeling, the use of remote sensing and the logistics of disaster management. It will appeal to scientists, engineers, first responders and health-care professionals, in addition to graduate students and researchers who have an interest in the prediction, prevention or mitigation of large-scale disasters.

  2. Large scale tracking algorithms.

    SciTech Connect

    Hansen, Ross L.; Love, Joshua Alan; Melgaard, David Kennett; Karelitz, David B.; Pitts, Todd Alan; Zollweg, Joshua David; Anderson, Dylan Z.; Nandy, Prabal; Whitlow, Gary L.; Bender, Daniel A.; Byrne, Raymond Harry

    2015-01-01

    Low signal-to-noise data processing algorithms for improved detection, tracking, discrimination and situational threat assessment are a key research challenge. As sensor technologies progress, the number of pixels will increase signi cantly. This will result in increased resolution, which could improve object discrimination, but unfortunately, will also result in a significant increase in the number of potential targets to track. Many tracking techniques, like multi-hypothesis trackers, suffer from a combinatorial explosion as the number of potential targets increase. As the resolution increases, the phenomenology applied towards detection algorithms also changes. For low resolution sensors, "blob" tracking is the norm. For higher resolution data, additional information may be employed in the detection and classfication steps. The most challenging scenarios are those where the targets cannot be fully resolved, yet must be tracked and distinguished for neighboring closely spaced objects. Tracking vehicles in an urban environment is an example of such a challenging scenario. This report evaluates several potential tracking algorithms for large-scale tracking in an urban environment.

  3. Large scale traffic simulations

    SciTech Connect

    Nagel, K.; Barrett, C.L.; Rickert, M.

    1997-04-01

    Large scale microscopic (i.e. vehicle-based) traffic simulations pose high demands on computational speed in at least two application areas: (i) real-time traffic forecasting, and (ii) long-term planning applications (where repeated {open_quotes}looping{close_quotes} between the microsimulation and the simulated planning of individual person`s behavior is necessary). As a rough number, a real-time simulation of an area such as Los Angeles (ca. 1 million travellers) will need a computational speed of much higher than 1 million {open_quotes}particle{close_quotes} (= vehicle) updates per second. This paper reviews how this problem is approached in different projects and how these approaches are dependent both on the specific questions and on the prospective user community. The approaches reach from highly parallel and vectorizable, single-bit implementations on parallel supercomputers for Statistical Physics questions, via more realistic implementations on coupled workstations, to more complicated driving dynamics implemented again on parallel supercomputers. 45 refs., 9 figs., 1 tab.

  4. Transposon-mediated Genome Manipulations in Vertebrates

    PubMed Central

    Ivics, Zoltán; Li, Meng Amy; Mátés, Lajos; Boeke, Jef D.; Bradley, Allan; Izsvák, Zsuzsanna

    2010-01-01

    Transposable elements are segments of DNA with the unique ability to move about in the genome. This inherent feature can be exploited to harness these elements as gene vectors for diverse genome manipulations. Transposon-based genetic strategies have been established in vertebrate species over the last decade, and current progress in this field indicates that transposable elements will serve as indispensable tools in the genetic toolkit of vertebrate models. In particular, transposons can be applied as vectors for somatic and germline transgenesis, and as insertional mutagens in both loss-of-function and gain-of-function forward mutagenesis screens. The major advantage of using transposons as genetic tools is that they facilitate analysis of gene function in an easy, controlled and scalable manner. Transposon-based technologies are beginning to be exploited to link sequence information to gene functions in vertebrate models. In this article, we provide an overview of transposon-based methods used in vertebrate model organisms, and highlight the most important considerations concerning genetic applications of the transposon systems. PMID:19478801

  5. Galaxy clustering on large scales.

    PubMed

    Efstathiou, G

    1993-06-01

    I describe some recent observations of large-scale structure in the galaxy distribution. The best constraints come from two-dimensional galaxy surveys and studies of angular correlation functions. Results from galaxy redshift surveys are much less precise but are consistent with the angular correlations, provided the distortions in mapping between real-space and redshift-space are relatively weak. The galaxy two-point correlation function, rich-cluster two-point correlation function, and galaxy-cluster cross-correlation function are all well described on large scales ( greater, similar 20h-1 Mpc, where the Hubble constant, H0 = 100h km.s-1.Mpc; 1 pc = 3.09 x 10(16) m) by the power spectrum of an initially scale-invariant, adiabatic, cold-dark-matter Universe with Gamma = Omegah approximately 0.2. I discuss how this fits in with the Cosmic Background Explorer (COBE) satellite detection of large-scale anisotropies in the microwave background radiation and other measures of large-scale structure in the Universe. PMID:11607400

  6. Very Large Scale Integration (VLSI).

    ERIC Educational Resources Information Center

    Yeaman, Andrew R. J.

    Very Large Scale Integration (VLSI), the state-of-the-art production techniques for computer chips, promises such powerful, inexpensive computing that, in the future, people will be able to communicate with computer devices in natural language or even speech. However, before full-scale VLSI implementation can occur, certain salient factors must be…

  7. Transposon transgenesis in Xenopus

    PubMed Central

    Yergeau, Donald A.; Kelley, Clair M.; Zhu, Haiqing; Kuliyev, Emin; Mead, Paul E.

    2010-01-01

    Transposon-mediated integration strategies in Xenopus offer simple and robust methods for the generation of germline transgenic animals. Co-injection of fertilized one-cell embryos with plasmid DNA harboring a transposon transgene and synthetic mRNA encoding the cognate transposase enzyme results in mosaic integration of the transposon at early cleavage stages that are frequently passed through the germline in the adult animal. Micro-injection of fertilized embryos is a routine procedure used by many laboratories that use Xenopus as a developmental model and, as such, the transposon transgenesis method can be performed without additional equipment or specialized methodologies. The methods for injecting Xenopus embryos are well documented in the literature so here we provide a step-by-step guide to other aspects of transposon transgenesis, including screening mosaic founders for germline transmission of the transgene and general husbandry considerations related to management of populations of transgenic frogs. PMID:20211730

  8. Microfluidic large-scale integration.

    PubMed

    Thorsen, Todd; Maerkl, Sebastian J; Quake, Stephen R

    2002-10-18

    We developed high-density microfluidic chips that contain plumbing networks with thousands of micromechanical valves and hundreds of individually addressable chambers. These fluidic devices are analogous to electronic integrated circuits fabricated using large-scale integration. A key component of these networks is the fluidic multiplexor, which is a combinatorial array of binary valve patterns that exponentially increases the processing power of a network by allowing complex fluid manipulations with a minimal number of inputs. We used these integrated microfluidic networks to construct the microfluidic analog of a comparator array and a microfluidic memory storage device whose behavior resembles random-access memory. PMID:12351675

  9. Large-scale mapping of mutations affecting zebrafish development

    PubMed Central

    Geisler, Robert; Rauch, Gerd-Jörg; Geiger-Rudolph, Silke; Albrecht, Andrea; van Bebber, Frauke; Berger, Andrea; Busch-Nentwich, Elisabeth; Dahm, Ralf; Dekens, Marcus PS; Dooley, Christopher; Elli, Alexandra F; Gehring, Ines; Geiger, Horst; Geisler, Maria; Glaser, Stefanie; Holley, Scott; Huber, Matthias; Kerr, Andy; Kirn, Anette; Knirsch, Martina; Konantz, Martina; Küchler, Axel M; Maderspacher, Florian; Neuhauss, Stephan C; Nicolson, Teresa; Ober, Elke A; Praeg, Elke; Ray, Russell; Rentzsch, Brit; Rick, Jens M; Rief, Eva; Schauerte, Heike E; Schepp, Carsten P; Schönberger, Ulrike; Schonthaler, Helia B; Seiler, Christoph; Sidi, Samuel; Söllner, Christian; Wehner, Anja; Weiler, Christian; Nüsslein-Volhard, Christiane

    2007-01-01

    Background Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers. Results We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the Tübingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM. Conclusion By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations. PMID:17212827

  10. Large scale topography of Io

    NASA Technical Reports Server (NTRS)

    Gaskell, R. W.; Synnott, S. P.

    1987-01-01

    To investigate the large scale topography of the Jovian satellite Io, both limb observations and stereographic techniques applied to landmarks are used. The raw data for this study consists of Voyager 1 images of Io, 800x800 arrays of picture elements each of which can take on 256 possible brightness values. In analyzing this data it was necessary to identify and locate landmarks and limb points on the raw images, remove the image distortions caused by the camera electronics and translate the corrected locations into positions relative to a reference geoid. Minimizing the uncertainty in the corrected locations is crucial to the success of this project. In the highest resolution frames, an error of a tenth of a pixel in image space location can lead to a 300 m error in true location. In the lowest resolution frames, the same error can lead to an uncertainty of several km.

  11. Challenges for Large Scale Simulations

    NASA Astrophysics Data System (ADS)

    Troyer, Matthias

    2010-03-01

    With computational approaches becoming ubiquitous the growing impact of large scale computing on research influences both theoretical and experimental work. I will review a few examples in condensed matter physics and quantum optics, including the impact of computer simulations in the search for supersolidity, thermometry in ultracold quantum gases, and the challenging search for novel phases in strongly correlated electron systems. While only a decade ago such simulations needed the fastest supercomputers, many simulations can now be performed on small workstation clusters or even a laptop: what was previously restricted to a few experts can now potentially be used by many. Only part of the gain in computational capabilities is due to Moore's law and improvement in hardware. Equally impressive is the performance gain due to new algorithms - as I will illustrate using some recently developed algorithms. At the same time modern peta-scale supercomputers offer unprecedented computational power and allow us to tackle new problems and address questions that were impossible to solve numerically only a few years ago. While there is a roadmap for future hardware developments to exascale and beyond, the main challenges are on the algorithmic and software infrastructure side. Among the problems that face the computational physicist are: the development of new algorithms that scale to thousands of cores and beyond, a software infrastructure that lifts code development to a higher level and speeds up the development of new simulation programs for large scale computing machines, tools to analyze the large volume of data obtained from such simulations, and as an emerging field provenance-aware software that aims for reproducibility of the complete computational workflow from model parameters to the final figures. Interdisciplinary collaborations and collective efforts will be required, in contrast to the cottage-industry culture currently present in many areas of computational

  12. Toward the Analysis of the Petunia MADS Box Gene Family by Reverse and Forward Transposon Insertion Mutagenesis Approaches: B, C, and D Floral Organ Identity Functions Require SEPALLATA-Like MADS Box Genes in Petunia

    PubMed Central

    Vandenbussche, Michiel; Zethof, Jan; Souer, Erik; Koes, Ronald; Tornielli, Giovanni B.; Pezzotti, Mario; Ferrario, Silvia; Angenent, Gerco C.; Gerats, Tom

    2003-01-01

    We have initiated a systematic functional analysis of the MADS box, intervening region, K domain, C domain-type MADS box gene family in petunia. The starting point for this has been a reverse-genetics approach, aiming to select for transposon insertions into any MADS box gene. We have developed and applied a family signature insertion screening protocol that is highly suited for this purpose, resulting in the isolation of 32 insertion mutants in 20 different MADS box genes. In addition, we identified three more MADS box gene insertion mutants using a candidate-gene approach. The defined insertion lines provide a sound foundation for a systematic functional analysis of the MADS box gene family in petunia. Here, we focus on the analysis of Floral Binding Protein2 (FBP2) and FBP5 genes that encode the E-function, which in Arabidopsis has been shown to be required for B and C floral organ identity functions. fbp2 mutants display sepaloid petals and ectopic inflorescences originating from the third floral whorl, whereas fbp5 mutants appear as wild type. In fbp2 fbp5 double mutants, reversion of floral organs to leaf-like organs is increased further. Strikingly, ovules are replaced by leaf-like structures in the carpel, indicating that in addition to the B- and C-functions, the D-function, which specifies ovule development, requires E-function activity. Finally, we compare our data with results obtained using cosuppression approaches and conclude that the latter might be less suited for assigning functions to individual members of the MADS box gene family. PMID:14576291

  13. Large Scale Homing in Honeybees

    PubMed Central

    Pahl, Mario; Zhu, Hong; Tautz, Jürgen; Zhang, Shaowu

    2011-01-01

    Honeybee foragers frequently fly several kilometres to and from vital resources, and communicate those locations to their nest mates by a symbolic dance language. Research has shown that they achieve this feat by memorizing landmarks and the skyline panorama, using the sun and polarized skylight as compasses and by integrating their outbound flight paths. In order to investigate the capacity of the honeybees' homing abilities, we artificially displaced foragers to novel release spots at various distances up to 13 km in the four cardinal directions. Returning bees were individually registered by a radio frequency identification (RFID) system at the hive entrance. We found that homing rate, homing speed and the maximum homing distance depend on the release direction. Bees released in the east were more likely to find their way back home, and returned faster than bees released in any other direction, due to the familiarity of global landmarks seen from the hive. Our findings suggest that such large scale homing is facilitated by global landmarks acting as beacons, and possibly the entire skyline panorama. PMID:21602920

  14. Large Scale Nanolaminate Deformable Mirror

    SciTech Connect

    Papavasiliou, A; Olivier, S; Barbee, T; Miles, R; Chang, K

    2005-11-30

    This work concerns the development of a technology that uses Nanolaminate foils to form light-weight, deformable mirrors that are scalable over a wide range of mirror sizes. While MEMS-based deformable mirrors and spatial light modulators have considerably reduced the cost and increased the capabilities of adaptive optic systems, there has not been a way to utilize the advantages of lithography and batch-fabrication to produce large-scale deformable mirrors. This technology is made scalable by using fabrication techniques and lithography that are not limited to the sizes of conventional MEMS devices. Like many MEMS devices, these mirrors use parallel plate electrostatic actuators. This technology replicates that functionality by suspending a horizontal piece of nanolaminate foil over an electrode by electroplated nickel posts. This actuator is attached, with another post, to another nanolaminate foil that acts as the mirror surface. Most MEMS devices are produced with integrated circuit lithography techniques that are capable of very small line widths, but are not scalable to large sizes. This technology is very tolerant of lithography errors and can use coarser, printed circuit board lithography techniques that can be scaled to very large sizes. These mirrors use small, lithographically defined actuators and thin nanolaminate foils allowing them to produce deformations over a large area while minimizing weight. This paper will describe a staged program to develop this technology. First-principles models were developed to determine design parameters. Three stages of fabrication will be described starting with a 3 x 3 device using conventional metal foils and epoxy to a 10-across all-metal device with nanolaminate mirror surfaces.

  15. Large-Scale Information Systems

    SciTech Connect

    D. M. Nicol; H. R. Ammerlahn; M. E. Goldsby; M. M. Johnson; D. E. Rhodes; A. S. Yoshimura

    2000-12-01

    Large enterprises are ever more dependent on their Large-Scale Information Systems (LSLS), computer systems that are distinguished architecturally by distributed components--data sources, networks, computing engines, simulations, human-in-the-loop control and remote access stations. These systems provide such capabilities as workflow, data fusion and distributed database access. The Nuclear Weapons Complex (NWC) contains many examples of LSIS components, a fact that motivates this research. However, most LSIS in use grew up from collections of separate subsystems that were not designed to be components of an integrated system. For this reason, they are often difficult to analyze and control. The problem is made more difficult by the size of a typical system, its diversity of information sources, and the institutional complexities associated with its geographic distribution across the enterprise. Moreover, there is no integrated approach for analyzing or managing such systems. Indeed, integrated development of LSIS is an active area of academic research. This work developed such an approach by simulating the various components of the LSIS and allowing the simulated components to interact with real LSIS subsystems. This research demonstrated two benefits. First, applying it to a particular LSIS provided a thorough understanding of the interfaces between the system's components. Second, it demonstrated how more rapid and detailed answers could be obtained to questions significant to the enterprise by interacting with the relevant LSIS subsystems through simulated components designed with those questions in mind. In a final, added phase of the project, investigations were made on extending this research to wireless communication networks in support of telemetry applications.

  16. A Multifunctional Mutagenesis System for Analysis of Gene Function in Zebrafish

    PubMed Central

    Quach, Helen Ngoc Bao; Tao, Shijie; Vrljicak, Pavle; Joshi, Adita; Ruan, Hua; Sukumaran, Rashmi; Varshney, Gaurav K.; LaFave, Matthew C.; Burgess, Shawn M.; Winkler, Christoph; Emelyanov, Alexander; Parinov, Sergey; Sampath, Karuna

    2015-01-01

    Since the sequencing of the human reference genome, many human disease-related genes have been discovered. However, understanding the functions of all the genes in the genome remains a challenge. The biological activities of these genes are usually investigated in model organisms such as mice and zebrafish. Large-scale mutagenesis screens to generate disruptive mutations are useful for identifying and understanding the activities of genes. Here, we report a multifunctional mutagenesis system in zebrafish using the maize Ds transposon. Integration of the Ds transposable element containing an mCherry reporter for protein trap events and an EGFP reporter for enhancer trap events produced a collection of transgenic lines marking distinct cell and tissue types, and mutagenized genes in the zebrafish genome by trapping and prematurely terminating endogenous protein coding sequences. We obtained 642 zebrafish lines with dynamic reporter gene expression. The characterized fish lines with specific expression patterns will be made available through the European Zebrafish Resource Center (EZRC), and a database of reporter expression is available online (http://fishtrap.warwick.ac.uk/). Our approach complements other efforts using zebrafish to facilitate functional genomic studies in this model of human development and disease. PMID:25840430

  17. Large-Scale Reform Comes of Age

    ERIC Educational Resources Information Center

    Fullan, Michael

    2009-01-01

    This article reviews the history of large-scale education reform and makes the case that large-scale or whole system reform policies and strategies are becoming increasingly evident. The review briefly addresses the pre 1997 period concluding that while the pressure for reform was mounting that there were very few examples of deliberate or…

  18. Large-scale infrared scene projectors

    NASA Astrophysics Data System (ADS)

    Murray, Darin A.

    1999-07-01

    Large-scale infrared scene projectors, typically have unique opto-mechanical characteristics associated to their application. This paper outlines two large-scale zoom lens assemblies with different environmental and package constraints. Various challenges and their respective solutions are discussed and presented.

  19. Synthesis of small and large scale dynamos

    NASA Astrophysics Data System (ADS)

    Subramanian, Kandaswamy

    Using a closure model for the evolution of magnetic correlations, we uncover an interesting plausible saturated state of the small-scale fluctuation dynamo (SSD) and a novel analogy between quantum mechanical tunnelling and the generation of large-scale fields. Large scale fields develop via the α-effect, but as magnetic helicity can only change on a resistive timescale, the time it takes to organize the field into large scales increases with magnetic Reynolds number. This is very similar to the results which obtain from simulations using the full MHD equations.

  20. Large-scale regions of antimatter

    SciTech Connect

    Grobov, A. V. Rubin, S. G.

    2015-07-15

    Amodified mechanism of the formation of large-scale antimatter regions is proposed. Antimatter appears owing to fluctuations of a complex scalar field that carries a baryon charge in the inflation era.

  1. Survey on large scale system control methods

    NASA Technical Reports Server (NTRS)

    Mercadal, Mathieu

    1987-01-01

    The problem inherent to large scale systems such as power network, communication network and economic or ecological systems were studied. The increase in size and flexibility of future spacecraft has put those dynamical systems into the category of large scale systems, and tools specific to the class of large systems are being sought to design control systems that can guarantee more stability and better performance. Among several survey papers, reference was found to a thorough investigation on decentralized control methods. Especially helpful was the classification made of the different existing approaches to deal with large scale systems. A very similar classification is used, even though the papers surveyed are somehow different from the ones reviewed in other papers. Special attention is brought to the applicability of the existing methods to controlling large mechanical systems like large space structures. Some recent developments are added to this survey.

  2. Large-scale inhomogeneities and galaxy statistics

    NASA Technical Reports Server (NTRS)

    Schaeffer, R.; Silk, J.

    1984-01-01

    The density fluctuations associated with the formation of large-scale cosmic pancake-like and filamentary structures are evaluated using the Zel'dovich approximation for the evolution of nonlinear inhomogeneities in the expanding universe. It is shown that the large-scale nonlinear density fluctuations in the galaxy distribution due to pancakes modify the standard scale-invariant correlation function xi(r) at scales comparable to the coherence length of adiabatic fluctuations. The typical contribution of pancakes and filaments to the J3 integral, and more generally to the moments of galaxy counts in a volume of approximately (15-40 per h Mpc)exp 3, provides a statistical test for the existence of large scale inhomogeneities. An application to several recent three dimensional data sets shows that despite large observational uncertainties over the relevant scales characteristic features may be present that can be attributed to pancakes in most, but not all, of the various galaxy samples.

  3. The large-scale distribution of galaxies

    NASA Technical Reports Server (NTRS)

    Geller, Margaret J.

    1989-01-01

    The spatial distribution of galaxies in the universe is characterized on the basis of the six completed strips of the Harvard-Smithsonian Center for Astrophysics redshift-survey extension. The design of the survey is briefly reviewed, and the results are presented graphically. Vast low-density voids similar to the void in Bootes are found, almost completely surrounded by thin sheets of galaxies. Also discussed are the implications of the results for the survey sampling problem, the two-point correlation function of the galaxy distribution, the possibility of detecting large-scale coherent flows, theoretical models of large-scale structure, and the identification of groups and clusters of galaxies.

  4. Management of large-scale technology

    NASA Technical Reports Server (NTRS)

    Levine, A.

    1985-01-01

    Two major themes are addressed in this assessment of the management of large-scale NASA programs: (1) how a high technology agency was a decade marked by a rapid expansion of funds and manpower in the first half and almost as rapid contraction in the second; and (2) how NASA combined central planning and control with decentralized project execution.

  5. A Large Scale Computer Terminal Output Controller.

    ERIC Educational Resources Information Center

    Tucker, Paul Thomas

    This paper describes the design and implementation of a large scale computer terminal output controller which supervises the transfer of information from a Control Data 6400 Computer to a PLATO IV data network. It discusses the cost considerations leading to the selection of educational television channels rather than telephone lines for…

  6. Large Scale Commodity Clusters for Lattice QCD

    SciTech Connect

    A. Pochinsky; W. Akers; R. Brower; J. Chen; P. Dreher; R. Edwards; S. Gottlieb; D. Holmgren; P. Mackenzie; J. Negele; D. Richards; J. Simone; W. Watson

    2002-06-01

    We describe the construction of large scale clusters for lattice QCD computing being developed under the umbrella of the U.S. DoE SciDAC initiative. We discuss the study of floating point and network performance that drove the design of the cluster, and present our plans for future multi-Terascale facilities.

  7. Evaluating Large-Scale Interactive Radio Programmes

    ERIC Educational Resources Information Center

    Potter, Charles; Naidoo, Gordon

    2009-01-01

    This article focuses on the challenges involved in conducting evaluations of interactive radio programmes in South Africa with large numbers of schools, teachers, and learners. It focuses on the role such large-scale evaluation has played during the South African radio learning programme's development stage, as well as during its subsequent…

  8. ARPACK: Solving large scale eigenvalue problems

    NASA Astrophysics Data System (ADS)

    Lehoucq, Rich; Maschhoff, Kristi; Sorensen, Danny; Yang, Chao

    2013-11-01

    ARPACK is a collection of Fortran77 subroutines designed to solve large scale eigenvalue problems. The package is designed to compute a few eigenvalues and corresponding eigenvectors of a general n by n matrix A. It is most appropriate for large sparse or structured matrices A where structured means that a matrix-vector product w

  9. Large-scale CFB combustion demonstration project

    SciTech Connect

    Nielsen, P.T.; Hebb, J.L.; Aquino, R.

    1998-07-01

    The Jacksonville Electric Authority's large-scale CFB demonstration project is described. Given the early stage of project development, the paper focuses on the project organizational structure, its role within the Department of Energy's Clean Coal Technology Demonstration Program, and the projected environmental performance. A description of the CFB combustion process in included.

  10. Large-scale CFB combustion demonstration project

    SciTech Connect

    Nielsen, P.T.; Hebb, J.L.; Aquino, R.

    1998-04-01

    The Jacksonville Electric Authority`s large-scale CFB demonstration project is described. Given the early stage of project development, the paper focuses on the project organizational structure, its role within the Department of Energy`s Clean Coal Technology Demonstration Program, and the projected environmental performance. A description of the CFB combustion process is included.

  11. Defining essential genes and identifying virulence factors of Porphyromonas gingivalis by massively-parallel sequencing of transposon libraries (Tn-seq)

    PubMed Central

    Klein, Brian A.; Duncan, Margaret J.; Hu, Linden T.

    2016-01-01

    Summary Porphyromonas gingivalis is a keystone pathogen in the development and progression of periodontal disease. Obstacles to the development of saturated transposon libraries have previously limited transposon mutant-based screens as well as essential gene studies. We have developed a system for efficient transposon mutagenesis of P. gingivalis using a modified mariner transposon. Tn-seq is a technique that allows for quantitative assessment of individual mutants within a transposon mutant library by sequencing the transposon-genome junctions and then compiling mutant presence by mapping to a base genome. Using Tn-seq, it is possible to quickly define all the insertional mutants in a library and thus identify non-essential genes under the conditions in which the library was produced. Identification of fitness of individual mutants under specific conditions can be performed by exposing the library to selective pressures. PMID:25636611

  12. Fishing for answers with transposons.

    PubMed

    Wadman, Shannon A; Clark, Karl J; Hackett, Perry B

    2005-01-01

    Transposons are one means that nature has used to introduce new genetic material into chromosomes of organisms from every kingdom. They have been extensively used in prokaryotic and lower eukaryotic systems, but until recently there was no transposon that had significant activity in vertebrates. The Sleeping Beauty (SB) transposon system was developed to direct the integration of precise DNA sequences into chromosomes. The SB system was derived from salmonid sequences that had been inactive for more than 10 million years. SB transposons have been used for two principle uses--as a vector for transgenesis and as a method for introducing various trap vectors into (gene-trap) or in the neighborhood of (enhancer-trap) genes to identify their functions. Results of these studies show that SB-mediated transgenesis is more efficient than that by injection of simple plasmids and that expression of transgenesis is stable and reliable following passage through the germline. PMID:15864468

  13. Fractals and cosmological large-scale structure

    NASA Technical Reports Server (NTRS)

    Luo, Xiaochun; Schramm, David N.

    1992-01-01

    Observations of galaxy-galaxy and cluster-cluster correlations as well as other large-scale structure can be fit with a 'limited' fractal with dimension D of about 1.2. This is not a 'pure' fractal out to the horizon: the distribution shifts from power law to random behavior at some large scale. If the observed patterns and structures are formed through an aggregation growth process, the fractal dimension D can serve as an interesting constraint on the properties of the stochastic motion responsible for limiting the fractal structure. In particular, it is found that the observed fractal should have grown from two-dimensional sheetlike objects such as pancakes, domain walls, or string wakes. This result is generic and does not depend on the details of the growth process.

  14. Condition Monitoring of Large-Scale Facilities

    NASA Technical Reports Server (NTRS)

    Hall, David L.

    1999-01-01

    This document provides a summary of the research conducted for the NASA Ames Research Center under grant NAG2-1182 (Condition-Based Monitoring of Large-Scale Facilities). The information includes copies of view graphs presented at NASA Ames in the final Workshop (held during December of 1998), as well as a copy of a technical report provided to the COTR (Dr. Anne Patterson-Hine) subsequent to the workshop. The material describes the experimental design, collection of data, and analysis results associated with monitoring the health of large-scale facilities. In addition to this material, a copy of the Pennsylvania State University Applied Research Laboratory data fusion visual programming tool kit was also provided to NASA Ames researchers.

  15. Large-scale extraction of proteins.

    PubMed

    Cunha, Teresa; Aires-Barros, Raquel

    2002-01-01

    The production of foreign proteins using selected host with the necessary posttranslational modifications is one of the key successes in modern biotechnology. This methodology allows the industrial production of proteins that otherwise are produced in small quantities. However, the separation and purification of these proteins from the fermentation media constitutes a major bottleneck for the widespread commercialization of recombinant proteins. The major production costs (50-90%) for typical biological product resides in the purification strategy. There is a need for efficient, effective, and economic large-scale bioseparation techniques, to achieve high purity and high recovery, while maintaining the biological activity of the molecule. Aqueous two-phase systems (ATPS) allow process integration as simultaneously separation and concentration of the target protein is achieved, with posterior removal and recycle of the polymer. The ease of scale-up combined with the high partition coefficients obtained allow its potential application in large-scale downstream processing of proteins produced by fermentation. The equipment and the methodology for aqueous two-phase extraction of proteins on a large scale using mixer-settlerand column contractors are described. The operation of the columns, either stagewise or differential, are summarized. A brief description of the methods used to account for mass transfer coefficients, hydrodynamics parameters of hold-up, drop size, and velocity, back mixing in the phases, and flooding performance, required for column design, is also provided. PMID:11876297

  16. Large scale processes in the solar nebula.

    NASA Astrophysics Data System (ADS)

    Boss, A. P.

    Most proposed chondrule formation mechanisms involve processes occurring inside the solar nebula, so the large scale (roughly 1 to 10 AU) structure of the nebula is of general interest for any chrondrule-forming mechanism. Chondrules and Ca, Al-rich inclusions (CAIs) might also have been formed as a direct result of the large scale structure of the nebula, such as passage of material through high temperature regions. While recent nebula models do predict the existence of relatively hot regions, the maximum temperatures in the inner planet region may not be high enough to account for chondrule or CAI thermal processing, unless the disk mass is considerably greater than the minimum mass necessary to restore the planets to solar composition. Furthermore, it does not seem to be possible to achieve both rapid heating and rapid cooling of grain assemblages in such a large scale furnace. However, if the accretion flow onto the nebula surface is clumpy, as suggested by observations of variability in young stars, then clump-disk impacts might be energetic enough to launch shock waves which could propagate through the nebula to the midplane, thermally processing any grain aggregates they encounter, and leaving behind a trail of chondrules.

  17. Transposon tagging of disease resistance genes

    SciTech Connect

    Michelmore, R.W. . Dept. of Physics)

    1989-01-01

    We are developing a transposon mutagenesis system for lettuce to clone genes for resistance to the fungal pathogen, Bremia lactucae. Activity of heterologous transposons is being studied in transgenic plants. Southern analysis of T{sub 1} and T{sub 2} plants containing Tam3 from Antirrhinum provided ambiguous results. Multiple endonuclease digests indicated that transposition had occurred; however, in no plant were all endonuclease digests consistent with a simple excision event. Southern or PCR analysis of over 50 plans containing Ac from maize have also failed to reveal clear evidence of transposition; this is contrast to experiments by others with the same constructs who have observed high rates of Ac excision in other plant species. Nearly all of 65 T{sub 2} families containing Ac interrupting a chimeric streptomycin resistance gene (Courtesy J. Jones, Sainsbury Lab., UK) clearly segregated for streptomycin resistance. Southern analyses, however, showed no evidence of transposition, indicating restoration of a functional message by other mechanisms, possibly mRNA processing. Transgenic plants have also been generated containing CaMV 35S or hsp70 promoters fused to transposase coding sequences or a Ds element interrupting a chimeric GUS gene (Courtesy M. Lassner, UC Davis). F{sub 1} plants containing both constructs were analyzed for transposition. Only two plants containing both constructs were obtained from 48 progeny, far fewer than expected, and neither showed evidence of transposition in Southerns and GUS assays. We are currently constructing further chimeric transposase fusions. To test for the stability of the targeted disease resistance genes, 50,000 F{sub 1} plants heterozygous for three resistance genes were generated; no mutants have been identified in the 5000 so far screened.

  18. DNA transposon Hermes inserts into DNA in nucleosome-free regions in vivo.

    PubMed

    Gangadharan, Sunil; Mularoni, Loris; Fain-Thornton, Jennifer; Wheelan, Sarah J; Craig, Nancy L

    2010-12-21

    Transposons are mobile genetic elements that are an important source of genetic variation and are useful tools for genome engineering, mutagenesis screens, and vectors for transgenesis including gene therapy. We have used second-generation sequencing to analyze ≈2 × 10(5) unique de novo transposon insertion sites of the transposon Hermes in the Saccharomyces cerevisiae genome from both in vitro transposition reactions by using purified yeast genomic DNA, to better characterize intrinsic sequence specificity, and sites recovered from in vivo transposition events, to characterize the effect of intracellular factors such as chromatin on target site selection. We find that Hermes transposon targeting in vivo is profoundly affected by chromatin structure: The subset of genome-wide target sites used in vivo is strongly associated (P < 2e-16 by Fisher's exact test) with nucleosome-free chromatin. Our characterization of the insertion site preferences of Hermes not only assists in the future use of this transposon as a molecular biology tool but also establishes methods to more fully determine targeting mechanisms of other transposons. We have also discovered a long-range sequence motif that defines S. cerevisiae nucleosome-free regions. PMID:21131571

  19. DNA transposon Hermes inserts into DNA in nucleosome-free regions in vivo

    PubMed Central

    Gangadharan, Sunil; Mularoni, Loris; Fain-Thornton, Jennifer; Wheelan, Sarah J.; Craig, Nancy L.

    2010-01-01

    Transposons are mobile genetic elements that are an important source of genetic variation and are useful tools for genome engineering, mutagenesis screens, and vectors for transgenesis including gene therapy. We have used second-generation sequencing to analyze ≈2 × 105 unique de novo transposon insertion sites of the transposon Hermes in the Saccharomyces cerevisiae genome from both in vitro transposition reactions by using purified yeast genomic DNA, to better characterize intrinsic sequence specificity, and sites recovered from in vivo transposition events, to characterize the effect of intracellular factors such as chromatin on target site selection. We find that Hermes transposon targeting in vivo is profoundly affected by chromatin structure: The subset of genome-wide target sites used in vivo is strongly associated (P < 2e-16 by Fisher's exact test) with nucleosome-free chromatin. Our characterization of the insertion site preferences of Hermes not only assists in the future use of this transposon as a molecular biology tool but also establishes methods to more fully determine targeting mechanisms of other transposons. We have also discovered a long-range sequence motif that defines S. cerevisiae nucleosome-free regions. PMID:21131571

  20. Colloquium: Large scale simulations on GPU clusters

    NASA Astrophysics Data System (ADS)

    Bernaschi, Massimo; Bisson, Mauro; Fatica, Massimiliano

    2015-06-01

    Graphics processing units (GPU) are currently used as a cost-effective platform for computer simulations and big-data processing. Large scale applications require that multiple GPUs work together but the efficiency obtained with cluster of GPUs is, at times, sub-optimal because the GPU features are not exploited at their best. We describe how it is possible to achieve an excellent efficiency for applications in statistical mechanics, particle dynamics and networks analysis by using suitable memory access patterns and mechanisms like CUDA streams, profiling tools, etc. Similar concepts and techniques may be applied also to other problems like the solution of Partial Differential Equations.

  1. Experimental Simulations of Large-Scale Collisions

    NASA Technical Reports Server (NTRS)

    Housen, Kevin R.

    2002-01-01

    This report summarizes research on the effects of target porosity on the mechanics of impact cratering. Impact experiments conducted on a centrifuge provide direct simulations of large-scale cratering on porous asteroids. The experiments show that large craters in porous materials form mostly by compaction, with essentially no deposition of material into the ejecta blanket that is a signature of cratering in less-porous materials. The ratio of ejecta mass to crater mass is shown to decrease with increasing crater size or target porosity. These results are consistent with the observation that large closely-packed craters on asteroid Mathilde appear to have formed without degradation to earlier craters.

  2. Nonthermal Components in the Large Scale Structure

    NASA Astrophysics Data System (ADS)

    Miniati, Francesco

    2004-12-01

    I address the issue of nonthermal processes in the large scale structure of the universe. After reviewing the properties of cosmic shocks and their role as particle accelerators, I discuss the main observational results, from radio to γ-ray and describe the processes that are thought be responsible for the observed nonthermal emissions. Finally, I emphasize the important role of γ-ray astronomy for the progress in the field. Non detections at these photon energies have already allowed us important conclusions. Future observations will tell us more about the physics of the intracluster medium, shocks dissipation and CR acceleration.

  3. Large-Scale PV Integration Study

    SciTech Connect

    Lu, Shuai; Etingov, Pavel V.; Diao, Ruisheng; Ma, Jian; Samaan, Nader A.; Makarov, Yuri V.; Guo, Xinxin; Hafen, Ryan P.; Jin, Chunlian; Kirkham, Harold; Shlatz, Eugene; Frantzis, Lisa; McClive, Timothy; Karlson, Gregory; Acharya, Dhruv; Ellis, Abraham; Stein, Joshua; Hansen, Clifford; Chadliev, Vladimir; Smart, Michael; Salgo, Richard; Sorensen, Rahn; Allen, Barbara; Idelchik, Boris

    2011-07-29

    This research effort evaluates the impact of large-scale photovoltaic (PV) and distributed generation (DG) output on NV Energy’s electric grid system in southern Nevada. It analyzes the ability of NV Energy’s generation to accommodate increasing amounts of utility-scale PV and DG, and the resulting cost of integrating variable renewable resources. The study was jointly funded by the United States Department of Energy and NV Energy, and conducted by a project team comprised of industry experts and research scientists from Navigant Consulting Inc., Sandia National Laboratories, Pacific Northwest National Laboratory and NV Energy.

  4. Large-scale planar lightwave circuits

    NASA Astrophysics Data System (ADS)

    Bidnyk, Serge; Zhang, Hua; Pearson, Matt; Balakrishnan, Ashok

    2011-01-01

    By leveraging advanced wafer processing and flip-chip bonding techniques, we have succeeded in hybrid integrating a myriad of active optical components, including photodetectors and laser diodes, with our planar lightwave circuit (PLC) platform. We have combined hybrid integration of active components with monolithic integration of other critical functions, such as diffraction gratings, on-chip mirrors, mode-converters, and thermo-optic elements. Further process development has led to the integration of polarization controlling functionality. Most recently, all these technological advancements have been combined to create large-scale planar lightwave circuits that comprise hundreds of optical elements integrated on chips less than a square inch in size.

  5. Neutrinos and large-scale structure

    SciTech Connect

    Eisenstein, Daniel J.

    2015-07-15

    I review the use of cosmological large-scale structure to measure properties of neutrinos and other relic populations of light relativistic particles. With experiments to measure the anisotropies of the cosmic microwave anisotropies and the clustering of matter at low redshift, we now have securely measured a relativistic background with density appropriate to the cosmic neutrino background. Our limits on the mass of the neutrino continue to shrink. Experiments coming in the next decade will greatly improve the available precision on searches for the energy density of novel relativistic backgrounds and the mass of neutrinos.

  6. Large scale phononic metamaterials for seismic isolation

    SciTech Connect

    Aravantinos-Zafiris, N.; Sigalas, M. M.

    2015-08-14

    In this work, we numerically examine structures that could be characterized as large scale phononic metamaterials. These novel structures could have band gaps in the frequency spectrum of seismic waves when their dimensions are chosen appropriately, thus raising the belief that they could be serious candidates for seismic isolation structures. Different and easy to fabricate structures were examined made from construction materials such as concrete and steel. The well-known finite difference time domain method is used in our calculations in order to calculate the band structures of the proposed metamaterials.

  7. Transposon tagging and the study of root development in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Tsugeki, R.; Olson, M. L.; Fedoroff, N. V.

    1998-01-01

    The maize Ac-Ds transposable element family has been used as the basis of transposon mutagenesis systems that function in a variety of plants, including Arabidopsis. We have developed modified transposons and methods which simplify the detection, cloning and analysis of insertion mutations. We have identified and are analyzing two plant lines in which genes expressed either in the root cap cells or in the quiescent cells, cortex/endodermal initial cells and columella cells of the root cap have been tagged with a transposon carrying a reporter gene. A gene expressed in root cap cells tagged with an enhancer-trap Ds was isolated and its corresponding EST cDNA was identified. Nucleotide and deduced amino acid sequences of the gene show no significant similarity to other genes in the database. Genetic ablation experiments have been done by fusing a root cap-specific promoter to the diphtheria toxin A-chain gene and introducing the fusion construct into Arabidopsis plants. We find that in addition to eliminating gravitropism, root cap ablation inhibits elongation of roots by lowering root meristematic activities.

  8. Large-scale Globally Propagating Coronal Waves

    NASA Astrophysics Data System (ADS)

    Warmuth, Alexander

    2015-09-01

    Large-scale, globally propagating wave-like disturbances have been observed in the solar chromosphere and by inference in the corona since the 1960s. However, detailed analysis of these phenomena has only been conducted since the late 1990s. This was prompted by the availability of high-cadence coronal imaging data from numerous spaced-based instruments, which routinely show spectacular globally propagating bright fronts. Coronal waves, as these perturbations are usually referred to, have now been observed in a wide range of spectral channels, yielding a wealth of information. Many findings have supported the "classical" interpretation of the disturbances: fast-mode MHD waves or shocks that are propagating in the solar corona. However, observations that seemed inconsistent with this picture have stimulated the development of alternative models in which "pseudo waves" are generated by magnetic reconfiguration in the framework of an expanding coronal mass ejection. This has resulted in a vigorous debate on the physical nature of these disturbances. This review focuses on demonstrating how the numerous observational findings of the last one and a half decades can be used to constrain our models of large-scale coronal waves, and how a coherent physical understanding of these disturbances is finally emerging.

  9. Local gravity and large-scale structure

    NASA Technical Reports Server (NTRS)

    Juszkiewicz, Roman; Vittorio, Nicola; Wyse, Rosemary F. G.

    1990-01-01

    The magnitude and direction of the observed dipole anisotropy of the galaxy distribution can in principle constrain the amount of large-scale power present in the spectrum of primordial density fluctuations. This paper confronts the data, provided by a recent redshift survey of galaxies detected by the IRAS satellite, with the predictions of two cosmological models with very different levels of large-scale power: the biased Cold Dark Matter dominated model (CDM) and a baryon-dominated model (BDM) with isocurvature initial conditions. Model predictions are investigated for the Local Group peculiar velocity, v(R), induced by mass inhomogeneities distributed out to a given radius, R, for R less than about 10,000 km/s. Several convergence measures for v(R) are developed, which can become powerful cosmological tests when deep enough samples become available. For the present data sets, the CDM and BDM predictions are indistinguishable at the 2 sigma level and both are consistent with observations. A promising discriminant between cosmological models is the misalignment angle between v(R) and the apex of the dipole anisotropy of the microwave background.

  10. New soybean variants in oil composition identified by large scale mutagenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To identify loci involved in the determination of fatty acid content in mature soybean seeds, we subject 3400 seed samples to fatty acid profiling by gas chromatography. In the mutagenized population, a wide range of variation in the content of each of the five major fatty acids was observed. 74 mu...

  11. Transposon tagging of disease resistance genes. Final report, May 1, 1988--April 30, 1993

    SciTech Connect

    Michelmore, R.

    1994-09-01

    The goal of this project was to develop a transposon mutagenesis system for lettuce and to clone and characterize disease resistance genes by transposon tagging. The majority of studies were conducted with the Ac/Ds System. Researchers made and tested several constructs as well as utilized constructions shown to be functional in other plant species. Researchers demonstrated movement of Ac and DS in lettuce; however, they transposed at much lower frequencies in lettuce than in other plant species. Therefore, further manipulation of the system, particularly for flower specific expression of transposase, is required before a routine transposon system is available for lettuce. Populations of lettuce were generated and screened to test for the stability of resistance genes and several spontaneous mutations were isolated. Researchers also identified a resistance gene mutant in plants transformed with a Ds element and chimeric transposase gene. This is currently being characterized in detail.

  12. Large-Scale Organization of Glycosylation Networks

    NASA Astrophysics Data System (ADS)

    Kim, Pan-Jun; Lee, Dong-Yup; Jeong, Hawoong

    2009-03-01

    Glycosylation is a highly complex process to produce a diverse repertoire of cellular glycans that are frequently attached to proteins and lipids. Glycans participate in fundamental biological processes including molecular trafficking and clearance, cell proliferation and apoptosis, developmental biology, immune response, and pathogenesis. N-linked glycans found on proteins are formed by sequential attachments of monosaccharides with the help of a relatively small number of enzymes. Many of these enzymes can accept multiple N-linked glycans as substrates, thus generating a large number of glycan intermediates and their intermingled pathways. Motivated by the quantitative methods developed in complex network research, we investigate the large-scale organization of such N-glycosylation pathways in a mammalian cell. The uncovered results give the experimentally-testable predictions for glycosylation process, and can be applied to the engineering of therapeutic glycoproteins.

  13. Large-scale databases of proper names.

    PubMed

    Conley, P; Burgess, C; Hage, D

    1999-05-01

    Few tools for research in proper names have been available--specifically, there is no large-scale corpus of proper names. Two corpora of proper names were constructed, one based on U.S. phone book listings, the other derived from a database of Usenet text. Name frequencies from both corpora were compared with human subjects' reaction times (RTs) to the proper names in a naming task. Regression analysis showed that the Usenet frequencies contributed to predictions of human RT, whereas phone book frequencies did not. In addition, semantic neighborhood density measures derived from the HAL corpus were compared with the subjects' RTs and found to be a better predictor of RT than was frequency in either corpus. These new corpora are freely available on line for download. Potentials for these corpora range from using the names as stimuli in experiments to using the corpus data in software applications. PMID:10495803

  14. Estimation of large-scale dimension densities.

    PubMed

    Raab, C; Kurths, J

    2001-07-01

    We propose a technique to calculate large-scale dimension densities in both higher-dimensional spatio-temporal systems and low-dimensional systems from only a few data points, where known methods usually have an unsatisfactory scaling behavior. This is mainly due to boundary and finite-size effects. With our rather simple method, we normalize boundary effects and get a significant correction of the dimension estimate. This straightforward approach is based on rather general assumptions. So even weak coherent structures obtained from small spatial couplings can be detected with this method, which is impossible by using the Lyapunov-dimension density. We demonstrate the efficiency of our technique for coupled logistic maps, coupled tent maps, the Lorenz attractor, and the Roessler attractor. PMID:11461376

  15. The challenge of large-scale structure

    NASA Astrophysics Data System (ADS)

    Gregory, S. A.

    1996-03-01

    The tasks that I have assumed for myself in this presentation include three separate parts. The first, appropriate to the particular setting of this meeting, is to review the basic work of the founding of this field; the appropriateness comes from the fact that W. G. Tifft made immense contributions that are not often realized by the astronomical community. The second task is to outline the general tone of the observational evidence for large scale structures. (Here, in particular, I cannot claim to be complete. I beg forgiveness from any workers who are left out by my oversight for lack of space and time.) The third task is to point out some of the major aspects of the field that may represent the clues by which some brilliant sleuth will ultimately figure out how galaxies formed.

  16. Engineering management of large scale systems

    NASA Technical Reports Server (NTRS)

    Sanders, Serita; Gill, Tepper L.; Paul, Arthur S.

    1989-01-01

    The organization of high technology and engineering problem solving, has given rise to an emerging concept. Reasoning principles for integrating traditional engineering problem solving with system theory, management sciences, behavioral decision theory, and planning and design approaches can be incorporated into a methodological approach to solving problems with a long range perspective. Long range planning has a great potential to improve productivity by using a systematic and organized approach. Thus, efficiency and cost effectiveness are the driving forces in promoting the organization of engineering problems. Aspects of systems engineering that provide an understanding of management of large scale systems are broadly covered here. Due to the focus and application of research, other significant factors (e.g., human behavior, decision making, etc.) are not emphasized but are considered.

  17. Large scale cryogenic fluid systems testing

    NASA Technical Reports Server (NTRS)

    1992-01-01

    NASA Lewis Research Center's Cryogenic Fluid Systems Branch (CFSB) within the Space Propulsion Technology Division (SPTD) has the ultimate goal of enabling the long term storage and in-space fueling/resupply operations for spacecraft and reusable vehicles in support of space exploration. Using analytical modeling, ground based testing, and on-orbit experimentation, the CFSB is studying three primary categories of fluid technology: storage, supply, and transfer. The CFSB is also investigating fluid handling, advanced instrumentation, and tank structures and materials. Ground based testing of large-scale systems is done using liquid hydrogen as a test fluid at the Cryogenic Propellant Tank Facility (K-site) at Lewis' Plum Brook Station in Sandusky, Ohio. A general overview of tests involving liquid transfer, thermal control, pressure control, and pressurization is given.

  18. Batteries for Large Scale Energy Storage

    SciTech Connect

    Soloveichik, Grigorii L.

    2011-07-15

    In recent years, with the deployment of renewable energy sources, advances in electrified transportation, and development in smart grids, the markets for large-scale stationary energy storage have grown rapidly. Electrochemical energy storage methods are strong candidate solutions due to their high energy density, flexibility, and scalability. This review provides an overview of mature and emerging technologies for secondary and redox flow batteries. New developments in the chemistry of secondary and flow batteries as well as regenerative fuel cells are also considered. Advantages and disadvantages of current and prospective electrochemical energy storage options are discussed. The most promising technologies in the short term are high-temperature sodium batteries with β”-alumina electrolyte, lithium-ion batteries, and flow batteries. Regenerative fuel cells and lithium metal batteries with high energy density require further research to become practical.

  19. Large scale water lens for solar concentration.

    PubMed

    Mondol, A S; Vogel, B; Bastian, G

    2015-06-01

    Properties of large scale water lenses for solar concentration were investigated. These lenses were built from readily available materials, normal tap water and hyper-elastic linear low density polyethylene foil. Exposed to sunlight, the focal lengths and light intensities in the focal spot were measured and calculated. Their optical properties were modeled with a raytracing software based on the lens shape. We have achieved a good match of experimental and theoretical data by considering wavelength dependent concentration factor, absorption and focal length. The change in light concentration as a function of water volume was examined via the resulting load on the foil and the corresponding change of shape. The latter was extracted from images and modeled by a finite element simulation. PMID:26072893

  20. Large Scale Quantum Simulations of Nuclear Pasta

    NASA Astrophysics Data System (ADS)

    Fattoyev, Farrukh J.; Horowitz, Charles J.; Schuetrumpf, Bastian

    2016-03-01

    Complex and exotic nuclear geometries collectively referred to as ``nuclear pasta'' are expected to naturally exist in the crust of neutron stars and in supernovae matter. Using a set of self-consistent microscopic nuclear energy density functionals we present the first results of large scale quantum simulations of pasta phases at baryon densities 0 . 03 < ρ < 0 . 10 fm-3, proton fractions 0 . 05

  1. Large-scale simulations of reionization

    SciTech Connect

    Kohler, Katharina; Gnedin, Nickolay Y.; Hamilton, Andrew J.S.; /JILA, Boulder

    2005-11-01

    We use cosmological simulations to explore the large-scale effects of reionization. Since reionization is a process that involves a large dynamic range--from galaxies to rare bright quasars--we need to be able to cover a significant volume of the universe in our simulation without losing the important small scale effects from galaxies. Here we have taken an approach that uses clumping factors derived from small scale simulations to approximate the radiative transfer on the sub-cell scales. Using this technique, we can cover a simulation size up to 1280h{sup -1} Mpc with 10h{sup -1} Mpc cells. This allows us to construct synthetic spectra of quasars similar to observed spectra of SDSS quasars at high redshifts and compare them to the observational data. These spectra can then be analyzed for HII region sizes, the presence of the Gunn-Peterson trough, and the Lyman-{alpha} forest.

  2. The XMM Large Scale Structure Survey

    NASA Astrophysics Data System (ADS)

    Pierre, Marguerite

    2005-10-01

    We propose to complete, by an additional 5 deg2, the XMM-LSS Survey region overlying the Spitzer/SWIRE field. This field already has CFHTLS and Integral coverage, and will encompass about 10 deg2. The resulting multi-wavelength medium-depth survey, which complements XMM and Chandra deep surveys, will provide a unique view of large-scale structure over a wide range of redshift, and will show active galaxies in the full range of environments. The complete coverage by optical and IR surveys provides high-quality photometric redshifts, so that cosmological results can quickly be extracted. In the spirit of a Legacy survey, we will make the raw X-ray data immediately public. Multi-band catalogues and images will also be made available on short time scales.

  3. Grid sensitivity capability for large scale structures

    NASA Technical Reports Server (NTRS)

    Nagendra, Gopal K.; Wallerstein, David V.

    1989-01-01

    The considerations and the resultant approach used to implement design sensitivity capability for grids into a large scale, general purpose finite element system (MSC/NASTRAN) are presented. The design variables are grid perturbations with a rather general linking capability. Moreover, shape and sizing variables may be linked together. The design is general enough to facilitate geometric modeling techniques for generating design variable linking schemes in an easy and straightforward manner. Test cases have been run and validated by comparison with the overall finite difference method. The linking of a design sensitivity capability for shape variables in MSC/NASTRAN with an optimizer would give designers a powerful, automated tool to carry out practical optimization design of real life, complicated structures.

  4. Large-Scale Astrophysical Visualization on Smartphones

    NASA Astrophysics Data System (ADS)

    Becciani, U.; Massimino, P.; Costa, A.; Gheller, C.; Grillo, A.; Krokos, M.; Petta, C.

    2011-07-01

    Nowadays digital sky surveys and long-duration, high-resolution numerical simulations using high performance computing and grid systems produce multidimensional astrophysical datasets in the order of several Petabytes. Sharing visualizations of such datasets within communities and collaborating research groups is of paramount importance for disseminating results and advancing astrophysical research. Moreover educational and public outreach programs can benefit greatly from novel ways of presenting these datasets by promoting understanding of complex astrophysical processes, e.g., formation of stars and galaxies. We have previously developed VisIVO Server, a grid-enabled platform for high-performance large-scale astrophysical visualization. This article reviews the latest developments on VisIVO Web, a custom designed web portal wrapped around VisIVO Server, then introduces VisIVO Smartphone, a gateway connecting VisIVO Web and data repositories for mobile astrophysical visualization. We discuss current work and summarize future developments.

  5. Estimation of large-scale dimension densities

    NASA Astrophysics Data System (ADS)

    Raab, Corinna; Kurths, Jürgen

    2001-07-01

    We propose a technique to calculate large-scale dimension densities in both higher-dimensional spatio-temporal systems and low-dimensional systems from only a few data points, where known methods usually have an unsatisfactory scaling behavior. This is mainly due to boundary and finite-size effects. With our rather simple method, we normalize boundary effects and get a significant correction of the dimension estimate. This straightforward approach is based on rather general assumptions. So even weak coherent structures obtained from small spatial couplings can be detected with this method, which is impossible by using the Lyapunov-dimension density. We demonstrate the efficiency of our technique for coupled logistic maps, coupled tent maps, the Lorenz attractor, and the Roessler attractor.

  6. Supporting large-scale computational science

    SciTech Connect

    Musick, R

    1998-10-01

    A study has been carried out to determine the feasibility of using commercial database management systems (DBMSs) to support large-scale computational science. Conventional wisdom in the past has been that DBMSs are too slow for such data. Several events over the past few years have muddied the clarity of this mindset: 1. 2. 3. 4. Several commercial DBMS systems have demonstrated storage and ad-hoc quer access to Terabyte data sets. Several large-scale science teams, such as EOSDIS [NAS91], high energy physics [MM97] and human genome [Kin93] have adopted (or make frequent use of) commercial DBMS systems as the central part of their data management scheme. Several major DBMS vendors have introduced their first object-relational products (ORDBMSs), which have the potential to support large, array-oriented data. In some cases, performance is a moot issue. This is true in particular if the performance of legacy applications is not reduced while new, albeit slow, capabilities are added to the system. The basic assessment is still that DBMSs do not scale to large computational data. However, many of the reasons have changed, and there is an expiration date attached to that prognosis. This document expands on this conclusion, identifies the advantages and disadvantages of various commercial approaches, and describes the studies carried out in exploring this area. The document is meant to be brief, technical and informative, rather than a motivational pitch. The conclusions within are very likely to become outdated within the next 5-7 years, as market forces will have a significant impact on the state of the art in scientific data management over the next decade.

  7. Supporting large-scale computational science

    SciTech Connect

    Musick, R., LLNL

    1998-02-19

    Business needs have driven the development of commercial database systems since their inception. As a result, there has been a strong focus on supporting many users, minimizing the potential corruption or loss of data, and maximizing performance metrics like transactions per second, or TPC-C and TPC-D results. It turns out that these optimizations have little to do with the needs of the scientific community, and in particular have little impact on improving the management and use of large-scale high-dimensional data. At the same time, there is an unanswered need in the scientific community for many of the benefits offered by a robust DBMS. For example, tying an ad-hoc query language such as SQL together with a visualization toolkit would be a powerful enhancement to current capabilities. Unfortunately, there has been little emphasis or discussion in the VLDB community on this mismatch over the last decade. The goal of the paper is to identify the specific issues that need to be resolved before large-scale scientific applications can make use of DBMS products. This topic is addressed in the context of an evaluation of commercial DBMS technology applied to the exploration of data generated by the Department of Energy`s Accelerated Strategic Computing Initiative (ASCI). The paper describes the data being generated for ASCI as well as current capabilities for interacting with and exploring this data. The attraction of applying standard DBMS technology to this domain is discussed, as well as the technical and business issues that currently make this an infeasible solution.

  8. Large-scale sequential quadratic programming algorithms

    SciTech Connect

    Eldersveld, S.K.

    1992-09-01

    The problem addressed is the general nonlinear programming problem: finding a local minimizer for a nonlinear function subject to a mixture of nonlinear equality and inequality constraints. The methods studied are in the class of sequential quadratic programming (SQP) algorithms, which have previously proved successful for problems of moderate size. Our goal is to devise an SQP algorithm that is applicable to large-scale optimization problems, using sparse data structures and storing less curvature information but maintaining the property of superlinear convergence. The main features are: 1. The use of a quasi-Newton approximation to the reduced Hessian of the Lagrangian function. Only an estimate of the reduced Hessian matrix is required by our algorithm. The impact of not having available the full Hessian approximation is studied and alternative estimates are constructed. 2. The use of a transformation matrix Q. This allows the QP gradient to be computed easily when only the reduced Hessian approximation is maintained. 3. The use of a reduced-gradient form of the basis for the null space of the working set. This choice of basis is more practical than an orthogonal null-space basis for large-scale problems. The continuity condition for this choice is proven. 4. The use of incomplete solutions of quadratic programming subproblems. Certain iterates generated by an active-set method for the QP subproblem are used in place of the QP minimizer to define the search direction for the nonlinear problem. An implementation of the new algorithm has been obtained by modifying the code MINOS. Results and comparisons with MINOS and NPSOL are given for the new algorithm on a set of 92 test problems.

  9. Introducing Large-Scale Innovation in Schools

    NASA Astrophysics Data System (ADS)

    Sotiriou, Sofoklis; Riviou, Katherina; Cherouvis, Stephanos; Chelioti, Eleni; Bogner, Franz X.

    2016-08-01

    Education reform initiatives tend to promise higher effectiveness in classrooms especially when emphasis is given to e-learning and digital resources. Practical changes in classroom realities or school organization, however, are lacking. A major European initiative entitled Open Discovery Space (ODS) examined the challenge of modernizing school education via a large-scale implementation of an open-scale methodology in using technology-supported innovation. The present paper describes this innovation scheme which involved schools and teachers all over Europe, embedded technology-enhanced learning into wider school environments and provided training to teachers. Our implementation scheme consisted of three phases: (1) stimulating interest, (2) incorporating the innovation into school settings and (3) accelerating the implementation of the innovation. The scheme's impact was monitored for a school year using five indicators: leadership and vision building, ICT in the curriculum, development of ICT culture, professional development support, and school resources and infrastructure. Based on about 400 schools, our study produced four results: (1) The growth in digital maturity was substantial, even for previously high scoring schools. This was even more important for indicators such as vision and leadership" and "professional development." (2) The evolution of networking is presented graphically, showing the gradual growth of connections achieved. (3) These communities became core nodes, involving numerous teachers in sharing educational content and experiences: One out of three registered users (36 %) has shared his/her educational resources in at least one community. (4) Satisfaction scores ranged from 76 % (offer of useful support through teacher academies) to 87 % (good environment to exchange best practices). Initiatives such as ODS add substantial value to schools on a large scale.

  10. Introducing Large-Scale Innovation in Schools

    NASA Astrophysics Data System (ADS)

    Sotiriou, Sofoklis; Riviou, Katherina; Cherouvis, Stephanos; Chelioti, Eleni; Bogner, Franz X.

    2016-02-01

    Education reform initiatives tend to promise higher effectiveness in classrooms especially when emphasis is given to e-learning and digital resources. Practical changes in classroom realities or school organization, however, are lacking. A major European initiative entitled Open Discovery Space (ODS) examined the challenge of modernizing school education via a large-scale implementation of an open-scale methodology in using technology-supported innovation. The present paper describes this innovation scheme which involved schools and teachers all over Europe, embedded technology-enhanced learning into wider school environments and provided training to teachers. Our implementation scheme consisted of three phases: (1) stimulating interest, (2) incorporating the innovation into school settings and (3) accelerating the implementation of the innovation. The scheme's impact was monitored for a school year using five indicators: leadership and vision building, ICT in the curriculum, development of ICT culture, professional development support, and school resources and infrastructure. Based on about 400 schools, our study produced four results: (1) The growth in digital maturity was substantial, even for previously high scoring schools. This was even more important for indicators such as vision and leadership" and "professional development." (2) The evolution of networking is presented graphically, showing the gradual growth of connections achieved. (3) These communities became core nodes, involving numerous teachers in sharing educational content and experiences: One out of three registered users (36 %) has shared his/her educational resources in at least one community. (4) Satisfaction scores ranged from 76 % (offer of useful support through teacher academies) to 87 % (good environment to exchange best practices). Initiatives such as ODS add substantial value to schools on a large scale.

  11. Generation of induced pluripotent stem cells from human foetal fibroblasts using the Sleeping Beauty transposon gene delivery system.

    PubMed

    Davis, Richard P; Nemes, Csilla; Varga, Eszter; Freund, Christian; Kosmidis, Georgios; Gkatzis, Konstantinos; de Jong, Danielle; Szuhai, Károly; Dinnyés, András; Mummery, Christine L

    2013-01-01

    Transposon gene delivery systems offer an alternative, non-viral-based approach to generate induced pluripotent stem cells (iPSCs). Here we used the Sleeping Beauty (SB) transposon to generate four human iPSC lines from foetal fibroblasts. In contrast to other gene delivery systems, the SB transposon does not exhibit an integration bias towards particular genetic elements, thereby reducing the risk of insertional mutagenesis. Furthermore, unlike the alternative transposon piggyBac, SB has no SB-like elements within the human genome, minimising the possibility of mobilising endogenous transposon elements. All iPSC lines exhibited the expected characteristics of pluripotent human cells, including the ability to differentiate to derivatives of all three germ layers in vitro. Re-expression of the SB transposase in the iPSCs after reprogramming resulted in the mobilisation of some of the transposons. These results indicate that the SB transposon system is a useful addition to methods for generating human iPSCs, both for basic and applied biomedical research, and in the context of future therapeutic application. PMID:23933400

  12. The Hermes transposon of Musca domestica and its use as a mutagen of Schizosaccharomyces pombe

    PubMed Central

    Park, Jung M.; Evertts, Adam G.; Levin, Henry L.

    2009-01-01

    Transposon mutagenesis allows for the discovery and characterization of genes by creating mutations that can be easily mapped and sequenced. Moreover, this method allows for a relatively unbiased approach to isolating genes of interest. Recently, a system of transposon based mutagenesis for Schizosaccharomyces pombe became available. This mutagenesis relies on Hermes, a DNA transposon from the house fly that readily integrates into the chromosomes of S. pombe. The Hermes system is distinct from the retrotransposons of S. pombe because it efficiently integrates into open reading frames. To mutagenize S. pombe, cells are transformed with a plasmid that contains a drug resistance marker flanked by the terminal inverted repeats of Hermes. The Hermes transposase expressed from a second plasmid excises the resistance marker with the inverted repeats and inserts this DNA into chromosomal sites. After S. pombe with these two plasmids grow 25 generations, approximately 2% of the cells contain insertions. Of the cells with insertions, 68% contain single integration events. The protocols listed here provide the detailed information necessary to mutagenize a strain of interest, screen for specific phenotypes, and sequence the positions of insertion. PMID:19450689

  13. The Hermes transposon of Musca domestica and its use as a mutagen of Schizosaccharomyces pombe.

    PubMed

    Park, Jung M; Evertts, Adam G; Levin, Henry L

    2009-11-01

    Transposon mutagenesis allows for the discovery and characterization of genes by creating mutations that can be easily mapped and sequenced. Moreover, this method allows for a relatively unbiased approach to isolating genes of interest. Recently, a system of transposon based mutagenesis for Schizosaccharomyces pombe became available. This mutagenesis relies on Hermes, a DNA transposon from the house fly that readily integrates into the chromosomes of S. pombe. The Hermes system is distinct from the retrotransposons of S. pombe because it efficiently integrates into open reading frames. To mutagenize S. pombe, cells are transformed with a plasmid that contains a drug resistance marker flanked by the terminal inverted repeats of Hermes. The Hermes transposase expressed from a second plasmid excises the resistance marker with the inverted repeats and inserts this DNA into chromosomal sites. After S. pombe with these two plasmids grow 25 generations, approximately 2% of the cells contain insertions. Of the cells with insertions, 68% contain single integration events. The protocols listed here provide the detailed information necessary to mutagenize a strain of interest, screen for specific phenotypes, and sequence the positions of insertion. PMID:19450689

  14. Gravity and large-scale nonlocal bias

    NASA Astrophysics Data System (ADS)

    Chan, Kwan Chuen; Scoccimarro, Román; Sheth, Ravi K.

    2012-04-01

    For Gaussian primordial fluctuations the relationship between galaxy and matter overdensities, bias, is most often assumed to be local at the time of observation in the large-scale limit. This hypothesis is however unstable under time evolution, we provide proofs under several (increasingly more realistic) sets of assumptions. In the simplest toy model galaxies are created locally and linearly biased at a single formation time, and subsequently move with the dark matter (no velocity bias) conserving their comoving number density (no merging). We show that, after this formation time, the bias becomes unavoidably nonlocal and nonlinear at large scales. We identify the nonlocal gravitationally induced fields in which the galaxy overdensity can be expanded, showing that they can be constructed out of the invariants of the deformation tensor (Galileons), the main signature of which is a quadrupole field in second-order perturbation theory. In addition, we show that this result persists if we include an arbitrary evolution of the comoving number density of tracers. We then include velocity bias, and show that new contributions appear; these are related to the breaking of Galilean invariance of the bias relation, a dipole field being the signature at second order. We test these predictions by studying the dependence of halo overdensities in cells of fixed dark matter density: measurements in simulations show that departures from the mean bias relation are strongly correlated with the nonlocal gravitationally induced fields identified by our formalism, suggesting that the halo distribution at the present time is indeed more closely related to the mass distribution at an earlier rather than present time. However, the nonlocality seen in the simulations is not fully captured by assuming local bias in Lagrangian space. The effects on nonlocal bias seen in the simulations are most important for the most biased halos, as expected from our predictions. Accounting for these

  15. Large-Scale Statistics for Cu Electromigration

    NASA Astrophysics Data System (ADS)

    Hauschildt, M.; Gall, M.; Hernandez, R.

    2009-06-01

    Even after the successful introduction of Cu-based metallization, the electromigration failure risk has remained one of the important reliability concerns for advanced process technologies. The observation of strong bimodality for the electron up-flow direction in dual-inlaid Cu interconnects has added complexity, but is now widely accepted. The failure voids can occur both within the via ("early" mode) or within the trench ("late" mode). More recently, bimodality has been reported also in down-flow electromigration, leading to very short lifetimes due to small, slit-shaped voids under vias. For a more thorough investigation of these early failure phenomena, specific test structures were designed based on the Wheatstone Bridge technique. The use of these structures enabled an increase of the tested sample size close to 675000, allowing a direct analysis of electromigration failure mechanisms at the single-digit ppm regime. Results indicate that down-flow electromigration exhibits bimodality at very small percentage levels, not readily identifiable with standard testing methods. The activation energy for the down-flow early failure mechanism was determined to be 0.83±0.02 eV. Within the small error bounds of this large-scale statistical experiment, this value is deemed to be significantly lower than the usually reported activation energy of 0.90 eV for electromigration-induced diffusion along Cu/SiCN interfaces. Due to the advantages of the Wheatstone Bridge technique, we were also able to expand the experimental temperature range down to 150° C, coming quite close to typical operating conditions up to 125° C. As a result of the lowered activation energy, we conclude that the down-flow early failure mode may control the chip lifetime at operating conditions. The slit-like character of the early failure void morphology also raises concerns about the validity of the Blech-effect for this mechanism. A very small amount of Cu depletion may cause failure even before a

  16. Large-Scale Identification of Virulence Genes from Streptococcus pneumoniae

    PubMed Central

    Polissi, Alessandra; Pontiggia, Andrea; Feger, Georg; Altieri, Mario; Mottl, Harald; Ferrari, Livia; Simon, Daniel

    1998-01-01

    Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitis in children. Apart from the capsule, the virulence factors of this pathogen are not completely understood. Recent technical advances in the field of bacterial pathogenesis (in vivo expression technology and signature-tagged mutagenesis [STM]) have allowed a large-scale identification of virulence genes. We have adapted to S. pneumoniae the STM technique, originally used for the discovery of Salmonella genes involved in pathogenicity. A library of pneumococcal chromosomal fragments (400 to 600 bp) was constructed in a suicide plasmid vector carrying unique DNA sequence tags and a chloramphenicol resistance marker. The recent clinical isolate G54 was transformed with this library. Chloramphenicol-resistant mutants were obtained by homologous recombination, resulting in genes inactivated by insertion of the suicide vector carrying a unique tag. In a mouse pneumonia model, 1.250 candidate clones were screened; 200 of these were not recovered from the lungs were therefore considered virulence-attenuated mutants. The regions flanking the chloramphenicol gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 200 mutants had insertions in 126 different genes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding proteases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequences that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were individually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the existence of virulence factors specific for pneumonia

  17. Large Scale Computer Simulation of Erthocyte Membranes

    NASA Astrophysics Data System (ADS)

    Harvey, Cameron; Revalee, Joel; Laradji, Mohamed

    2007-11-01

    The cell membrane is crucial to the life of the cell. Apart from partitioning the inner and outer environment of the cell, they also act as a support of complex and specialized molecular machinery, important for both the mechanical integrity of the cell, and its multitude of physiological functions. Due to its relative simplicity, the red blood cell has been a favorite experimental prototype for investigations of the structural and functional properties of the cell membrane. The erythrocyte membrane is a composite quasi two-dimensional structure composed essentially of a self-assembled fluid lipid bilayer and a polymerized protein meshwork, referred to as the cytoskeleton or membrane skeleton. In the case of the erythrocyte, the polymer meshwork is mainly composed of spectrin, anchored to the bilayer through specialized proteins. Using a coarse-grained model, recently developed by us, of self-assembled lipid membranes with implicit solvent and using soft-core potentials, we simulated large scale red-blood-cells bilayers with dimensions ˜ 10-1 μm^2, with explicit cytoskeleton. Our aim is to investigate the renormalization of the elastic properties of the bilayer due to the underlying spectrin meshwork.

  18. Large-scale carbon fiber tests

    NASA Technical Reports Server (NTRS)

    Pride, R. A.

    1980-01-01

    A realistic release of carbon fibers was established by burning a minimum of 45 kg of carbon fiber composite aircraft structural components in each of five large scale, outdoor aviation jet fuel fire tests. This release was quantified by several independent assessments with various instruments developed specifically for these tests. The most likely values for the mass of single carbon fibers released ranged from 0.2 percent of the initial mass of carbon fiber for the source tests (zero wind velocity) to a maximum of 0.6 percent of the initial carbon fiber mass for dissemination tests (5 to 6 m/s wind velocity). Mean fiber lengths for fibers greater than 1 mm in length ranged from 2.5 to 3.5 mm. Mean diameters ranged from 3.6 to 5.3 micrometers which was indicative of significant oxidation. Footprints of downwind dissemination of the fire released fibers were measured to 19.1 km from the fire.

  19. Curvature constraints from large scale structure

    NASA Astrophysics Data System (ADS)

    Di Dio, Enea; Montanari, Francesco; Raccanelli, Alvise; Durrer, Ruth; Kamionkowski, Marc; Lesgourgues, Julien

    2016-06-01

    We modified the CLASS code in order to include relativistic galaxy number counts in spatially curved geometries; we present the formalism and study the effect of relativistic corrections on spatial curvature. The new version of the code is now publicly available. Using a Fisher matrix analysis, we investigate how measurements of the spatial curvature parameter ΩK with future galaxy surveys are affected by relativistic effects, which influence observations of the large scale galaxy distribution. These effects include contributions from cosmic magnification, Doppler terms and terms involving the gravitational potential. As an application, we consider angle and redshift dependent power spectra, which are especially well suited for model independent cosmological constraints. We compute our results for a representative deep, wide and spectroscopic survey, and our results show the impact of relativistic corrections on spatial curvature parameter estimation. We show that constraints on the curvature parameter may be strongly biased if, in particular, cosmic magnification is not included in the analysis. Other relativistic effects turn out to be subdominant in the studied configuration. We analyze how the shift in the estimated best-fit value for the curvature and other cosmological parameters depends on the magnification bias parameter, and find that significant biases are to be expected if this term is not properly considered in the analysis.

  20. Large scale digital atlases in neuroscience

    NASA Astrophysics Data System (ADS)

    Hawrylycz, M.; Feng, D.; Lau, C.; Kuan, C.; Miller, J.; Dang, C.; Ng, L.

    2014-03-01

    Imaging in neuroscience has revolutionized our current understanding of brain structure, architecture and increasingly its function. Many characteristics of morphology, cell type, and neuronal circuitry have been elucidated through methods of neuroimaging. Combining this data in a meaningful, standardized, and accessible manner is the scope and goal of the digital brain atlas. Digital brain atlases are used today in neuroscience to characterize the spatial organization of neuronal structures, for planning and guidance during neurosurgery, and as a reference for interpreting other data modalities such as gene expression and connectivity data. The field of digital atlases is extensive and in addition to atlases of the human includes high quality brain atlases of the mouse, rat, rhesus macaque, and other model organisms. Using techniques based on histology, structural and functional magnetic resonance imaging as well as gene expression data, modern digital atlases use probabilistic and multimodal techniques, as well as sophisticated visualization software to form an integrated product. Toward this goal, brain atlases form a common coordinate framework for summarizing, accessing, and organizing this knowledge and will undoubtedly remain a key technology in neuroscience in the future. Since the development of its flagship project of a genome wide image-based atlas of the mouse brain, the Allen Institute for Brain Science has used imaging as a primary data modality for many of its large scale atlas projects. We present an overview of Allen Institute digital atlases in neuroscience, with a focus on the challenges and opportunities for image processing and computation.

  1. Food appropriation through large scale land acquisitions

    NASA Astrophysics Data System (ADS)

    Rulli, Maria Cristina; D'Odorico, Paolo

    2014-05-01

    The increasing demand for agricultural products and the uncertainty of international food markets has recently drawn the attention of governments and agribusiness firms toward investments in productive agricultural land, mostly in the developing world. The targeted countries are typically located in regions that have remained only marginally utilized because of lack of modern technology. It is expected that in the long run large scale land acquisitions (LSLAs) for commercial farming will bring the technology required to close the existing crops yield gaps. While the extent of the acquired land and the associated appropriation of freshwater resources have been investigated in detail, the amount of food this land can produce and the number of people it could feed still need to be quantified. Here we use a unique dataset of land deals to provide a global quantitative assessment of the rates of crop and food appropriation potentially associated with LSLAs. We show how up to 300-550 million people could be fed by crops grown in the acquired land, should these investments in agriculture improve crop production and close the yield gap. In contrast, about 190-370 million people could be supported by this land without closing of the yield gap. These numbers raise some concern because the food produced in the acquired land is typically exported to other regions, while the target countries exhibit high levels of malnourishment. Conversely, if used for domestic consumption, the crops harvested in the acquired land could ensure food security to the local populations.

  2. Large-scale wind turbine structures

    NASA Technical Reports Server (NTRS)

    Spera, David A.

    1988-01-01

    The purpose of this presentation is to show how structural technology was applied in the design of modern wind turbines, which were recently brought to an advanced stage of development as sources of renewable power. Wind turbine structures present many difficult problems because they are relatively slender and flexible; subject to vibration and aeroelastic instabilities; acted upon by loads which are often nondeterministic; operated continuously with little maintenance in all weather; and dominated by life-cycle cost considerations. Progress in horizontal-axis wind turbines (HAWT) development was paced by progress in the understanding of structural loads, modeling of structural dynamic response, and designing of innovative structural response. During the past 15 years a series of large HAWTs was developed. This has culminated in the recent completion of the world's largest operating wind turbine, the 3.2 MW Mod-5B power plane installed on the island of Oahu, Hawaii. Some of the applications of structures technology to wind turbine will be illustrated by referring to the Mod-5B design. First, a video overview will be presented to provide familiarization with the Mod-5B project and the important components of the wind turbine system. Next, the structural requirements for large-scale wind turbines will be discussed, emphasizing the difficult fatigue-life requirements. Finally, the procedures used to design the structure will be presented, including the use of the fracture mechanics approach for determining allowable fatigue stresses.

  3. Large-scale wind turbine structures

    NASA Astrophysics Data System (ADS)

    Spera, David A.

    1988-05-01

    The purpose of this presentation is to show how structural technology was applied in the design of modern wind turbines, which were recently brought to an advanced stage of development as sources of renewable power. Wind turbine structures present many difficult problems because they are relatively slender and flexible; subject to vibration and aeroelastic instabilities; acted upon by loads which are often nondeterministic; operated continuously with little maintenance in all weather; and dominated by life-cycle cost considerations. Progress in horizontal-axis wind turbines (HAWT) development was paced by progress in the understanding of structural loads, modeling of structural dynamic response, and designing of innovative structural response. During the past 15 years a series of large HAWTs was developed. This has culminated in the recent completion of the world's largest operating wind turbine, the 3.2 MW Mod-5B power plane installed on the island of Oahu, Hawaii. Some of the applications of structures technology to wind turbine will be illustrated by referring to the Mod-5B design. First, a video overview will be presented to provide familiarization with the Mod-5B project and the important components of the wind turbine system. Next, the structural requirements for large-scale wind turbines will be discussed, emphasizing the difficult fatigue-life requirements. Finally, the procedures used to design the structure will be presented, including the use of the fracture mechanics approach for determining allowable fatigue stresses.

  4. An informal paper on large-scale dynamic systems

    NASA Technical Reports Server (NTRS)

    Ho, Y. C.

    1975-01-01

    Large scale systems are defined as systems requiring more than one decision maker to control the system. Decentralized control and decomposition are discussed for large scale dynamic systems. Information and many-person decision problems are analyzed.

  5. Plant regeneration methods for rapid generation of a large scale Ds transposant population in rice.

    PubMed

    Xuan, Yuan Hu; Huang, Jin; Yi, Gihwan; Park, Dong-Soo; Park, Soo Kwon; Eun, Moo Young; Yun, Doh Won; Lee, Gang-Seob; Kim, Tae Ho; Han, Chang-deok

    2013-01-01

    To mutagenize rice genomes, a two-element system is utilized. This system comprises an immobile Ac element driven by the CaMV 35S promoter, and a gene trap Ds carrying a partial intron with alternative splice acceptors fused to the GUS coding region. Rapid, large-scale generation of a Ds transposant population was achieved using a plant regeneration procedure involving the tissue culture of seed-derived calli carrying Ac and Ds elements. During tissue cultures, Ds mobility accompanies changes in methylation patterns of a terminal region of Ds, where over 70% of plants contained independent Ds insertions. In the transposon population, around 12% of plants expressed GUS at the early seedling stage. A flanking-sequence-tag (FST) database has been established by cloning over 19,968 Ds insertion sites and the Ds map shows relatively uniform distribution across the rice chromosomes. PMID:23918423

  6. CRISPR/Cas9-targeted mutagenesis in Caenorhabditis elegans.

    PubMed

    Waaijers, Selma; Portegijs, Vincent; Kerver, Jana; Lemmens, Bennie B L G; Tijsterman, Marcel; van den Heuvel, Sander; Boxem, Mike

    2013-11-01

    The generation of genetic mutants in Caenorhabditis elegans has long relied on the selection of mutations in large-scale screens. Directed mutagenesis of specific loci in the genome would greatly speed up analysis of gene function. Here, we adapt the CRISPR/Cas9 system to generate mutations at specific sites in the C. elegans genome. PMID:23979586

  7. Sensitivity technologies for large scale simulation.

    SciTech Connect

    Collis, Samuel Scott; Bartlett, Roscoe Ainsworth; Smith, Thomas Michael; Heinkenschloss, Matthias; Wilcox, Lucas C.; Hill, Judith C.; Ghattas, Omar; Berggren, Martin Olof; Akcelik, Volkan; Ober, Curtis Curry; van Bloemen Waanders, Bart Gustaaf; Keiter, Eric Richard

    2005-01-01

    Sensitivity analysis is critically important to numerous analysis algorithms, including large scale optimization, uncertainty quantification,reduced order modeling, and error estimation. Our research focused on developing tools, algorithms and standard interfaces to facilitate the implementation of sensitivity type analysis into existing code and equally important, the work was focused on ways to increase the visibility of sensitivity analysis. We attempt to accomplish the first objective through the development of hybrid automatic differentiation tools, standard linear algebra interfaces for numerical algorithms, time domain decomposition algorithms and two level Newton methods. We attempt to accomplish the second goal by presenting the results of several case studies in which direct sensitivities and adjoint methods have been effectively applied, in addition to an investigation of h-p adaptivity using adjoint based a posteriori error estimation. A mathematical overview is provided of direct sensitivities and adjoint methods for both steady state and transient simulations. Two case studies are presented to demonstrate the utility of these methods. A direct sensitivity method is implemented to solve a source inversion problem for steady state internal flows subject to convection diffusion. Real time performance is achieved using novel decomposition into offline and online calculations. Adjoint methods are used to reconstruct initial conditions of a contamination event in an external flow. We demonstrate an adjoint based transient solution. In addition, we investigated time domain decomposition algorithms in an attempt to improve the efficiency of transient simulations. Because derivative calculations are at the root of sensitivity calculations, we have developed hybrid automatic differentiation methods and implemented this approach for shape optimization for gas dynamics using the Euler equations. The hybrid automatic differentiation method was applied to a first

  8. International space station. Large scale integration approach

    NASA Astrophysics Data System (ADS)

    Cohen, Brad

    The International Space Station is the most complex large scale integration program in development today. The approach developed for specification, subsystem development, and verification lay a firm basis on which future programs of this nature can be based. International Space Station is composed of many critical items, hardware and software, built by numerous International Partners, NASA Institutions, and U.S. Contractors and is launched over a period of five years. Each launch creates a unique configuration that must be safe, survivable, operable, and support ongoing assembly (assemblable) to arrive at the assembly complete configuration in 2003. The approaches to integrating each of the modules into a viable spacecraft and continue the assembly is a challenge in itself. Added to this challenge are the severe schedule constraints and lack of an "Iron Bird", which prevents assembly and checkout of each on-orbit configuration prior to launch. This paper will focus on the following areas: 1) Specification development process explaining how the requirements and specifications were derived using a modular concept driven by launch vehicle capability. Each module is composed of components of subsystems versus completed subsystems. 2) Approach to stage (each stage consists of the launched module added to the current on-orbit spacecraft) specifications. Specifically, how each launched module and stage ensures support of the current and future elements of the assembly. 3) Verification approach, due to the schedule constraints, is primarily analysis supported by testing. Specifically, how are the interfaces ensured to mate and function on-orbit when they cannot be mated before launch. 4) Lessons learned. Where can we improve this complex system design and integration task?

  9. Large Scale Flame Spread Environmental Characterization Testing

    NASA Technical Reports Server (NTRS)

    Clayman, Lauren K.; Olson, Sandra L.; Gokoghi, Suleyman A.; Brooker, John E.; Ferkul, Paul V.; Kacher, Henry F.

    2013-01-01

    Under the Advanced Exploration Systems (AES) Spacecraft Fire Safety Demonstration Project (SFSDP), as a risk mitigation activity in support of the development of a large-scale fire demonstration experiment in microgravity, flame-spread tests were conducted in normal gravity on thin, cellulose-based fuels in a sealed chamber. The primary objective of the tests was to measure pressure rise in a chamber as sample material, burning direction (upward/downward), total heat release, heat release rate, and heat loss mechanisms were varied between tests. A Design of Experiments (DOE) method was imposed to produce an array of tests from a fixed set of constraints and a coupled response model was developed. Supplementary tests were run without experimental design to additionally vary select parameters such as initial chamber pressure. The starting chamber pressure for each test was set below atmospheric to prevent chamber overpressure. Bottom ignition, or upward propagating burns, produced rapid acceleratory turbulent flame spread. Pressure rise in the chamber increases as the amount of fuel burned increases mainly because of the larger amount of heat generation and, to a much smaller extent, due to the increase in gaseous number of moles. Top ignition, or downward propagating burns, produced a steady flame spread with a very small flat flame across the burning edge. Steady-state pressure is achieved during downward flame spread as the pressure rises and plateaus. This indicates that the heat generation by the flame matches the heat loss to surroundings during the longer, slower downward burns. One heat loss mechanism included mounting a heat exchanger directly above the burning sample in the path of the plume to act as a heat sink and more efficiently dissipate the heat due to the combustion event. This proved an effective means for chamber overpressure mitigation for those tests producing the most total heat release and thusly was determined to be a feasible mitigation

  10. Synchronization of coupled large-scale Boolean networks

    SciTech Connect

    Li, Fangfei

    2014-03-15

    This paper investigates the complete synchronization and partial synchronization of two large-scale Boolean networks. First, the aggregation algorithm towards large-scale Boolean network is reviewed. Second, the aggregation algorithm is applied to study the complete synchronization and partial synchronization of large-scale Boolean networks. Finally, an illustrative example is presented to show the efficiency of the proposed results.

  11. Large-Scale Spacecraft Fire Safety Tests

    NASA Technical Reports Server (NTRS)

    Urban, David; Ruff, Gary A.; Ferkul, Paul V.; Olson, Sandra; Fernandez-Pello, A. Carlos; T'ien, James S.; Torero, Jose L.; Cowlard, Adam J.; Rouvreau, Sebastien; Minster, Olivier; Toth, Balazs; Legros, Guillaume; Eigenbrod, Christian; Smirnov, Nickolay; Fujita, Osamu; Jomaas, Grunde

    2014-01-01

    An international collaborative program is underway to address open issues in spacecraft fire safety. Because of limited access to long-term low-gravity conditions and the small volume generally allotted for these experiments, there have been relatively few experiments that directly study spacecraft fire safety under low-gravity conditions. Furthermore, none of these experiments have studied sample sizes and environment conditions typical of those expected in a spacecraft fire. The major constraint has been the size of the sample, with prior experiments limited to samples of the order of 10 cm in length and width or smaller. This lack of experimental data forces spacecraft designers to base their designs and safety precautions on 1-g understanding of flame spread, fire detection, and suppression. However, low-gravity combustion research has demonstrated substantial differences in flame behavior in low-gravity. This, combined with the differences caused by the confined spacecraft environment, necessitates practical scale spacecraft fire safety research to mitigate risks for future space missions. To address this issue, a large-scale spacecraft fire experiment is under development by NASA and an international team of investigators. This poster presents the objectives, status, and concept of this collaborative international project (Saffire). The project plan is to conduct fire safety experiments on three sequential flights of an unmanned ISS re-supply spacecraft (the Orbital Cygnus vehicle) after they have completed their delivery of cargo to the ISS and have begun their return journeys to earth. On two flights (Saffire-1 and Saffire-3), the experiment will consist of a flame spread test involving a meter-scale sample ignited in the pressurized volume of the spacecraft and allowed to burn to completion while measurements are made. On one of the flights (Saffire-2), 9 smaller (5 x 30 cm) samples will be tested to evaluate NASAs material flammability screening tests

  12. Tyrosine Recombinase Retrotransposons and Transposons.

    PubMed

    Poulter, Russell T M; Butler, Margi I

    2015-04-01

    Retrotransposons carrying tyrosine recombinases (YR) are widespread in eukaryotes. The first described tyrosine recombinase mobile element, DIRS1, is a retroelement from the slime mold Dictyostelium discoideum. The YR elements are bordered by terminal repeats related to their replication via free circular dsDNA intermediates. Site-specific recombination is believed to integrate the circle without creating duplications of the target sites. Recently a large number of YR retrotransposons have been described, including elements from fungi (mucorales and basidiomycetes), plants (green algae) and a wide range of animals including nematodes, insects, sea urchins, fish, amphibia and reptiles. YR retrotransposons can be divided into three major groups: the DIRS elements, PAT-like and the Ngaro elements. The three groups form distinct clades on phylogenetic trees based on alignments of reverse transcriptase/ribonuclease H (RT/RH) and YR sequences, and also having some structural distinctions. A group of eukaryote DNA transposons, cryptons, also carry tyrosine recombinases. These DNA transposons do not encode a reverse transcriptase. They have been detected in several pathogenic fungi and oomycetes. Sequence comparisons suggest that the crypton YRs are related to those of the YR retrotransposons. We suggest that the YR retrotransposons arose from the combination of a crypton-like YR DNA transposon and the RT/RH encoding sequence of a retrotransposon. This acquisition must have occurred at a very early point in the evolution of eukaryotes. PMID:26104693

  13. Identification of a Virulence-Associated Determinant, Dihydrolipoamide Dehydrogenase (lpd), in Mycoplasma gallisepticum through In Vivo Screening of Transposon Mutants

    PubMed Central

    Hudson, P.; Gorton, T. S.; Papazisi, L.; Cecchini, K.; Frasca, S.; Geary, S. J.

    2006-01-01

    To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7. PMID:16428737

  14. Transposon tagging of disease resistance genes. Progress report, May 1, 1988--1992

    SciTech Connect

    Michelmore, R.

    1994-06-01

    Our goal is to clone genes in lettuce determining resistance to downy mildew. One approach involves the mobilization of transposons into resistance genes to mutate and tag the target gene. Because transposons have yet to be isolated and characterized from lettuce, the majority of our experiments have involved Ac from corn as this is increasingly the best characterized transposon. Over the past several years, various labs have contributed to a detailed understanding of the biology of Ac in corn and heterologous plant species. We have collaborated closely with several of these labs, exchanged materials and incorporated their advances into our analysis of transposition in lettuce. The original proposal described the development of a transposon mutagenesis system for lettuce and its subsequent use to tag disease resistance genes. The development phase involved characterization and manipulation of Ac transposition, identification of suitable whole plant selectable markers for the construction of chimeric non-autonomous elements, and investigation of the stability of resistance genes. Investigation of Ac transposition in lettuce has received the majority of our attention. Initially, we made a simple construct with wildtype Ac and introduced it into lettuce. No transposition was observed; although other labs demonstrated that the same construct was functional in tomato. We then focused on assaying for Ac transposition with constructs of increasing sophistication that had been demonstrated by others to be functional in other species. The latest constructs for transposon mutagenesis clearly demonstrated transposition in lettuce. This allowed us to generate seed stocks that we will start to screen for insertional inactivation of resistance genes this year.

  15. Large-scale assembly of colloidal particles

    NASA Astrophysics Data System (ADS)

    Yang, Hongta

    This study reports a simple, roll-to-roll compatible coating technology for producing three-dimensional highly ordered colloidal crystal-polymer composites, colloidal crystals, and macroporous polymer membranes. A vertically beveled doctor blade is utilized to shear align silica microsphere-monomer suspensions to form large-area composites in a single step. The polymer matrix and the silica microspheres can be selectively removed to create colloidal crystals and self-standing macroporous polymer membranes. The thickness of the shear-aligned crystal is correlated with the viscosity of the colloidal suspension and the coating speed, and the correlations can be qualitatively explained by adapting the mechanisms developed for conventional doctor blade coating. Five important research topics related to the application of large-scale three-dimensional highly ordered macroporous films by doctor blade coating are covered in this study. The first topic describes the invention in large area and low cost color reflective displays. This invention is inspired by the heat pipe technology. The self-standing macroporous polymer films exhibit brilliant colors which originate from the Bragg diffractive of visible light form the three-dimensional highly ordered air cavities. The colors can be easily changed by tuning the size of the air cavities to cover the whole visible spectrum. When the air cavities are filled with a solvent which has the same refractive index as that of the polymer, the macroporous polymer films become completely transparent due to the index matching. When the solvent trapped in the cavities is evaporated by in-situ heating, the sample color changes back to brilliant color. This process is highly reversible and reproducible for thousands of cycles. The second topic reports the achievement of rapid and reversible vapor detection by using 3-D macroporous photonic crystals. Capillary condensation of a condensable vapor in the interconnected macropores leads to the

  16. Population generation for large-scale simulation

    NASA Astrophysics Data System (ADS)

    Hannon, Andrew C.; King, Gary; Morrison, Clayton; Galstyan, Aram; Cohen, Paul

    2005-05-01

    Computer simulation is used to research phenomena ranging from the structure of the space-time continuum to population genetics and future combat.1-3 Multi-agent simulations in particular are now commonplace in many fields.4, 5 By modeling populations whose complex behavior emerges from individual interactions, these simulations help to answer questions about effects where closed form solutions are difficult to solve or impossible to derive.6 To be useful, simulations must accurately model the relevant aspects of the underlying domain. In multi-agent simulation, this means that the modeling must include both the agents and their relationships. Typically, each agent can be modeled as a set of attributes drawn from various distributions (e.g., height, morale, intelligence and so forth). Though these can interact - for example, agent height is related to agent weight - they are usually independent. Modeling relations between agents, on the other hand, adds a new layer of complexity, and tools from graph theory and social network analysis are finding increasing application.7, 8 Recognizing the role and proper use of these techniques, however, remains the subject of ongoing research. We recently encountered these complexities while building large scale social simulations.9-11 One of these, the Hats Simulator, is designed to be a lightweight proxy for intelligence analysis problems. Hats models a "society in a box" consisting of many simple agents, called hats. Hats gets its name from the classic spaghetti western, in which the heroes and villains are known by the color of the hats they wear. The Hats society also has its heroes and villains, but the challenge is to identify which color hat they should be wearing based on how they behave. There are three types of hats: benign hats, known terrorists, and covert terrorists. Covert terrorists look just like benign hats but act like terrorists. Population structure can make covert hat identification significantly more

  17. Multitree Algorithms for Large-Scale Astrostatistics

    NASA Astrophysics Data System (ADS)

    March, William B.; Ozakin, Arkadas; Lee, Dongryeol; Riegel, Ryan; Gray, Alexander G.

    2012-03-01

    this number every week, resulting in billions of objects. At such scales, even linear-time analysis operations present challenges, particularly since statistical analyses are inherently interactive processes, requiring that computations complete within some reasonable human attention span. The quadratic (or worse) runtimes of straightforward implementations become quickly unbearable. Examples of applications. These analysis subroutines occur ubiquitously in astrostatistical work. We list just a few examples. The need to cross-match objects across different catalogs has led to various algorithms, which at some point perform an AllNN computation. 2-point and higher-order spatial correlations for the basis of spatial statistics, and are utilized in astronomy to compare the spatial structures of two datasets, such as an observed sample and a theoretical sample, for example, forming the basis for two-sample hypothesis testing. Friends-of-friends clustering is often used to identify halos in data from astrophysical simulations. Minimum spanning tree properties have also been proposed as statistics of large-scale structure. Comparison of the distributions of different kinds of objects requires accurate density estimation, for which KDE is the overall statistical method of choice. The prediction of redshifts from optical data requires accurate regression, for which kernel regression is a powerful method. The identification of objects of various types in astronomy, such as stars versus galaxies, requires accurate classification, for which KDA is a powerful method. Overview. In this chapter, we will briefly sketch the main ideas behind recent fast algorithms which achieve, for example, linear runtimes for pairwise-distance problems, or similarly dramatic reductions in computational growth. In some cases, the runtime orders for these algorithms are mathematically provable statements, while in others we have only conjectures backed by experimental observations for the time being

  18. Probes of large-scale structure in the universe

    NASA Technical Reports Server (NTRS)

    Suto, Yasushi; Gorski, Krzysztof; Juszkiewicz, Roman; Silk, Joseph

    1988-01-01

    A general formalism is developed which shows that the gravitational instability theory for the origin of the large-scale structure of the universe is now capable of critically confronting observational results on cosmic background radiation angular anisotropies, large-scale bulk motions, and large-scale clumpiness in the galaxy counts. The results indicate that presently advocated cosmological models will have considerable difficulty in simultaneously explaining the observational results.

  19. Signature-tagged mutagenesis of Vibrio vulnificus

    PubMed Central

    YAMAMOTO, Mai; KASHIMOTO, Takashige; TONG, Ping; XIAO, Jianbo; SUGIYAMA, Michiko; INOUE, Miyuki; MATSUNAGA, Rie; HOSOHARA, Kohei; NAKATA, Kazue; YOKOTA, Kenji; OGUMA, Keiji; YAMAMOTO, Koichiro

    2015-01-01

    Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus. PMID:25755021

  20. Chemical Mutagens, Transposons, and Transgenes to Interrogate Gene Function in Drosophila melanogaster

    PubMed Central

    Venken, Koen J.T.; Bellen, Hugo J.

    2014-01-01

    The study of genetics, genes, and chromosomal inheritance was initiated by Thomas Morgan in when the first visible mutations were identified in fruit flies. The field expanded upon the work initiated by Herman Muller in 1926 when he used X-rays to develop the first balancer chromosomes. Today, balancers are still invaluable to maintain mutations and transgenes but the arsenal of tools has expanded vastly and numerous new methods have been developed, many relying on the availability of the genome sequence and transposable elements. Forward genetic screens based on chemical mutagenesis or transposable elements have resulted in the unbiased identification of many novel players involved in processes probed by specific phenotypic assays. Reverse genetic approaches have relied on the availability of a carefully selected set of transposon insertions spread throughout the genome to allow the manipulation of the region in the vicinity of each insertion. Lastly, the ability to transform Drosophila with single copy transgenes using transposons or site-specific integration using the ΦC31 integrase has allowed numerous manipulations, including the ability to create and integrate genomic rescue constructs, generate duplications, RNAi knock-out technology, binary expression systems like the GAL4/UAS system as well as other methods. Here, we will discuss the most useful methodologies to interrogate the fruit fly genome in vivo focusing on chemical mutagenesis, transposons and transgenes. Genome engineering approaches based on nucleases and RNAi technology are discussed in following chapters. PMID:24583113

  1. Generation of an inducible and optimized piggyBac transposon system†

    PubMed Central

    Cadiñanos, Juan; Bradley, Allan

    2007-01-01

    Genomic studies in the mouse have been slowed by the lack of transposon-mediated mutagenesis. However, since the resurrection of Sleeping Beauty (SB), the possibility of performing forward genetics in mice has been reinforced. Recently, piggyBac (PB), a functional transposon from insects, was also described to work in mammals. As the activity of PB is higher than that of SB11 and SB12, two hyperactive SB transposases, we have characterized and improved the PB system in mouse ES cells. We have generated a mouse codon-optimized version of the PB transposase coding sequence (CDS) which provides transposition levels greater than the original. We have also found that the promoter sequence predicted in the 5′-terminal repeat of the PB transposon is active in the mammalian context. Finally, we have engineered inducible versions of the optimized piggyBac transposase fused with ERT2. One of them, when induced, provides higher levels of transposition than the native piggyBac CDS, whereas in the absence of induction its activity is indistinguishable from background. We expect that these tools, adaptable to perform mouse-germline mutagenesis, will facilitate the identification of genes involved in pathological and physiological processes, such as cancer or ES cell differentiation. PMID:17576687

  2. INTERNATIONAL WORKSHOP ON LARGE-SCALE REFORESTATION: PROCEEDINGS

    EPA Science Inventory

    The purpose of the workshop was to identify major operational and ecological considerations needed to successfully conduct large-scale reforestation projects throughout the forested regions of the world. Large-scale" for this workshop means projects where, by human effort, approx...

  3. Using Large-Scale Assessment Scores to Determine Student Grades

    ERIC Educational Resources Information Center

    Miller, Tess

    2013-01-01

    Many Canadian provinces provide guidelines for teachers to determine students' final grades by combining a percentage of students' scores from provincial large-scale assessments with their term scores. This practice is thought to hold students accountable by motivating them to put effort into completing the large-scale assessment, thereby…

  4. The Challenge of Large-Scale Literacy Improvement

    ERIC Educational Resources Information Center

    Levin, Ben

    2010-01-01

    This paper discusses the challenge of making large-scale improvements in literacy in schools across an entire education system. Despite growing interest and rhetoric, there are very few examples of sustained, large-scale change efforts around school-age literacy. The paper reviews 2 instances of such efforts, in England and Ontario. After…

  5. Fungicide-induced transposon movement in Monilinia fructicola.

    PubMed

    Chen, Fengping; Everhart, Sydney E; Bryson, P Karen; Luo, Chaoxi; Song, Xi; Liu, Xili; Schnabel, Guido

    2015-12-01

    Repeated applications of fungicides with a single mode of action are believed to select for pre-existing resistant strains in a pathogen population, while the impact of sub-lethal doses of such fungicides on sensitive members of the population is unknown. In this study, in vitro evidence is presented that continuous exposure of Monilinia fructicola mycelium to some fungicides can induce genetic change in form of transposon transposition. Three fungicide-sensitive M. fructicola isolates were exposed in 12 weekly transfers of mycelia to a dose gradient of demethylation inhibitor fungicide (DMI) SYP-Z048 and quinone outside inhibitor fungicide (QoI) azoxystrobin in solo or mixture treatments. Evidence of mutagenesis was assessed by monitoring Mftc1, a multicopy transposable element of M. fructicola, by PCR and Southern blot analysis. Movement of Mftc1 was observed following azoxystrobin and azoxystrobin plus SYP-Z048 treatments in two of the three isolates, but not in the non-fungicide-treated controls. Interestingly, the upstream promoter region of MfCYP51 was a prime target for Mftc1 transposition in these isolates. Transposition of Mftc1 was verified by Southern blot in two of three isolates from another, similar experiment following prolonged, sublethal azoxystrobin exposure, although in these isolates movement of Mftc1 in the upstream MfCYP51 promoter region was not observed. More research is warranted to determine whether fungicide-induced mutagenesis may also happen under field conditions. PMID:26537535

  6. Distribution probability of large-scale landslides in central Nepal

    NASA Astrophysics Data System (ADS)

    Timilsina, Manita; Bhandary, Netra P.; Dahal, Ranjan Kumar; Yatabe, Ryuichi

    2014-12-01

    Large-scale landslides in the Himalaya are defined as huge, deep-seated landslide masses that occurred in the geological past. They are widely distributed in the Nepal Himalaya. The steep topography and high local relief provide high potential for such failures, whereas the dynamic geology and adverse climatic conditions play a key role in the occurrence and reactivation of such landslides. The major geoscientific problems related with such large-scale landslides are 1) difficulties in their identification and delineation, 2) sources of small-scale failures, and 3) reactivation. Only a few scientific publications have been published concerning large-scale landslides in Nepal. In this context, the identification and quantification of large-scale landslides and their potential distribution are crucial. Therefore, this study explores the distribution of large-scale landslides in the Lesser Himalaya. It provides simple guidelines to identify large-scale landslides based on their typical characteristics and using a 3D schematic diagram. Based on the spatial distribution of landslides, geomorphological/geological parameters and logistic regression, an equation of large-scale landslide distribution is also derived. The equation is validated by applying it to another area. For the new area, the area under the receiver operating curve of the landslide distribution probability in the new area is 0.699, and a distribution probability value could explain > 65% of existing landslides. Therefore, the regression equation can be applied to areas of the Lesser Himalaya of central Nepal with similar geological and geomorphological conditions.

  7. Transposon tagging in diploid strawberry.

    PubMed

    Veilleux, Richard E; Mills, Kerri P; Baxter, Aaron J; Upham, Kendall T; Ferguson, Tammy J; Holt, Sarah Hudson; Lu, Nan; Ruiz-Rojas, Juan J; Pantazis, Christopher J; Davis, Cherish M; Lindsay, Robert C; Powell, Frankie L; Dan, Yinghui; Dickerman, Allan W; Oosumi, Teruko; Shulaev, Vladimir

    2012-10-01

    Fragaria vesca was transformed with a transposon tagging construct harbouring amino terminally deleted maize transposase and EGFP (Ac element), NPTII, CaMV 35S promoter (P35S) driving transposase and mannopine synthase promoter (Pmas) driving EGFP (Ds element). Of 180 primary transgenics, 48 were potential launch pads, 72 were multiple insertions or chimaeras, and 60 exhibited somatic transposition. T₁ progeny of 32 putative launch pads were screened by multiplex PCR for transposition. Evidence of germ-line transposition occurred in 13 putative launch pads; however, the transposition frequency was too low in three for efficient recovery of transposants. The transposition frequency in the remaining launch pads ranged from 16% to 40%. After self-pollination of the T₀ launch pads, putative transposants in the T₁ generation were identified by multiplex PCR. Sequencing of hiTAIL-PCR products derived from nested primers within the Ds end sequences (either P35S at the left border or the inverted repeat at the right border) of T₁ plants revealed transposition of the Ds element to distant sites in the strawberry genome. From more than 2400 T₁ plants screened, 103 unique transposants have been identified, among which 17 were somatic transpositions observed in the T₀ generation. Ds insertion sites were dispersed among various gene elements [exons (15%), introns (23%), promoters (30%), 3' UTRs (17%) as well as intergenically (15%)]. Three-primer (one on either side of the Ds insertion and one within the Ds T-DNA) PCR could be used to identify homozygous T₂ transposon-tagged plants. The mutant collection has been catalogued in an on-line database. PMID:22845757

  8. Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons

    SciTech Connect

    Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.; Lamson, Jacob S.; He, Jennifer; Hoover, Cindi A.; Blow, Matthew J.; Bristow, James; Butland, Gareth; Arkin, Adam P.; Deutschbauer, Adam

    2015-05-12

    Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with any transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative D-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes

  9. Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons

    DOE PAGESBeta

    Wetmore, Kelly M.; Price, Morgan N.; Waters, Robert J.; Lamson, Jacob S.; He, Jennifer; Hoover, Cindi A.; Blow, Matthew J.; Bristow, James; Butland, Gareth; Arkin, Adam P.; et al

    2015-05-12

    Transposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with anymore » transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative D-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling. A large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are

  10. Needs, opportunities, and options for large scale systems research

    SciTech Connect

    Thompson, G.L.

    1984-10-01

    The Office of Energy Research was recently asked to perform a study of Large Scale Systems in order to facilitate the development of a true large systems theory. It was decided to ask experts in the fields of electrical engineering, chemical engineering and manufacturing/operations research for their ideas concerning large scale systems research. The author was asked to distribute a questionnaire among these experts to find out their opinions concerning recent accomplishments and future research directions in large scale systems research. He was also requested to convene a conference which included three experts in each area as panel members to discuss the general area of large scale systems research. The conference was held on March 26--27, 1984 in Pittsburgh with nine panel members, and 15 other attendees. The present report is a summary of the ideas presented and the recommendations proposed by the attendees.

  11. Large scale suppression of scalar power on a spatial condensation

    NASA Astrophysics Data System (ADS)

    Kouwn, Seyen; Kwon, O.-Kab; Oh, Phillial

    2015-03-01

    We consider a deformed single-field inflation model in terms of three SO(3) symmetric moduli fields. We find that spatially linear solutions for the moduli fields induce a phase transition during the early stage of the inflation and the suppression of scalar power spectrum at large scales. This suppression can be an origin of anomalies for large-scale perturbation modes in the cosmological observation.

  12. Interpretation of large-scale deviations from the Hubble flow

    NASA Astrophysics Data System (ADS)

    Grinstein, B.; Politzer, H. David; Rey, S.-J.; Wise, Mark B.

    1987-03-01

    The theoretical expectation for large-scale streaming velocities relative to the Hubble flow is expressed in terms of statistical correlation functions. Only for objects that trace the mass would these velocities have a simple cosmological interpretation. If some biasing effects the objects' formation, then nonlinear gravitational evolution is essential to predicting the expected large-scale velocities, which also depend on the nature of the biasing.

  13. Large-scale identification of serotype 4 Streptococcus pneumoniae virulence factors

    PubMed Central

    Hava, David L.; Camilli, Andrew

    2009-01-01

    Summary Streptococcus pneumoniae (the pneumococcus) is carried in the nasopharynx of healthy individuals, but can spread to other host sites and lead to pneumonia, bacteraemia, otitis media and meningitis. Although it is logical to think a priori that differential gene expression would contribute to the ability of this pathogen to colonize different sites, in fact very few genes have been demonstrated to play tissue specific roles in virulence or carriage. Using signature-tagged mutagenesis to screen 6149 mariner-transposon insertion strains, we identified 387 mutants attenuated for infection in a murine model of pneumonia. Among these mutants are ones with disruptions in a number of putative tissue-specific transcriptional regulators, surface proteins, metabolic proteins and proteins of unknown function, most of which had not previously been associated with virulence. A subset of these, including most of those with insertions in putative transcriptional regulators, was examined for phenotypes in murine models of bacteraemia and nasopharyngeal carriage. Four classes of mutants defective in infection models of the: (I) lung, (II) lung and blood, (III) lung and nasopharynx, and (IV) all three tissues were identified, thus demonstrating the existence of tissue-specific pneumococcal virulence factors. Included in these strains were two with disruptions in a genetic locus that putatively codes for a transcriptional regulator, three surface proteins and three sortase homologues. Mutation analysis revealed that three of the seven genes in this locus are virulence factors that are specific to mucosal surfaces. PMID:12207705

  14. Large-scale motions in a plane wall jet

    NASA Astrophysics Data System (ADS)

    Gnanamanickam, Ebenezer; Jonathan, Latim; Shibani, Bhatt

    2015-11-01

    The dynamic significance of large-scale motions in turbulent boundary layers have been the focus of several recent studies, primarily focussing on canonical flows - zero pressure gradient boundary layers, flows within pipes and channels. This work presents an investigation into the large-scale motions in a boundary layer that is used as the prototypical flow field for flows with large-scale mixing and reactions, the plane wall jet. An experimental investigation is carried out in a plane wall jet facility designed to operate at friction Reynolds numbers Reτ > 1000 , which allows for the development of a significant logarithmic region. The streamwise turbulent intensity across the boundary layer is decomposed into small-scale (less than one integral length-scale δ) and large-scale components. The small-scale energy has a peak in the near-wall region associated with the near-wall turbulent cycle as in canonical boundary layers. However, eddies of large-scales are the dominating eddies having significantly higher energy, than the small-scales across almost the entire boundary layer even at the low to moderate Reynolds numbers under consideration. The large-scales also appear to amplitude and frequency modulate the smaller scales across the entire boundary layer.

  15. Large-scale filament formation inhibits the activity of CTP synthetase

    PubMed Central

    Barry, Rachael M; Bitbol, Anne-Florence; Lorestani, Alexander; Charles, Emeric J; Habrian, Chris H; Hansen, Jesse M; Li, Hsin-Jung; Baldwin, Enoch P; Wingreen, Ned S; Kollman, Justin M; Gitai, Zemer

    2014-01-01

    CTP Synthetase (CtpS) is a universally conserved and essential metabolic enzyme. While many enzymes form small oligomers, CtpS forms large-scale filamentous structures of unknown function in prokaryotes and eukaryotes. By simultaneously monitoring CtpS polymerization and enzymatic activity, we show that polymerization inhibits activity, and CtpS's product, CTP, induces assembly. To understand how assembly inhibits activity, we used electron microscopy to define the structure of CtpS polymers. This structure suggests that polymerization sterically hinders a conformational change necessary for CtpS activity. Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation. This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels. We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable. DOI: http://dx.doi.org/10.7554/eLife.03638.001 PMID:25030911

  16. Genome-Wide Analysis of Transposon and Retroviral Insertions Reveals Preferential Integrations in Regions of DNA Flexibility.

    PubMed

    Vrljicak, Pavle; Tao, Shijie; Varshney, Gaurav K; Quach, Helen Ngoc Bao; Joshi, Adita; LaFave, Matthew C; Burgess, Shawn M; Sampath, Karuna

    2016-01-01

    DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift, and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence the integration of heterologous DNA in genomes, and have implications for targeted genome engineering. PMID:26818075

  17. Genome-Wide Analysis of Transposon and Retroviral Insertions Reveals Preferential Integrations in Regions of DNA Flexibility

    PubMed Central

    Vrljicak, Pavle; Tao, Shijie; Varshney, Gaurav K.; Quach, Helen Ngoc Bao; Joshi, Adita; LaFave, Matthew C.; Burgess, Shawn M.; Sampath, Karuna

    2016-01-01

    DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift, and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence the integration of heterologous DNA in genomes, and have implications for targeted genome engineering. PMID:26818075

  18. The large-scale landslide risk classification in catchment scale

    NASA Astrophysics Data System (ADS)

    Liu, Che-Hsin; Wu, Tingyeh; Chen, Lien-Kuang; Lin, Sheng-Chi

    2013-04-01

    The landslide disasters caused heavy casualties during Typhoon Morakot, 2009. This disaster is defined as largescale landslide due to the casualty numbers. This event also reflects the survey on large-scale landslide potential is so far insufficient and significant. The large-scale landslide potential analysis provides information about where should be focused on even though it is very difficult to distinguish. Accordingly, the authors intend to investigate the methods used by different countries, such as Hong Kong, Italy, Japan and Switzerland to clarify the assessment methodology. The objects include the place with susceptibility of rock slide and dip slope and the major landslide areas defined from historical records. Three different levels of scales are confirmed necessarily from country to slopeland, which are basin, catchment, and slope scales. Totally ten spots were classified with high large-scale landslide potential in the basin scale. The authors therefore focused on the catchment scale and employ risk matrix to classify the potential in this paper. The protected objects and large-scale landslide susceptibility ratio are two main indexes to classify the large-scale landslide risk. The protected objects are the constructions and transportation facilities. The large-scale landslide susceptibility ratio is based on the data of major landslide area and dip slope and rock slide areas. Totally 1,040 catchments are concerned and are classified into three levels, which are high, medium, and low levels. The proportions of high, medium, and low levels are 11%, 51%, and 38%, individually. This result represents the catchments with high proportion of protected objects or large-scale landslide susceptibility. The conclusion is made and it be the base material for the slopeland authorities when considering slopeland management and the further investigation.

  19. Dynamic Large-Scale Chromosomal Rearrangements Fuel Rapid Adaptation in Yeast Populations

    PubMed Central

    Chang, Shang-Lin; Lai, Huei-Yi; Tung, Shu-Yun; Leu, Jun-Yi

    2013-01-01

    Large-scale genome rearrangements have been observed in cells adapting to various selective conditions during laboratory evolution experiments. However, it remains unclear whether these types of mutations can be stably maintained in populations and how they impact the evolutionary trajectories. Here we show that chromosomal rearrangements contribute to extremely high copper tolerance in a set of natural yeast strains isolated from Evolution Canyon (EC), Israel. The chromosomal rearrangements in EC strains result in segmental duplications in chromosomes 7 and 8, which increase the copy number of genes involved in copper regulation, including the crucial transcriptional activator CUP2 and the metallothionein CUP1. The copy number of CUP2 is correlated with the level of copper tolerance, indicating that increasing dosages of a single transcriptional activator by chromosomal rearrangements has a profound effect on a regulatory pathway. By gene expression analysis and functional assays, we identified three previously unknown downstream targets of CUP2: PHO84, SCM4, and CIN2, all of which contributed to copper tolerance in EC strains. Finally, we conducted an evolution experiment to examine how cells maintained these changes in a fluctuating environment. Interestingly, the rearranged chromosomes were reverted back to the wild-type configuration at a high frequency and the recovered chromosome became fixed in less selective conditions. Our results suggest that transposon-mediated chromosomal rearrangements can be highly dynamic and can serve as a reversible mechanism during early stages of adaptive evolution. PMID:23358723

  20. EINSTEIN'S SIGNATURE IN COSMOLOGICAL LARGE-SCALE STRUCTURE

    SciTech Connect

    Bruni, Marco; Hidalgo, Juan Carlos; Wands, David

    2014-10-10

    We show how the nonlinearity of general relativity generates a characteristic nonGaussian signal in cosmological large-scale structure that we calculate at all perturbative orders in a large-scale limit. Newtonian gravity and general relativity provide complementary theoretical frameworks for modeling large-scale structure in ΛCDM cosmology; a relativistic approach is essential to determine initial conditions, which can then be used in Newtonian simulations studying the nonlinear evolution of the matter density. Most inflationary models in the very early universe predict an almost Gaussian distribution for the primordial metric perturbation, ζ. However, we argue that it is the Ricci curvature of comoving-orthogonal spatial hypersurfaces, R, that drives structure formation at large scales. We show how the nonlinear relation between the spatial curvature, R, and the metric perturbation, ζ, translates into a specific nonGaussian contribution to the initial comoving matter density that we calculate for the simple case of an initially Gaussian ζ. Our analysis shows the nonlinear signature of Einstein's gravity in large-scale structure.

  1. Unsaturated Hydraulic Conductivity for Evaporation in Large scale Heterogeneous Soils

    NASA Astrophysics Data System (ADS)

    Sun, D.; Zhu, J.

    2014-12-01

    In this study we aim to provide some practical guidelines of how the commonly used simple averaging schemes (arithmetic, geometric, or harmonic mean) perform in simulating large scale evaporation in a large scale heterogeneous landscape. Previous studies on hydraulic property upscaling focusing on steady state flux exchanges illustrated that an effective hydraulic property is usually more difficult to define for evaporation. This study focuses on upscaling hydraulic properties of large scale transient evaporation dynamics using the idea of the stream tube approach. Specifically, the two main objectives are: (1) if the three simple averaging schemes (i.e., arithmetic, geometric and harmonic means) of hydraulic parameters are appropriate in representing large scale evaporation processes, and (2) how the applicability of these simple averaging schemes depends on the time scale of evaporation processes in heterogeneous soils. Multiple realizations of local evaporation processes are carried out using HYDRUS-1D computational code (Simunek et al, 1998). The three averaging schemes of soil hydraulic parameters were used to simulate the cumulative flux exchange, which is then compared with the large scale average cumulative flux. The sensitivity of the relative errors to the time frame of evaporation processes is also discussed.

  2. Systematic Mutagenesis of the Escherichia coli Genome†

    PubMed Central

    Kang, Yisheng; Durfee, Tim; Glasner, Jeremy D.; Qiu, Yu; Frisch, David; Winterberg, Kelly M.; Blattner, Frederick R.

    2004-01-01

    A high-throughput method has been developed for the systematic mutagenesis of the Escherichia coli genome. The system is based on in vitro transposition of a modified Tn5 element, the Sce-poson, into linear fragments of each open reading frame. The transposon introduces both positive (kanamycin resistance) and negative (I-SceI recognition site) selectable markers for isolation of mutants and subsequent allele replacement, respectively. Reaction products are then introduced into the genome by homologous recombination via the λRed proteins. The method has yielded insertion alleles for 1976 genes during a first pass through the genome including, unexpectedly, a number of known and putative essential genes. Sce-poson insertions can be easily replaced by markerless mutations by using the I-SceI homing endonuclease to select against retention of the transposon as demonstrated by the substitution of amber and/or in-frame deletions in six different genes. This allows a Sce-poson-containing gene to be specifically targeted for either designed or random modifications, as well as permitting the stepwise engineering of strains with multiple mutations. The promiscuous nature of Tn5 transposition also enables a targeted gene to be dissected by using randomly inserted Sce-posons as shown by a lacZ allelic series. Finally, assessment of the insertion sites by an iterative weighted matrix algorithm reveals that these hyperactive Tn5 complexes generally recognize a highly degenerate asymmetric motif on one end of the target site helping to explain the randomness of Tn5 transposition. PMID:15262929

  3. Computer Simulation of Mutagenesis.

    ERIC Educational Resources Information Center

    North, J. C.; Dent, M. T.

    1978-01-01

    A FORTRAN program is described which simulates point-substitution mutations in the DNA strands of typical organisms. Its objective is to help students to understand the significance and structure of the genetic code, and the mechanisms and effect of mutagenesis. (Author/BB)

  4. 2004 Mutagenesis Gordon Conference

    SciTech Connect

    Dr. Sue Jinks-Robertson

    2005-09-16

    Mutations are genetic alterations that drive biological evolution and cause many, if not all, human diseases. Mutation originates via two distinct mechanisms: ''vertical'' variation is de novo change of one or few bases, whereas ''horizontal'' variation occurs by genetic recombination, which creates new mosaics of pre-existing sequences. The Mutagenesis Conference has traditionally focused on the generation of mutagenic intermediates during normal DNA synthesis or in response to environmental insults, as well as the diverse repair mechanisms that prevent the fixation of such intermediates as permanent mutations. While the 2004 Conference will continue to focus on the molecular mechanisms of mutagenesis, there will be increased emphasis on the biological consequences of mutations, both in terms of evolutionary processes and in terms of human disease. The meeting will open with two historical accounts of mutation research that recapitulate the intellectual framework of this field and thereby place the current research paradigms into perspective. The two introductory keynote lectures will be followed by sessions on: (1) mutagenic systems, (2) hypermutable sequences, (3) mechanisms of mutation, (4) mutation avoidance systems, (5) mutation in human hereditary and infectious diseases, (6) mutation rates in evolution and genotype-phenotype relationships, (7) ecology, mutagenesis and the modeling of evolution and (8) genetic diversity of the human population and models for human mutagenesis. The Conference will end with a synthesis of the meeting as the keynote closing lecture.

  5. High-resolution definition of the Vibrio cholerae essential gene set with hidden Markov model–based analyses of transposon-insertion sequencing data

    PubMed Central

    Chao, Michael C.; Pritchard, Justin R.; Zhang, Yanjia J.; Rubin, Eric J.; Livny, Jonathan; Davis, Brigid M.; Waldor, Matthew K.

    2013-01-01

    The coupling of high-density transposon mutagenesis to high-throughput DNA sequencing (transposon-insertion sequencing) enables simultaneous and genome-wide assessment of the contributions of individual loci to bacterial growth and survival. We have refined analysis of transposon-insertion sequencing data by normalizing for the effect of DNA replication on sequencing output and using a hidden Markov model (HMM)-based filter to exploit heretofore unappreciated information inherent in all transposon-insertion sequencing data sets. The HMM can smooth variations in read abundance and thereby reduce the effects of read noise, as well as permit fine scale mapping that is independent of genomic annotation and enable classification of loci into several functional categories (e.g. essential, domain essential or ‘sick’). We generated a high-resolution map of genomic loci (encompassing both intra- and intergenic sequences) that are required or beneficial for in vitro growth of the cholera pathogen, Vibrio cholerae. This work uncovered new metabolic and physiologic requirements for V. cholerae survival, and by combining transposon-insertion sequencing and transcriptomic data sets, we also identified several novel noncoding RNA species that contribute to V. cholerae growth. Our findings suggest that HMM-based approaches will enhance extraction of biological meaning from transposon-insertion sequencing genomic data. PMID:23901011

  6. Do Large-Scale Topological Features Correlate with Flare Properties?

    NASA Astrophysics Data System (ADS)

    DeRosa, Marc L.; Barnes, Graham

    2016-05-01

    In this study, we aim to identify whether the presence or absence of particular topological features in the large-scale coronal magnetic field are correlated with whether a flare is confined or eruptive. To this end, we first determine the locations of null points, spine lines, and separatrix surfaces within the potential fields associated with the locations of several strong flares from the current and previous sunspot cycles. We then validate the topological skeletons against large-scale features in observations, such as the locations of streamers and pseudostreamers in coronagraph images. Finally, we characterize the topological environment in the vicinity of the flaring active regions and identify the trends involving their large-scale topologies and the properties of the associated flares.

  7. A relativistic signature in large-scale structure

    NASA Astrophysics Data System (ADS)

    Bartolo, Nicola; Bertacca, Daniele; Bruni, Marco; Koyama, Kazuya; Maartens, Roy; Matarrese, Sabino; Sasaki, Misao; Verde, Licia; Wands, David

    2016-09-01

    In General Relativity, the constraint equation relating metric and density perturbations is inherently nonlinear, leading to an effective non-Gaussianity in the dark matter density field on large scales-even if the primordial metric perturbation is Gaussian. Intrinsic non-Gaussianity in the large-scale dark matter overdensity in GR is real and physical. However, the variance smoothed on a local physical scale is not correlated with the large-scale curvature perturbation, so that there is no relativistic signature in the galaxy bias when using the simplest model of bias. It is an open question whether the observable mass proxies such as luminosity or weak lensing correspond directly to the physical mass in the simple halo bias model. If not, there may be observables that encode this relativistic signature.

  8. Toward Improved Support for Loosely Coupled Large Scale Simulation Workflows

    SciTech Connect

    Boehm, Swen; Elwasif, Wael R; Naughton, III, Thomas J; Vallee, Geoffroy R

    2014-01-01

    High-performance computing (HPC) workloads are increasingly leveraging loosely coupled large scale simula- tions. Unfortunately, most large-scale HPC platforms, including Cray/ALPS environments, are designed for the execution of long-running jobs based on coarse-grained launch capabilities (e.g., one MPI rank per core on all allocated compute nodes). This assumption limits capability-class workload campaigns that require large numbers of discrete or loosely coupled simulations, and where time-to-solution is an untenable pacing issue. This paper describes the challenges related to the support of fine-grained launch capabilities that are necessary for the execution of loosely coupled large scale simulations on Cray/ALPS platforms. More precisely, we present the details of an enhanced runtime system to support this use case, and report on initial results from early testing on systems at Oak Ridge National Laboratory.

  9. Acoustic Studies of the Large Scale Ocean Circulation

    NASA Technical Reports Server (NTRS)

    Menemenlis, Dimitris

    1999-01-01

    Detailed knowledge of ocean circulation and its transport properties is prerequisite to an understanding of the earth's climate and of important biological and chemical cycles. Results from two recent experiments, THETIS-2 in the Western Mediterranean and ATOC in the North Pacific, illustrate the use of ocean acoustic tomography for studies of the large scale circulation. The attraction of acoustic tomography is its ability to sample and average the large-scale oceanic thermal structure, synoptically, along several sections, and at regular intervals. In both studies, the acoustic data are compared to, and then combined with, general circulation models, meteorological analyses, satellite altimetry, and direct measurements from ships. Both studies provide complete regional descriptions of the time-evolving, three-dimensional, large scale circulation, albeit with large uncertainties. The studies raise serious issues about existing ocean observing capability and provide guidelines for future efforts.

  10. Coupling between convection and large-scale circulation

    NASA Astrophysics Data System (ADS)

    Becker, T.; Stevens, B. B.; Hohenegger, C.

    2014-12-01

    The ultimate drivers of convection - radiation, tropospheric humidity and surface fluxes - are altered both by the large-scale circulation and by convection itself. A quantity to which all drivers of convection contribute is moist static energy, or gross moist stability, respectively. Therefore, a variance analysis of the moist static energy budget in radiative-convective equilibrium helps understanding the interaction of precipitating convection and the large-scale environment. In addition, this method provides insights concerning the impact of convective aggregation on this coupling. As a starting point, the interaction is analyzed with a general circulation model, but a model intercomparison study using a hierarchy of models is planned. Effective coupling parameters will be derived from cloud resolving models and these will in turn be related to assumptions used to parameterize convection in large-scale models.

  11. Large-scale current systems in the dayside Venus ionosphere

    NASA Technical Reports Server (NTRS)

    Luhmann, J. G.; Elphic, R. C.; Brace, L. H.

    1981-01-01

    The occasional observation of large-scale horizontal magnetic fields within the dayside ionosphere of Venus by the flux gate magnetometer on the Pioneer Venus orbiter suggests the presence of large-scale current systems. Using the measured altitude profiles of the magnetic field and the electron density and temperature, together with the previously reported neutral atmosphere density and composition, it is found that the local ionosphere can be described at these times by a simple steady state model which treats the unobserved quantities, such as the electric field, as parameters. When the model is appropriate, the altitude profiles of the ion and electron velocities and the currents along the satellite trajectory can be inferred. These results elucidate the configurations and sources of the ionospheric current systems which produce the observed large-scale magnetic fields, and in particular illustrate the effect of ion-neutral coupling in the determination of the current system at low altitudes.

  12. Magnetic Helicity and Large Scale Magnetic Fields: A Primer

    NASA Astrophysics Data System (ADS)

    Blackman, Eric G.

    2015-05-01

    Magnetic fields of laboratory, planetary, stellar, and galactic plasmas commonly exhibit significant order on large temporal or spatial scales compared to the otherwise random motions within the hosting system. Such ordered fields can be measured in the case of planets, stars, and galaxies, or inferred indirectly by the action of their dynamical influence, such as jets. Whether large scale fields are amplified in situ or a remnant from previous stages of an object's history is often debated for objects without a definitive magnetic activity cycle. Magnetic helicity, a measure of twist and linkage of magnetic field lines, is a unifying tool for understanding large scale field evolution for both mechanisms of origin. Its importance stems from its two basic properties: (1) magnetic helicity is typically better conserved than magnetic energy; and (2) the magnetic energy associated with a fixed amount of magnetic helicity is minimized when the system relaxes this helical structure to the largest scale available. Here I discuss how magnetic helicity has come to help us understand the saturation of and sustenance of large scale dynamos, the need for either local or global helicity fluxes to avoid dynamo quenching, and the associated observational consequences. I also discuss how magnetic helicity acts as a hindrance to turbulent diffusion of large scale fields, and thus a helper for fossil remnant large scale field origin models in some contexts. I briefly discuss the connection between large scale fields and accretion disk theory as well. The goal here is to provide a conceptual primer to help the reader efficiently penetrate the literature.

  13. Survey of decentralized control methods. [for large scale dynamic systems

    NASA Technical Reports Server (NTRS)

    Athans, M.

    1975-01-01

    An overview is presented of the types of problems that are being considered by control theorists in the area of dynamic large scale systems with emphasis on decentralized control strategies. Approaches that deal directly with decentralized decision making for large scale systems are discussed. It is shown that future advances in decentralized system theory are intimately connected with advances in the stochastic control problem with nonclassical information pattern. The basic assumptions and mathematical tools associated with the latter are summarized, and recommendations concerning future research are presented.

  14. Clearing and Labeling Techniques for Large-Scale Biological Tissues

    PubMed Central

    Seo, Jinyoung; Choe, Minjin; Kim, Sung-Yon

    2016-01-01

    Clearing and labeling techniques for large-scale biological tissues enable simultaneous extraction of molecular and structural information with minimal disassembly of the sample, facilitating the integration of molecular, cellular and systems biology across different scales. Recent years have witnessed an explosive increase in the number of such methods and their applications, reflecting heightened interest in organ-wide clearing and labeling across many fields of biology and medicine. In this review, we provide an overview and comparison of existing clearing and labeling techniques and discuss challenges and opportunities in the investigations of large-scale biological systems. PMID:27239813

  15. The Evolution of Baryons in Cosmic Large Scale Structure

    NASA Astrophysics Data System (ADS)

    Snedden, Ali; Arielle Phillips, Lara; Mathews, Grant James; Coughlin, Jared; Suh, In-Saeng; Bhattacharya, Aparna

    2015-01-01

    The environments of galaxies play a critical role in their formation and evolution. We study these environments using cosmological simulations with star formation and supernova feedback included. From these simulations, we parse the large scale structure into clusters, filaments and voids using a segmentation algorithm adapted from medical imaging. We trace the star formation history, gas phase and metal evolution of the baryons in the intergalactic medium as function of structure. We find that our algorithm reproduces the baryon fraction in the intracluster medium and that the majority of star formation occurs in cold, dense filaments. We present the consequences this large scale environment has for galactic halos and galaxy evolution.

  16. Corridors Increase Plant Species Richness at Large Scales

    SciTech Connect

    Damschen, Ellen I.; Haddad, Nick M.; Orrock,John L.; Tewksbury, Joshua J.; Levey, Douglas J.

    2006-09-01

    Habitat fragmentation is one of the largest threats to biodiversity. Landscape corridors, which are hypothesized to reduce the negative consequences of fragmentation, have become common features of ecological management plans worldwide. Despite their popularity, there is little evidence documenting the effectiveness of corridors in preserving biodiversity at large scales. Using a large-scale replicated experiment, we showed that habitat patches connected by corridors retain more native plant species than do isolated patches, that this difference increases over time, and that corridors do not promote invasion by exotic species. Our results support the use of corridors in biodiversity conservation.

  17. Large-scale ER-damper for seismic protection

    NASA Astrophysics Data System (ADS)

    McMahon, Scott; Makris, Nicos

    1997-05-01

    A large scale electrorheological (ER) damper has been designed, constructed, and tested. The damper consists of a main cylinder and a piston rod that pushes an ER-fluid through a number of stationary annular ducts. This damper is a scaled- up version of a prototype ER-damper which has been developed and extensively studied in the past. In this paper, results from comprehensive testing of the large-scale damper are presented, and the proposed theory developed for predicting the damper response is validated.

  18. Clearing and Labeling Techniques for Large-Scale Biological Tissues.

    PubMed

    Seo, Jinyoung; Choe, Minjin; Kim, Sung-Yon

    2016-06-30

    Clearing and labeling techniques for large-scale biological tissues enable simultaneous extraction of molecular and structural information with minimal disassembly of the sample, facilitating the integration of molecular, cellular and systems biology across different scales. Recent years have witnessed an explosive increase in the number of such methods and their applications, reflecting heightened interest in organ-wide clearing and labeling across many fields of biology and medicine. In this review, we provide an overview and comparison of existing clearing and labeling techniques and discuss challenges and opportunities in the investigations of large-scale biological systems. PMID:27239813

  19. Contribution of peculiar shear motions to large-scale structure

    NASA Technical Reports Server (NTRS)

    Mueler, Hans-Reinhard; Treumann, Rudolf A.

    1994-01-01

    Self-gravitating shear flow instability simulations in a cold dark matter-dominated expanding Einstein-de Sitter universe have been performed. When the shear flow speed exceeds a certain threshold, self-gravitating Kelvin-Helmoholtz instability occurs, forming density voids and excesses along the shear flow layer which serve as seeds for large-scale structure formation. A possible mechanism for generating shear peculiar motions are velocity fluctuations induced by the density perturbations of the postinflation era. In this scenario, short scales grow earlier than large scales. A model of this kind may contribute to the cellular structure of the luminous mass distribution in the universe.

  20. Large-scale liquid scintillation detectors for solar neutrinos

    NASA Astrophysics Data System (ADS)

    Benziger, Jay B.; Calaprice, Frank P.

    2016-04-01

    Large-scale liquid scintillation detectors are capable of providing spectral yields of the low energy solar neutrinos. These detectors require > 100 tons of liquid scintillator with high optical and radiopurity. In this paper requirements for low-energy neutrino detection by liquid scintillation are specified and the procedures to achieve low backgrounds in large-scale liquid scintillation detectors for solar neutrinos are reviewed. The designs, operations and achievements of Borexino, KamLAND and SNO+ in measuring the low-energy solar neutrino fluxes are reviewed.

  1. Genetic Signature of Histiocytic Sarcoma Revealed by a Sleeping Beauty Transposon Genetic Screen in Mice

    PubMed Central

    Been, Raha A.; Linden, Michael A.; Hager, Courtney J.; DeCoursin, Krista J.; Abrahante, Juan E.; Landman, Sean R.; Steinbach, Michael; Sarver, Aaron L.; Largaespada, David A.; Starr, Timothy K.

    2014-01-01

    Histiocytic sarcoma is a rare, aggressive neoplasm that responds poorly to therapy. Histiocytic sarcoma is thought to arise from macrophage precursor cells via genetic changes that are largely undefined. To improve our understanding of the etiology of histiocytic sarcoma we conducted a forward genetic screen in mice using the Sleeping Beauty transposon as a mutagen to identify genetic drivers of histiocytic sarcoma. Sleeping Beauty mutagenesis was targeted to myeloid lineage cells using the Lysozyme2 promoter. Mice with activated Sleeping Beauty mutagenesis had significantly shortened lifespan and the majority of these mice developed tumors resembling human histiocytic sarcoma. Analysis of transposon insertions identified 27 common insertion sites containing 28 candidate cancer genes. Several of these genes are known drivers of hematological neoplasms, like Raf1, Fli1, and Mitf, while others are well-known cancer genes, including Nf1, Myc, Jak2, and Pten. Importantly, several new potential drivers of histiocytic sarcoma were identified and could serve as targets for therapy for histiocytic sarcoma patients. PMID:24827933

  2. Decomposition and coordination of large-scale operations optimization

    NASA Astrophysics Data System (ADS)

    Cheng, Ruoyu

    Nowadays, highly integrated manufacturing has resulted in more and more large-scale industrial operations. As one of the most effective strategies to ensure high-level operations in modern industry, large-scale engineering optimization has garnered a great amount of interest from academic scholars and industrial practitioners. Large-scale optimization problems frequently occur in industrial applications, and many of them naturally present special structure or can be transformed to taking special structure. Some decomposition and coordination methods have the potential to solve these problems at a reasonable speed. This thesis focuses on three classes of large-scale optimization problems: linear programming, quadratic programming, and mixed-integer programming problems. The main contributions include the design of structural complexity analysis for investigating scaling behavior and computational efficiency of decomposition strategies, novel coordination techniques and algorithms to improve the convergence behavior of decomposition and coordination methods, as well as the development of a decentralized optimization framework which embeds the decomposition strategies in a distributed computing environment. The complexity study can provide fundamental guidelines to practical applications of the decomposition and coordination methods. In this thesis, several case studies imply the viability of the proposed decentralized optimization techniques for real industrial applications. A pulp mill benchmark problem is used to investigate the applicability of the LP/QP decentralized optimization strategies, while a truck allocation problem in the decision support of mining operations is used to study the MILP decentralized optimization strategies.

  3. A bibliographical surveys of large-scale systems

    NASA Technical Reports Server (NTRS)

    Corliss, W. R.

    1970-01-01

    A limited, partly annotated bibliography was prepared on the subject of large-scale system control. Approximately 400 references are divided into thirteen application areas, such as large societal systems and large communication systems. A first-author index is provided.

  4. The Large-Scale Structure of Scientific Method

    ERIC Educational Resources Information Center

    Kosso, Peter

    2009-01-01

    The standard textbook description of the nature of science describes the proposal, testing, and acceptance of a theoretical idea almost entirely in isolation from other theories. The resulting model of science is a kind of piecemeal empiricism that misses the important network structure of scientific knowledge. Only the large-scale description of…

  5. Individual Skill Differences and Large-Scale Environmental Learning

    ERIC Educational Resources Information Center

    Fields, Alexa W.; Shelton, Amy L.

    2006-01-01

    Spatial skills are known to vary widely among normal individuals. This project was designed to address whether these individual differences are differentially related to large-scale environmental learning from route (ground-level) and survey (aerial) perspectives. Participants learned two virtual environments (route and survey) with limited…

  6. Mixing Metaphors: Building Infrastructure for Large Scale School Turnaround

    ERIC Educational Resources Information Center

    Peurach, Donald J.; Neumerski, Christine M.

    2015-01-01

    The purpose of this analysis is to increase understanding of the possibilities and challenges of building educational infrastructure--the basic, foundational structures, systems, and resources--to support large-scale school turnaround. Building educational infrastructure often exceeds the capacity of schools, districts, and state education…

  7. Large-scale drift and Rossby wave turbulence

    NASA Astrophysics Data System (ADS)

    Harper, K. L.; Nazarenko, S. V.

    2016-08-01

    We study drift/Rossby wave turbulence described by the large-scale limit of the Charney–Hasegawa–Mima equation. We define the zonal and meridional regions as Z:= \\{{k} :| {k}y| \\gt \\sqrt{3}{k}x\\} and M:= \\{{k} :| {k}y| \\lt \\sqrt{3}{k}x\\} respectively, where {k}=({k}x,{k}y) is in a plane perpendicular to the magnetic field such that k x is along the isopycnals and k y is along the plasma density gradient. We prove that the only types of resonant triads allowed are M≤ftrightarrow M+Z and Z≤ftrightarrow Z+Z. Therefore, if the spectrum of weak large-scale drift/Rossby turbulence is initially in Z it will remain in Z indefinitely. We present a generalised Fjørtoft’s argument to find transfer directions for the quadratic invariants in the two-dimensional {k}-space. Using direct numerical simulations, we test and confirm our theoretical predictions for weak large-scale drift/Rossby turbulence, and establish qualitative differences with cases when turbulence is strong. We demonstrate that the qualitative features of the large-scale limit survive when the typical turbulent scale is only moderately greater than the Larmor/Rossby radius.

  8. Large Scale Field Campaign Contributions to Soil Moisture Remote Sensing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Large-scale field experiments have been an essential component of soil moisture remote sensing for over two decades. They have provided test beds for both the technology and science necessary to develop and refine satellite mission concepts. The high degree of spatial variability of soil moisture an...

  9. Large-scale V/STOL testing. [in wind tunnels

    NASA Technical Reports Server (NTRS)

    Koenig, D. G.; Aiken, T. N.; Aoyagi, K.; Falarski, M. D.

    1977-01-01

    Several facets of large-scale testing of V/STOL aircraft configurations are discussed with particular emphasis on test experience in the Ames 40- by 80-foot wind tunnel. Examples of powered-lift test programs are presented in order to illustrate tradeoffs confronting the planner of V/STOL test programs. It is indicated that large-scale V/STOL wind-tunnel testing can sometimes compete with small-scale testing in the effort required (overall test time) and program costs because of the possibility of conducting a number of different tests with a single large-scale model where several small-scale models would be required. The benefits of both high- and full-scale Reynolds numbers, more detailed configuration simulation, and number and type of onboard measurements increase rapidly with scale. Planning must be more detailed at large scale in order to balance the trade-offs between the increased costs, as number of measurements and model configuration variables increase and the benefits of larger amounts of information coming out of one test.

  10. Current Scientific Issues in Large Scale Atmospheric Dynamics

    NASA Technical Reports Server (NTRS)

    Miller, T. L. (Compiler)

    1986-01-01

    Topics in large scale atmospheric dynamics are discussed. Aspects of atmospheric blocking, the influence of transient baroclinic eddies on planetary-scale waves, cyclogenesis, the effects of orography on planetary scale flow, small scale frontal structure, and simulations of gravity waves in frontal zones are discussed.

  11. Large-Scale Machine Learning for Classification and Search

    ERIC Educational Resources Information Center

    Liu, Wei

    2012-01-01

    With the rapid development of the Internet, nowadays tremendous amounts of data including images and videos, up to millions or billions, can be collected for training machine learning models. Inspired by this trend, this thesis is dedicated to developing large-scale machine learning techniques for the purpose of making classification and nearest…

  12. Considerations for Managing Large-Scale Clinical Trials.

    ERIC Educational Resources Information Center

    Tuttle, Waneta C.; And Others

    1989-01-01

    Research management strategies used effectively in a large-scale clinical trial to determine the health effects of exposure to Agent Orange in Vietnam are discussed, including pre-project planning, organization according to strategy, attention to scheduling, a team approach, emphasis on guest relations, cross-training of personnel, and preparing…

  13. Ecosystem resilience despite large-scale altered hydro climatic conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Climate change is predicted to increase both drought frequency and duration, and when coupled with substantial warming, will establish a new hydroclimatological paradigm for many regions. Large-scale, warm droughts have recently impacted North America, Africa, Europe, Amazonia, and Australia result...

  14. Lessons from Large-Scale Renewable Energy Integration Studies: Preprint

    SciTech Connect

    Bird, L.; Milligan, M.

    2012-06-01

    In general, large-scale integration studies in Europe and the United States find that high penetrations of renewable generation are technically feasible with operational changes and increased access to transmission. This paper describes other key findings such as the need for fast markets, large balancing areas, system flexibility, and the use of advanced forecasting.

  15. Probabilistic Cuing in Large-Scale Environmental Search

    ERIC Educational Resources Information Center

    Smith, Alastair D.; Hood, Bruce M.; Gilchrist, Iain D.

    2010-01-01

    Finding an object in our environment is an important human ability that also represents a critical component of human foraging behavior. One type of information that aids efficient large-scale search is the likelihood of the object being in one location over another. In this study we investigated the conditions under which individuals respond to…

  16. Extracting Useful Semantic Information from Large Scale Corpora of Text

    ERIC Educational Resources Information Center

    Mendoza, Ray Padilla, Jr.

    2012-01-01

    Extracting and representing semantic information from large scale corpora is at the crux of computer-assisted knowledge generation. Semantic information depends on collocation extraction methods, mathematical models used to represent distributional information, and weighting functions which transform the space. This dissertation provides a…

  17. Efficient On-Demand Operations in Large-Scale Infrastructures

    ERIC Educational Resources Information Center

    Ko, Steven Y.

    2009-01-01

    In large-scale distributed infrastructures such as clouds, Grids, peer-to-peer systems, and wide-area testbeds, users and administrators typically desire to perform "on-demand operations" that deal with the most up-to-date state of the infrastructure. However, the scale and dynamism present in the operating environment make it challenging to…

  18. Large-Scale Environmental Influences on Aquatic Animal Health

    EPA Science Inventory

    In the latter portion of the 20th century, North America experienced numerous large-scale mortality events affecting a broad diversity of aquatic animals. Short-term forensic investigations of these events have sometimes characterized a causative agent or condition, but have rare...

  19. Newton iterative methods for large scale nonlinear systems

    SciTech Connect

    Walker, H.F.; Turner, K.

    1993-01-01

    Objective is to develop robust, efficient Newton iterative methods for general large scale problems well suited for discretizations of partial differential equations, integral equations, and other continuous problems. A concomitant objective is to develop improved iterative linear algebra methods. We first outline research on Newton iterative methods and then review work on iterative linear algebra methods. (DLC)

  20. Moon-based Earth Observation for Large Scale Geoscience Phenomena

    NASA Astrophysics Data System (ADS)

    Guo, Huadong; Liu, Guang; Ding, Yixing

    2016-07-01

    The capability of Earth observation for large-global-scale natural phenomena needs to be improved and new observing platform are expected. We have studied the concept of Moon as an Earth observation in these years. Comparing with manmade satellite platform, Moon-based Earth observation can obtain multi-spherical, full-band, active and passive information,which is of following advantages: large observation range, variable view angle, long-term continuous observation, extra-long life cycle, with the characteristics of longevity ,consistency, integrity, stability and uniqueness. Moon-based Earth observation is suitable for monitoring the large scale geoscience phenomena including large scale atmosphere change, large scale ocean change,large scale land surface dynamic change,solid earth dynamic change,etc. For the purpose of establishing a Moon-based Earth observation platform, we already have a plan to study the five aspects as follows: mechanism and models of moon-based observing earth sciences macroscopic phenomena; sensors' parameters optimization and methods of moon-based Earth observation; site selection and environment of moon-based Earth observation; Moon-based Earth observation platform; and Moon-based Earth observation fundamental scientific framework.

  1. Assuring Quality in Large-Scale Online Course Development

    ERIC Educational Resources Information Center

    Parscal, Tina; Riemer, Deborah

    2010-01-01

    Student demand for online education requires colleges and universities to rapidly expand the number of courses and programs offered online while maintaining high quality. This paper outlines two universities respective processes to assure quality in large-scale online programs that integrate instructional design, eBook custom publishing, Quality…

  2. Large-scale search for dark-matter axions

    SciTech Connect

    Hagmann, C.A., LLNL; Kinion, D.; Stoeffl, W.; Van Bibber, K.; Daw, E.J.; McBride, J.; Peng, H.; Rosenberg, L.J.; Xin, H.; Laveigne, J.; Sikivie, P.; Sullivan, N.S.; Tanner, D.B.; Moltz, D.M.; Powell, J.; Clarke, J.; Nezrick, F.A.; Turner, M.S.; Golubev, N.A.; Kravchuk, L.V.

    1998-01-01

    Early results from a large-scale search for dark matter axions are presented. In this experiment, axions constituting our dark-matter halo may be resonantly converted to monochromatic microwave photons in a high-Q microwave cavity permeated by a strong magnetic field. Sensitivity at the level of one important axion model (KSVZ) has been demonstrated.

  3. DESIGN OF LARGE-SCALE AIR MONITORING NETWORKS

    EPA Science Inventory

    The potential effects of air pollution on human health have received much attention in recent years. In the U.S. and other countries, there are extensive large-scale monitoring networks designed to collect data to inform the public of exposure risks to air pollution. A major crit...

  4. Over-driven control for large-scale MR dampers

    NASA Astrophysics Data System (ADS)

    Friedman, A. J.; Dyke, S. J.; Phillips, B. M.

    2013-04-01

    As semi-active electro-mechanical control devices increase in scale for use in real-world civil engineering applications, their dynamics become increasingly complicated. Control designs that are able to take these characteristics into account will be more effective in achieving good performance. Large-scale magnetorheological (MR) dampers exhibit a significant time lag in their force-response to voltage inputs, reducing the efficacy of typical controllers designed for smaller scale devices where the lag is negligible. A new control algorithm is presented for large-scale MR devices that uses over-driving and back-driving of the commands to overcome the challenges associated with the dynamics of these large-scale MR dampers. An illustrative numerical example is considered to demonstrate the controller performance. Via simulations of the structure using several seismic ground motions, the merits of the proposed control strategy to achieve reductions in various response parameters are examined and compared against several accepted control algorithms. Experimental evidence is provided to validate the improved capabilities of the proposed controller in achieving the desired control force levels. Through real-time hybrid simulation (RTHS), the proposed controllers are also examined and experimentally evaluated in terms of their efficacy and robust performance. The results demonstrate that the proposed control strategy has superior performance over typical control algorithms when paired with a large-scale MR damper, and is robust for structural control applications.

  5. Large-scale search for dark-matter axions

    SciTech Connect

    Kinion, D; van Bibber, K

    2000-08-30

    We review the status of two ongoing large-scale searches for axions which may constitute the dark matter of our Milky Way halo. The experiments are based on the microwave cavity technique proposed by Sikivie, and marks a ''second-generation'' to the original experiments performed by the Rochester-Brookhaven-Fermilab collaboration, and the University of Florida group.

  6. Large-Scale Innovation and Change in UK Higher Education

    ERIC Educational Resources Information Center

    Brown, Stephen

    2013-01-01

    This paper reflects on challenges universities face as they respond to change. It reviews current theories and models of change management, discusses why universities are particularly difficult environments in which to achieve large scale, lasting change and reports on a recent attempt by the UK JISC to enable a range of UK universities to employ…

  7. Global smoothing and continuation for large-scale molecular optimization

    SciTech Connect

    More, J.J.; Wu, Zhijun

    1995-10-01

    We discuss the formulation of optimization problems that arise in the study of distance geometry, ionic systems, and molecular clusters. We show that continuation techniques based on global smoothing are applicable to these molecular optimization problems, and we outline the issues that must be resolved in the solution of large-scale molecular optimization problems.

  8. Large-Scale Networked Virtual Environments: Architecture and Applications

    ERIC Educational Resources Information Center

    Lamotte, Wim; Quax, Peter; Flerackers, Eddy

    2008-01-01

    Purpose: Scalability is an important research topic in the context of networked virtual environments (NVEs). This paper aims to describe the ALVIC (Architecture for Large-scale Virtual Interactive Communities) approach to NVE scalability. Design/methodology/approach: The setup and results from two case studies are shown: a 3-D learning environment…

  9. Measurement, Sampling, and Equating Errors in Large-Scale Assessments

    ERIC Educational Resources Information Center

    Wu, Margaret

    2010-01-01

    In large-scale assessments, such as state-wide testing programs, national sample-based assessments, and international comparative studies, there are many steps involved in the measurement and reporting of student achievement. There are always sources of inaccuracies in each of the steps. It is of interest to identify the source and magnitude of…

  10. Large Scale Survey Data in Career Development Research

    ERIC Educational Resources Information Center

    Diemer, Matthew A.

    2008-01-01

    Large scale survey datasets have been underutilized but offer numerous advantages for career development scholars, as they contain numerous career development constructs with large and diverse samples that are followed longitudinally. Constructs such as work salience, vocational expectations, educational expectations, work satisfaction, and…