WorldWideScience

Sample records for assemble spliced alignments

  1. Automated Eukaryotic Gene Structure Annotation Using EVidenceModeler and the Program to Assemble Spliced Alignments

    Energy Technology Data Exchange (ETDEWEB)

    Haas, B J; Salzberg, S L; Zhu, W; Pertea, M; Allen, J E; Orvis, J; White, O; Buell, C R; Wortman, J R

    2007-12-10

    EVidenceModeler (EVM) is presented as an automated eukaryotic gene structure annotation tool that reports eukaryotic gene structures as a weighted consensus of all available evidence. EVM, when combined with the Program to Assemble Spliced Alignments (PASA), yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms. Our experiments on both rice and human genome sequences demonstrate that EVM produces automated gene structure annotation approaching the quality of manual curation.

  2. BBMap: A Fast, Accurate, Splice-Aware Aligner

    Energy Technology Data Exchange (ETDEWEB)

    Bushnell, Brian

    2014-03-17

    Alignment of reads is one of the primary computational tasks in bioinformatics. Of paramount importance to resequencing, alignment is also crucial to other areas - quality control, scaffolding, string-graph assembly, homology detection, assembly evaluation, error-correction, expression quantification, and even as a tool to evaluate other tools. An optimal aligner would greatly improve virtually any sequencing process, but optimal alignment is prohibitively expensive for gigabases of data. Here, we will present BBMap [1], a fast splice-aware aligner for short and long reads. We will demonstrate that BBMap has superior speed, sensitivity, and specificity to alternative high-throughput aligners bowtie2 [2], bwa [3], smalt, [4] GSNAP [5], and BLASR [6].

  3. AGORA: Assembly Guided by Optical Restriction Alignment

    Directory of Open Access Journals (Sweden)

    Lin Henry C

    2012-08-01

    Full Text Available Abstract Background Genome assembly is difficult due to repeated sequences within the genome, which create ambiguities and cause the final assembly to be broken up into many separate sequences (contigs. Long range linking information, such as mate-pairs or mapping data, is necessary to help assembly software resolve repeats, thereby leading to a more complete reconstruction of genomes. Prior work has used optical maps for validating assemblies and scaffolding contigs, after an initial assembly has been produced. However, optical maps have not previously been used within the genome assembly process. Here, we use optical map information within the popular de Bruijn graph assembly paradigm to eliminate paths in the de Bruijn graph which are not consistent with the optical map and help determine the correct reconstruction of the genome. Results We developed a new algorithm called AGORA: Assembly Guided by Optical Restriction Alignment. AGORA is the first algorithm to use optical map information directly within the de Bruijn graph framework to help produce an accurate assembly of a genome that is consistent with the optical map information provided. Our simulations on bacterial genomes show that AGORA is effective at producing assemblies closely matching the reference sequences. Additionally, we show that noise in the optical map can have a strong impact on the final assembly quality for some complex genomes, and we also measure how various characteristics of the starting de Bruijn graph may impact the quality of the final assembly. Lastly, we show that a proper choice of restriction enzyme for the optical map may substantially improve the quality of the final assembly. Conclusions Our work shows that optical maps can be used effectively to assemble genomes within the de Bruijn graph assembly framework. Our experiments also provide insights into the characteristics of the mapping data that most affect the performance of our algorithm, indicating the

  4. An in vivo genetic screen for genes involved in spliced leader trans-splicing indicates a crucial role for continuous de novo spliced leader RNP assembly.

    Science.gov (United States)

    Philippe, Lucas; Pandarakalam, George C; Fasimoye, Rotimi; Harrison, Neale; Connolly, Bernadette; Pettitt, Jonathan; Müller, Berndt

    2017-08-21

    Spliced leader (SL) trans-splicing is a critical element of gene expression in a number of eukaryotic groups. This process is arguably best understood in nematodes, where biochemical and molecular studies in Caenorhabditis elegans and Ascaris suum have identified key steps and factors involved. Despite this, the precise details of SL trans-splicing have yet to be elucidated. In part, this is because the systematic identification of the molecules involved has not previously been possible due to the lack of a specific phenotype associated with defects in this process. We present here a novel GFP-based reporter assay that can monitor SL1 trans-splicing in living C. elegans. Using this assay, we have identified mutants in sna-1 that are defective in SL trans-splicing, and demonstrate that reducing function of SNA-1, SNA-2 and SUT-1, proteins that associate with SL1 RNA and related SmY RNAs, impairs SL trans-splicing. We further demonstrate that the Sm proteins and pICln, SMN and Gemin5, which are involved in small nuclear ribonucleoprotein assembly, have an important role in SL trans-splicing. Taken together these results provide the first in vivo evidence for proteins involved in SL trans-splicing, and indicate that continuous replacement of SL ribonucleoproteins consumed during trans-splicing reactions is essential for effective trans-splicing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Making Macroscopic Assemblies of Aligned Carbon Nanotubes

    Science.gov (United States)

    Smalley, Richard E.; Colbert, Daniel T.; Smith, Ken A.; Walters, Deron A.; Casavant, Michael J.; Qin, Xiaochuan; Yakobson, Boris; Hauge, Robert H.; Saini, Rajesh Kumar; Chiung, Wan-Ting; hide

    2005-01-01

    A method of aligning and assembling single-wall carbon nanotubes (SWNTs) to fabricate macroscopic structures has been invented. The method entails suspending SWNTs in a fluid, orienting the SWNTs by use of a magnetic and/or electric field, and then removing the aligned SWNTs from suspension in such a way as to assemble them while maintaining the alignment. SWNTs are essentially tubular extensions of fullerene molecules. It is desirable to assemble aligned SWNTs into macroscopic structures because the common alignment of the SWNTs in such a structure makes it possible to exploit, on a macroscopic scale, the unique mechanical, chemical, and electrical properties that individual oriented SWNTs exhibit at the molecular level. Because of their small size and high electrical conductivity, carbon nanotubes, and especially SWNTs, are useful for making electrical connectors in integrated circuits. Carbon nanotubes can be used as antennas at optical frequencies, and as probes in scanning tunneling microscopes, atomic-force microscopes, and the like. Carbon nanotubes can be used with or instead of carbon black in tires. Carbon nanotubes are useful as supports for catalysts. Ropes of SWNTs are metallic and, as such, are potentially useful in some applications in which electrical conductors are needed - for example, they could be used as additives in formulating electrically conductive paints. Finally, macroscopic assemblies of aligned SWNTs can serve as templates for the growth of more and larger structures of the same type. The great variety of tubular fullerene molecules and of the structures that could be formed by assembling them in various ways precludes a complete description of the present method within the limits of this article. It must suffice to present a typical example of the use of one of many possible variants of the method to form a membrane comprising SWNTs aligned substantially parallel to each other in the membrane plane. The apparatus used in this variant

  6. Assembly of pre-mRNA splicing complex is cap dependent.

    Science.gov (United States)

    Patzelt, E; Thalmann, E; Hartmuth, K; Blaas, D; Kuechler, E

    1987-01-01

    To study the influence of the ubiquitous cap structure of nuclear pre-mRNAs on the assembly of a functional splicing complex, the in vitro splicing of a truncated human metallothionein pre-mRNA was examined in the presence of the cap analogue m7GTP. Significant inhibition of splicing was observed at a concentration as low as 5 microM m7GTP. Analysis of the splicing reaction on glycerol density gradients showed two complexes sedimenting at 45S and 22S. When the reaction was carried out in presence of m7GTP a marked decrease of the material sedimenting at 45S, representing the active splicing complex, was observed. When capped pre-mRNA was replaced by uncapped pre-mRNA, complex formation was significantly reduced. These data indicate that the cap structure plays an important yet unknown role in the assembly of spliceosomes. Images PMID:3644239

  7. Assembly of splicing complexes on exon 11 of the human insulin receptor gene does not correlate with splicing efficiency in-vitro

    Directory of Open Access Journals (Sweden)

    Caples Matt

    2004-07-01

    Full Text Available Abstract Background Incorporation of exon 11 of the insulin receptor gene is both developmentally and hormonally-regulated. Previously, we have shown the presence of enhancer and silencer elements that modulate the incorporation of the small 36-nucleotide exon. In this study, we investigated the role of inherent splice site strength in the alternative splicing decision and whether recognition of the splice sites is the major determinant of exon incorporation. Results We found that mutation of the flanking sub-optimal splice sites to consensus sequences caused the exon to be constitutively spliced in-vivo. These findings are consistent with the exon-definition model for splicing. In-vitro splicing of RNA templates containing exon 11 and portions of the upstream intron recapitulated the regulation seen in-vivo. Unexpectedly, we found that the splice sites are occupied and spliceosomal complex A was assembled on all templates in-vitro irrespective of splicing efficiency. Conclusion These findings demonstrate that the exon-definition model explains alternative splicing of exon 11 in the IR gene in-vivo but not in-vitro. The in-vitro results suggest that the regulation occurs at a later step in spliceosome assembly on this exon.

  8. Evaluation of tools for long read RNA-seq splice-aware alignment.

    Science.gov (United States)

    Križanovic, Krešimir; Echchiki, Amina; Roux, Julien; Šikic, Mile

    2017-10-23

    High-throughput sequencing has transformed the study of gene expression levels through RNA-seq, a technique that is now routinely used by various fields, such as genetic research or diagnostics. The advent of third generation sequencing technologies providing significantly longer reads opens up new possibilities. However, the high error rates common to these technologies set new bioinformatics challenges for the gapped alignment of reads to their genomic origin. In this study, we have explored how currently available RNA-seq splice-aware alignment tools cope with increased read lengths and error rates. All tested tools were initially developed for short NGS reads, but some have claimed support for long Pacific Biosciences (PacBio) or even Oxford Nanopore Technologies (ONT) MinION reads. The tools were tested on synthetic and real datasets from two technologies (PacBio and ONT MinION). Alignment quality and resource usage were compared across different aligners. The effect of error correction of long reads was explored, both using self-correction and correction with an external short reads dataset. A tool was developed for evaluating RNA-seq alignment results. This tool can be used to compare the alignment of simulated reads to their genomic origin, or to compare the alignment of real reads to a set of annotated transcripts. Our tests show that while some RNA-seq aligners were unable to cope with long error-prone reads, others produced overall good results. We further show that alignment accuracy can be improved using error-corrected reads. https://github.com/kkrizanovic/RNAseqEval, https://figshare.com/projects/RNAseq_benchmark/24391. mile.sikic@fer.hr. Supplementary data are available at Bioinformatics online.

  9. Optical assembly and alignment for the National Ignition Facility project

    Science.gov (United States)

    Hurst, Patricia A.; Grasz, Erna L.; Wong, Henry; Schmitt, Ed H.; Simmons, Matt R.

    1998-06-01

    The National Ignition Facility (NIF) will use about 8,000 large optics to carry a high-power laser through a stadium- size building, and will do so on a very tight schedule and budget. The collocated Optics Assembly Building (OAB) will assemble and align, in a clean-room environment, the NIF's large optics, which are the biggest optics ever assembled in such an environment. In addition, the OAB must allow for just-in-time processing and clean transfer to the areas where the optics will be used. By using a mixture of off- the-shelf and newly designed equipment and by working with industry, we have developed innovative handling systems to perform the clean assembly and precise alignment required for the full variety of optics, as well as for postassembly inspection. We have also developed a set of loading mechanisms that safely get the clean optics to their places in the main NIF building.

  10. Waferscale assembly of Field-Aligned nanotube Networks (FANs)

    DEFF Research Database (Denmark)

    Dimaki, Maria; Bøggild, Peter

    2006-01-01

    frequencies of the electrical field used to attract the nanotubes to the electrodes. Preliminary data of response to visible light irradiation as well as changes in the humidity indicate that the field aligned networks could be used as sensor components that may well integrate with CMOS due to mild assembly...

  11. Aligned and Electrospun Piezoelectric Polymer Fiber Assembly and Scaffold

    Science.gov (United States)

    Scott-Carnell, Lisa A. (Inventor); Siochi, Emilie J. (Inventor); Holloway, Nancy M. (Inventor); Leong, Kam W. (Inventor); Kulangara, Karina (Inventor)

    2015-01-01

    A scaffold assembly and related methods of manufacturing and/or using the scaffold for stem cell culture and tissue engineering applications are disclosed which at least partially mimic a native biological environment by providing biochemical, topographical, mechanical and electrical cues by using an electroactive material. The assembly includes at least one layer of substantially aligned, electrospun polymer fiber having an operative connection for individual voltage application. A method of cell tissue engineering and/or stem cell differentiation uses the assembly seeded with a sample of cells suspended in cell culture media, incubates and applies voltage to one or more layers, and thus produces cells and/or a tissue construct. In another aspect, the invention provides a method of manufacturing the assembly including the steps of providing a first pre-electroded substrate surface; electrospinning a first substantially aligned polymer fiber layer onto the first surface; providing a second pre-electroded substrate surface; electrospinning a second substantially aligned polymer fiber layer onto the second surface; and, retaining together the layered surfaces with a clamp and/or an adhesive compound.

  12. Aligned and Electrospun Piezoelectric Polymer Fiber Assembly and Scaffold

    Science.gov (United States)

    Scott Carnell, Lisa A. (Inventor); Siochi, Emilie J. (Inventor); Holloway, Nancy M. (Inventor); Leong, Kam W. (Inventor); Kulangara, Karina (Inventor)

    2017-01-01

    A method of manufacturing and/or using a scaffold assembly for stem cell culture and tissue engineering applications is disclosed. The scaffold at least partially mimics a native biological environment by providing biochemical, topographical, mechanical and electrical cues by using an electroactive material. The assembly includes at least one layer of substantially aligned, electrospun polymer fiber having an operative connection for individual voltage application. A method of cell tissue engineering and/or stem cell differentiation that uses the assembly seeded with a sample of cells suspended in cell culture media, incubates and applies voltage to one or more layers, and thus produces cells and/or a tissue construct. In another aspect, the invention provides a method of manufacturing the assembly including the steps of providing a first pre-electroded substrate surface; electrospinning a first substantially aligned polymer fiber layer onto the first surface; providing a second pre-electroded substrate surface; electrospinning a second substantially aligned polymer fiber layer onto the second surface; and, retaining together the layered surfaces with a clamp and/or an adhesive compound.

  13. Alignment measurements uncertainties for large assemblies using probabilistic analysis techniques

    CERN Document Server

    AUTHOR|(CDS)2090816; Almond, Heather

    Big science and ambitious industrial projects continually push forward with technical requirements beyond the grasp of conventional engineering techniques. Example of those are ultra-high precision requirements in the field of celestial telescopes, particle accelerators and aerospace industry. Such extreme requirements are limited largely by the capability of the metrology used, namely, it’s uncertainty in relation to the alignment tolerance required. The current work was initiated as part of Maria Curie European research project held at CERN, Geneva aiming to answer those challenges as related to future accelerators requiring alignment of 2 m large assemblies to tolerances in the 10 µm range. The thesis has found several gaps in current knowledge limiting such capability. Among those was the lack of application of state of the art uncertainty propagation methods in alignment measurements metrology. Another major limiting factor found was the lack of uncertainty statements in the thermal errors compensatio...

  14. Error analysis on assembly and alignment of laser optical unit

    Directory of Open Access Journals (Sweden)

    Zhao Xiong

    2015-07-01

    Full Text Available As one of largest optical units used in high-power laser inertial confinement fusion facility, the large-aperture transport mirror’s misalignment error can have a very negative impact on the targeting performance of laser beams. In this article, we have carried out a fundamental analysis on the mounting and misalignment errors of transport mirror. An integrated simulated assembly station is proposed to align the mirror precisely, and the design of transport mirror unit is optimized to satisfy the stringent specifications. Finally, methods that integrated theoretical modeling, numerical simulation, and field experiments are used to evaluate the mirror’s alignment, and the results indicate a more robust and precise alignment performance of new design.

  15. Assembly Test of Elastic Averaging Technique to Improve Mechanical Alignment for Accelerating Structure Assemblies in CLIC

    CERN Document Server

    Huopana, J

    2010-01-01

    The CLIC (Compact LInear Collider) is being studied at CERN as a potential multi-TeV e+e- collider [1]. The manufacturing and assembly tolerances for the required RF-components are important for the final efficiency and for the operation of CLIC. The proper function of an accelerating structure is very sensitive to errors in shape and location of the accelerating cavity. This causes considerable issues in the field of mechanical design and manufacturing. Currently the design of the accelerating structures is a disk design. Alternatively it is possible to create the accelerating assembly from quadrants, which favour the mass manufacturing. The functional shape inside of the accelerating structure remains the same and a single assembly uses less parts. The alignment of these quadrants has been previously made kinematic by using steel pins or spheres to align the pieces together. This method proved to be a quite tedious and time consuming method of assembly. To limit the number of different error sources, a meth...

  16. Assembly, loading, and alignment of an analytical ultracentrifuge sample cell.

    Science.gov (United States)

    Balbo, Andrea; Zhao, Huaying; Brown, Patrick H; Schuck, Peter

    2009-11-05

    The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions. This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions

  17. In Vitro Seamless Stack Enzymatic Assembly of DNA Molecules Based on a Strategy Involving Splicing of Restriction Sites.

    Science.gov (United States)

    Yu, Dong; Tan, Yanning; Sun, Zhizhong; Sun, Xuewu; Sheng, Xiabing; Zhou, Tianshun; Liu, Ling; Mo, Yi; Jiang, Beibei; Ouyang, Ning; Yin, Xiaolin; Duan, Meijuan; Yuan, Dingyang

    2017-10-27

    The standard binary enzymatic assembly, which operates by inserting one DNA fragment into a plasmid, has a higher assembly success rate than the polynary enzymatic assembly, which inserts two or more fragments into the plasmid. However, it often leaves a nucleotide scar at the junction site. When a large DNA molecule is assembled stepwise into a backbone plasmid in a random piecewise manner, the scars will damage the structure of the original DNA sequence in the final assembled plasmids. Here, we propose an in vitro Seamless Stack Enzymatic Assembly (SSEA) method, a novel binary enzymatic assembly method involving a seamless strategy of splicing restriction sites via a stepwise process of multiple enzymatic reactions that does not leave nucleotide scars at the junction sites. We have demonstrated the success and versatility of this method through the assembly of 1) a 4.98 kb DNA molecule in the 5' → 3' direction using BamHI to generate the sticky end of the assembly entrance, 2) a 7.09 kb DNA molecule in the 3' → 5' direction using SmaI to generate the blunt end of the assembly entrance, and 3) an 11.88 kb DNA molecule by changing the assembly entrance.

  18. Proteomic analysis of Entamoeba histolytica in vivo assembled pre-mRNA splicing complexes.

    Science.gov (United States)

    Valdés, Jesús; Nozaki, Tomoyoshi; Sato, Emi; Chiba, Yoko; Nakada-Tsukui, Kumiko; Villegas-Sepúlveda, Nicolás; Winkler, Robert; Azuara-Liceaga, Elisa; Mendoza-Figueroa, María Saraí; Watanabe, Natsuki; Santos, Herbert J; Saito-Nakano, Yumiko; Galindo-Rosales, José Manuel

    2014-12-05

    The genome of the human intestinal parasite Entamoeba histolytica contains nearly 3000 introns and bioinformatic predictions indicate that major and minor spliceosomes occur in Entamoeba. However, except for the U2-, U4-, U5- and U6 snRNAs, no other splicing factor has been cloned and characterized. Here, we HA-tagged cloned the snRNP component U1A and assessed its expression and nuclear localization. Because the snRNP-free U1A form interacts with polyadenylate-binding protein, HA-U1A immunoprecipitates could identify early and late splicing complexes. Avoiding Entamoeba's endonucleases and ensuring the precipitation of RNA-binding proteins, parasite cultures were UV cross-linked prior to nuclear fraction immunoprecipitations with HA antibodies, and precipitates were subjected to tandem mass spectrometry (MS/MS) analyses. To discriminate their nuclear roles (chromatin-, co-transcriptional-, splicing-related), MS/MS analyses were carried out with proteins eluted with MS2-GST-sepharose from nuclear extracts of an MS2 aptamer-tagged Rabx13 intron amoeba transformant. Thus, we probed thirty-six Entamoeba proteins corresponding to 32 cognate splicing-specific factors, including 13 DExH/D helicases required for all stages of splicing, and 12 different splicing-related helicases were identified also. Furthermore 50 additional proteins, possibly involved in co-transcriptional processes were identified, revealing the complexity of co-transcriptional splicing in Entamoeba. Some of these later factors were not previously found in splicing complex analyses. Numerous facts about the splicing of the nearly 3000 introns of the Entamoeba genome have not been unraveled, particularly the splicing factors and their activities. Considering that many of such introns are located in metabolic genes, the knowledge of the splicing cues has the potential to be used to attack or control the parasite. We have found numerous new splicing-related factors which could have therapeutic benefit. We

  19. Unidirectional molecular assembly alignment on graphene enabled by nanomechanical symmetry breaking.

    Science.gov (United States)

    Hong, Liu; Nishihara, Taishi; Hijikata, Yuh; Miyauchi, Yuhei; Itami, Kenichiro

    2018-02-05

    Precise fabrication of molecular assemblies on a solid surface has long been of central interest in surface science. Their perfectly oriented growth only along a desired in-plane direction, however, remains a challenge, because of the thermodynamical equivalence of multiple axis directions on a solid-surface lattice. Here we demonstrate the successful fabrication of an in-plane, unidirectional molecular assembly on graphene. Our methodology relies on nanomechanical symmetry breaking effects under atomic force microscopy tip scanning, which has never been used in molecular alignment. Individual one-dimensional (1D) molecular assemblies were aligned along a selected symmetry axis of the graphene lattice under finely-tuned scanning conditions after removing initially-adsorbed molecules. Experimental statistics and computational simulations suggest that the anisotropic tip scanning locally breaks the directional equivalence of the graphene surface, which enables nucleation of the unidirectional 1D assemblies. Our findings will open new opportunities in the molecular alignment control on various atomically flat surfaces.

  20. Testing to evaluate synergistic effects from LOCA environments. Test IX. Simultaneous mode; cables, splice assemblies, and electrical insulation samples

    Energy Technology Data Exchange (ETDEWEB)

    Thome, F.V.

    1978-04-01

    This test was conducted to complement Test VIII which was a sequential test of cables, cable splices, and insulation samples. In this test, the generic LOCA environments (radiation, temperature, pressure, chemical spray) were simulated and simultaneously applied to the test items. There were no failures of any assemblies and all were able to function at rated current and voltage throughout the entire test. An additional parameter, dissipation factor, was monitored in this test and when used in conjunction with capacitance, provided a better indication of insulation degradation.

  1. SeqLib: a C ++ API for rapid BAM manipulation, sequence alignment and sequence assembly.

    Science.gov (United States)

    Wala, Jeremiah; Beroukhim, Rameen

    2017-03-01

    We present SeqLib, a C ++ API and command line tool that provides a rapid and user-friendly interface to BAM/SAM/CRAM files, global sequence alignment operations and sequence assembly. Four C libraries perform core operations in SeqLib: HTSlib for BAM access, BWA-MEM and BLAT for sequence alignment and Fermi for error correction and sequence assembly. Benchmarking indicates that SeqLib has lower CPU and memory requirements than leading C ++ sequence analysis APIs. We demonstrate an example of how minimal SeqLib code can extract, error-correct and assemble reads from a CRAM file and then align with BWA-MEM. SeqLib also provides additional capabilities, including chromosome-aware interval queries and read plotting. Command line tools are available for performing integrated error correction, micro-assemblies and alignment. SeqLib is available on Linux and OSX for the C ++98 standard and later at github.com/walaj/SeqLib. SeqLib is released under the Apache2 license. Additional capabilities for BLAT alignment are available under the BLAT license. jwala@broadinstitue.org ; rameen@broadinstitute.org.

  2. Construction of rugged, ultrastable optical assemblies with optical component alignment at the few microradian level.

    Science.gov (United States)

    Killow, Christian J; Fitzsimons, Ewan D; Hough, James; Perreur-Lloyd, Michael; Robertson, David I; Rowan, Sheila; Ward, Henry

    2013-01-10

    A method for constructing quasimonolithic, precision-aligned optical assemblies is presented. Hydroxide-catalysis bonding is used, adapted to allow optimization of component fine alignment prior to the bond setting. We demonstrate the technique by bonding a fused silica mirror substrate to a fused silica baseplate. In-plane component placement at the submicrometer level is achieved, resulting in angular control of a reflected laser beam at the sub-10-μrad level. Within the context of the LISA Pathfinder mission, the technique has been demonstrated as suitable for use in space-flight applications. It is expected that there will also be applications in a wide range of areas where accuracy, stability, and strength of optical assemblies are important. © 2013 Optical Society of America

  3. Laser micro-machining of hydrophobic-hydrophilic patterns for fluid driven self-alignment in micro-assembly

    NARCIS (Netherlands)

    Römer, Gerardus Richardus, Bernardus, Engelina; Jorritsma, Mark; Arnaldo del Cerro, D.; Chang, Bo; Liimatainen, Ville; Zhou, Quan; Huis in 't Veld, Bert

    2011-01-01

    Fluid driven self-alignment is a low cost alternative to fast but relatively inaccurate robotic pickand-place assembly of micro-fabricated components. This fluidic self-alignment technique relies on a hydrophobic-hydrophilic pattern on the surface of the receiving substrate, which confines a fluid

  4. Shifting meiotic to mitotic spindle assembly in oocytes disrupts chromosome alignment.

    Science.gov (United States)

    Bennabi, Isma; Quéguiner, Isabelle; Kolano, Agnieszka; Boudier, Thomas; Mailly, Philippe; Verlhac, Marie-Hélène; Terret, Marie-Emilie

    2018-01-12

    Mitotic spindles assemble from two centrosomes, which are major microtubule-organizing centers (MTOCs) that contain centrioles. Meiotic spindles in oocytes, however, lack centrioles. In mouse oocytes, spindle microtubules are nucleated from multiple acentriolar MTOCs that are sorted and clustered prior to completion of spindle assembly in an "inside-out" mechanism, ending with establishment of the poles. We used HSET (kinesin-14) as a tool to shift meiotic spindle assembly toward a mitotic "outside-in" mode and analyzed the consequences on the fidelity of the division. We show that HSET levels must be tightly gated in meiosis I and that even slight overexpression of HSET forces spindle morphogenesis to become more mitotic-like: rapid spindle bipolarization and pole assembly coupled with focused poles. The unusual length of meiosis I is not sufficient to correct these early spindle morphogenesis defects, resulting in severe chromosome alignment abnormalities. Thus, the unique "inside-out" mechanism of meiotic spindle assembly is essential to prevent chromosomal misalignment and production of aneuploidy gametes. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

  5. Alignment, Assembly and Testing of High Energy X-Ray Optics

    Science.gov (United States)

    Gubarev, Mikhail; Ramsey, Brian

    2005-01-01

    We are developing grazing-incidence x-ray imaging optics for a balloon-borne hard x-ray telescope (HERO). The HERO payload, scheduled for launch in May 2005, currently consists of 8 mirror modules each containing 12 mirror shells fabricated using electroform-nickel replication off super-polished cylindrical mandrels. An optical system developed for aligning and assembling the shells in the modules will be described. Sources for systematic errors associated with this process will be discussed and results from on-ground x-ray testing of each module will be presented.

  6. Photoconduction properties in aligned assemblies of colloidal CdSe/CdS nanorods.

    Science.gov (United States)

    Persano, Anna; De Giorgi, Milena; Fiore, Angela; Cingolani, Roberto; Manna, Liberato; Cola, Adriano; Krahne, Roman

    2010-03-23

    We report on photoconduction and optical properties of aligned assemblies of core-shell CdSe/CdS nanorods prepared by a seeded growth approach. We fabricate oriented layers of nanorods by drop casting the nanorods from a solution on substrates with prepatterned, micrometer-spaced electrodes and obtain nanorod alignment due to the coffee stain effect. The photoconductivity of the nanorod layers can be improved significantly by an annealing process under vacuum conditions. The spectral response of the photocurrent shows distinct features that can be assigned to the electronic level structure of the core-shell nanorods and that relate well to the spectra obtained by absorption measurements. We study assemblies of nanorods oriented parallel and perpendicular to the applied electric field by the combined use of photocurrent and photoluminescence spectroscopy. We obtain consistent results which show that charge carrier separation and transport are more efficient for nanorods oriented parallel to the electric field. We also investigate the light polarization sensitivity of the photocurrent for the oriented nanorod layers and observe higher conductivity in the case of perpendicular polarization with respect to the long axis of the nanorods.

  7. Assembly of vorticity-aligned hard-sphere colloidal strings in a simple shear flow

    KAUST Repository

    Cheng, X.

    2011-12-23

    Colloidal suspensions self-assemble into equilibrium structures ranging from face- and body-centered cubic crystals to binary ionic crystals, and even kagome lattices. When driven out of equilibrium by hydrodynamic interactions, even more diverse structures can be accessed. However, mechanisms underlying out-of-equilibrium assembly are much less understood, though such processes are clearly relevant in many natural and industrial systems. Even in the simple case of hard-sphere colloidal particles under shear, there are conflicting predictions about whether particles link up into string-like structures along the shear flow direction. Here, using confocal microscopy, we measure the shear-induced suspension structure. Surprisingly, rather than flow-aligned strings, we observe log-rolling strings of particles normal to the plane of shear. By employing Stokesian dynamics simulations, we address the mechanism leading to this out-of-equilibrium structure and show that it emerges from a delicate balance between hydrodynamic and interparticle interactions. These results demonstrate a method for assembling large-scale particle structures using shear flows.

  8. Assembly of vorticity-aligned hard-sphere colloidal strings in a simple shear flow

    Science.gov (United States)

    Cheng, Xiang; Xu, Xinliang; Rice, Stuart A.; Dinner, Aaron R.; Cohen, Itai

    2012-01-01

    Colloidal suspensions self-assemble into equilibrium structures ranging from face- and body-centered cubic crystals to binary ionic crystals, and even kagome lattices. When driven out of equilibrium by hydrodynamic interactions, even more diverse structures can be accessed. However, mechanisms underlying out-of-equilibrium assembly are much less understood, though such processes are clearly relevant in many natural and industrial systems. Even in the simple case of hard-sphere colloidal particles under shear, there are conflicting predictions about whether particles link up into string-like structures along the shear flow direction. Here, using confocal microscopy, we measure the shear-induced suspension structure. Surprisingly, rather than flow-aligned strings, we observe log-rolling strings of particles normal to the plane of shear. By employing Stokesian dynamics simulations, we address the mechanism leading to this out-of-equilibrium structure and show that it emerges from a delicate balance between hydrodynamic and interparticle interactions. These results demonstrate a method for assembling large-scale particle structures using shear flows. PMID:22198839

  9. A self-assembled, π-stacked complex as a finely-tunable magnetic aligner for biomolecular NMR applications.

    Science.gov (United States)

    Sato, Sota; Takeuchi, Ryosuke; Yagi-Utsumi, Maho; Yamaguchi, Takumi; Yamaguchi, Yoshiki; Kato, Koichi; Fujita, Makoto

    2015-02-14

    The number of molecules constituting 1D intermolecular aggregates of the π-stacked self-assembled complex was controlled by altering the concentration and temperature. Enhanced magnetic orientation was observed for the aggregates with larger aggregation numbers. It was demonstrated that the designable complex aggregates serve as magnetic aligners to induce magnetic alignment upon an analyte protein coexisting in the solution, resulting in the observation of residual dipolar coupling (RDC) of the analyte.

  10. RNA-Seq analysis and annotation of a draft blueberry genome assembly identifies candidate genes involved in fruit ripening, biosynthesis of bioactive compounds, and stage-specific alternative splicing.

    Science.gov (United States)

    Gupta, Vikas; Estrada, April D; Blakley, Ivory; Reid, Rob; Patel, Ketan; Meyer, Mason D; Andersen, Stig Uggerhøj; Brown, Allan F; Lila, Mary Ann; Loraine, Ann E

    2015-01-01

    Blueberries are a rich source of antioxidants and other beneficial compounds that can protect against disease. Identifying genes involved in synthesis of bioactive compounds could enable the breeding of berry varieties with enhanced health benefits. Toward this end, we annotated a previously sequenced draft blueberry genome assembly using RNA-Seq data from five stages of berry fruit development and ripening. Genome-guided assembly of RNA-Seq read alignments combined with output from ab initio gene finders produced around 60,000 gene models, of which more than half were similar to proteins from other species, typically the grape Vitis vinifera. Comparison of gene models to the PlantCyc database of metabolic pathway enzymes identified candidate genes involved in synthesis of bioactive compounds, including bixin, an apocarotenoid with potential disease-fighting properties, and defense-related cyanogenic glycosides, which are toxic. Cyanogenic glycoside (CG) biosynthetic enzymes were highly expressed in green fruit, and a candidate CG detoxification enzyme was up-regulated during fruit ripening. Candidate genes for ethylene, anthocyanin, and 400 other biosynthetic pathways were also identified. Homology-based annotation using Blast2GO and InterPro assigned Gene Ontology terms to around 15,000 genes. RNA-Seq expression profiling showed that blueberry growth, maturation, and ripening involve dynamic gene expression changes, including coordinated up- and down-regulation of metabolic pathway enzymes and transcriptional regulators. Analysis of RNA-seq alignments identified developmentally regulated alternative splicing, promoter use, and 3' end formation. We report genome sequence, gene models, functional annotations, and RNA-Seq expression data that provide an important new resource enabling high throughput studies in blueberry.

  11. Production and cross-sectional characterization of aligned co-electrospun hollow microfibrous bulk assemblies

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Feng-Lei [Centre for Imaging Sciences, The University of Manchester, Manchester M13 9PT (United Kingdom); The School of Materials, The University of Manchester, Manchester M13 9PL (United Kingdom); CRUK-EPSRC Cancer Imaging Centre in Cambridge and Manchester (United Kingdom); Parker, Geoff J.M., E-mail: geoff.parker@manchester.ac.uk [Centre for Imaging Sciences, The University of Manchester, Manchester M13 9PT (United Kingdom); CRUK-EPSRC Cancer Imaging Centre in Cambridge and Manchester (United Kingdom); Eichhorn, Stephen J. [College of Engineering, Mathematics and Physical Sciences, University of Exeter, Exeter EX4 4QF (United Kingdom); Hubbard Cristinacce, Penny L. [Centre for Imaging Sciences, The University of Manchester, Manchester M13 9PT (United Kingdom); School of Psychological Sciences, University of Manchester, Manchester M13 9PT (United Kingdom)

    2015-11-15

    The development of co-electrospun (co-ES) hollow microfibrous assemblies of an appreciable thickness is critical for many practical applications, including filtration membranes and tissue-mimicking scaffolds. In this study, thick uniaxially aligned hollow microfibrous assemblies forming fiber bundles and strips were prepared by co-ES of polycaprolactone (PCL) and polyethylene oxide (PEO) as shell and core materials, respectively. Hollow microfiber bundles were deposited on a fixed rotating disc, which resulted in non-controllable cross-sectional shapes on a macroscopic scale. In comparison, fiber strips were produced with tuneable thickness and width by additionally employing an x–y translation stage in co-ES. Scanning electron microscopy (SEM) images of cross-sections of fiber assemblies were analyzed to investigate the effects of production time (from 0.5 h to 12 h), core flow rate (from 0.8 mL/h to 2.0 mL/h) and/or translation speed (from 0.2 mm/s to 5 mm/s) on the pores and porosity. We observed significant changes in pore size and shape with core flow rate but the influence of production time varied; five strips produced under the same conditions had reasonably good size and porosity reproducibility; pore sizes didn't vary significantly from strip bottom to surface, although the porosity gradually decreased and then returned to the initial level. - Highlights: • Hollow microfibrous assemblies based on co-electrospinning are demonstrated. • The thickness and width of co-electrospun strips were controllable. • Cross-sections of fibres had non-normally distributed pore sizes and shapes. • Cross-sections were significantly influenced by production time and flow rate. • Co-electrospun strips had reasonably good reproducible cross-sections.

  12. Directed self-assembly enabled fully self-aligned via processing (Conference Presentation)

    Science.gov (United States)

    Rincon-Delgadillo, Paulina A.; Murdoch, Gayle; Gronheid, Roel; Kim, Ryoung-Han; Bömmels, Jürgen

    2017-03-01

    Until recent years, dimensional scaling allowed for the fabrication of smaller and faster devices with increasing capacity. Currently, the limited area and the high density of the features in such devices have made self-aligned contacts/vias (SAC or SAV) a standard technique to overcome the decreasing distance between electrically functional elements in integrated circuits. In SAV schemes, the use of hard masks to define the effective transferred patterns allows for more relaxed via patterning conditions and overlay requirements. In this work, we explore a DSA-based fully self-aligned vias (FSAV) flow to further improve on traditional SAV processes. We use directed self-assembly (DSA) of block copolymers (BCP) to generate topographic features between the metal lines, which in combination with SAV, extend the benefits of this method to both X-Y directions, while maximizing the distance between the contacts and the adjacent not-connected metal lines to avoid potential shorts, as shown in Figure 1. In order to achieve this, patterned metal and/or dielectric lines are selectively functionalized using homopolymer brushes, to form a 1:1 chemical nano-pattern of specific surface energy, such that, when a BCP layer is coated and annealed on these samples, each block will align to the metal (or dielectric) lines underneath, as shown on Figure 2. We subsequently use one of the blocks as a template to generate topographic features between the metal lines. In addition, different hard masks are characterized to find the optimal material for the current scheme. Finally, we define the design rules for integration of the proposed flow into electrical devices.

  13. Assembly and alignment method for optimized spatial resolution of off-axis three-mirror fore optics of hyperspectral imager.

    Science.gov (United States)

    Kim, Youngsoo; Hong, Jinsuk; Choi, Byungin; Lee, Jong-Ung; Kim, Yeonsoo; Kim, Hyunsook

    2017-08-21

    A fore optics for the hyperspectral spectrometer is designed, manufactured, assembled, and aligned. The optics has a telecentric off-axis three-mirror configuration with a field of view wider than 14 degrees and an f-number as small as 2.3. The primary mirror (M1) and the secondary mirror (M2) are axially symmetric aspheric surfaces to minimize the sensitivity. The tertiary mirror (M3) is a decentered aspheric surface to minimize the coma and astigmatism aberration. The M2 also has a hole for the slit to maintain the optical performance while maximizing the telecentricity. To ensure the spatial resolution performance of the optical system, an alignment procedure is established to assemble and align the entrance slit of the spectrometer to the rear end of the fore optics. It has a great advantage to confirm and maintain the alignment integrity of the fore optics module throughout the alignment procedure. To perform the alignment procedure successfully, the precision movement control requirements are calculated and applied. As a result, the alignment goal of the RMS wave front error (WFE) to be smaller than 90 nm at all fields is achieved.

  14. Advanced topographic laser altimeter system (ATLAS) receiver telescope assembly (RTA) and transmitter alignment and test

    Science.gov (United States)

    Hagopian, John; Bolcar, Matthew; Chambers, John; Crane, Allen; Eegholm, Bente; Evans, Tyler; Hetherington, Samuel; Mentzell, Eric; Thompson, Patrick L.; Ramos-Izquierdo, Luis; Vaughnn, David

    2016-09-01

    The sole instrument on NASA's ICESat-2 spacecraft shown in Figure 1 will be the Advanced Topographic Laser Altimeter System (ATLAS)1. The ATLAS is a Light Detection and Ranging (LIDAR) instrument; it measures the time of flight of the six transmitted laser beams to the Earth and back to determine altitude for geospatial mapping of global ice. The ATLAS laser beam is split into 6 main beams by a Diffractive Optical Element (DOE) that are reflected off of the earth and imaged by an 800 mm diameter Receiver Telescope Assembly (RTA). The RTA is composed of a 2-mirror telescope and Aft Optics Assembly (AOA) that collects and focuses the light from the 6 probe beams into 6 science fibers. Each fiber optic has a field of view on the earth that subtends 83 micro Radians. The light collected by each fiber is detected by a photomultiplier and timing related to a master clock to determine time of flight and therefore distance. The collection of the light from the 6 laser spots projected to the ground allows for dense cross track sampling to provide for slope measurements of ice fields. NASA LIDAR instruments typically utilize telescopes that are not diffraction limited since they function as a light collector rather than imaging function. The more challenging requirements of the ATLAS instrument require better performance of the telescope at the ¼ wave level to provide for improved sampling and signal to noise. NASA Goddard Space Flight Center (GSFC) contracted the build of the telescope to General Dynamics (GD). GD fabricated and tested the flight and flight spare telescope and then integrated the government supplied AOA for testing of the RTA before and after vibration qualification. The RTA was then delivered to GSFC for independent verification and testing over expected thermal vacuum conditions. The testing at GSFC included a measurement of the RTA wavefront error and encircled energy in several orientations to determine the expected zero gravity figure, encircled

  15. Comprehensive analysis of alternative splicing in rice and comparative analyses with Arabidopsis

    Directory of Open Access Journals (Sweden)

    Mount Stephen M

    2006-12-01

    Full Text Available Abstract Background Recently, genomic sequencing efforts were finished for Oryza sativa (cultivated rice and Arabidopsis thaliana (Arabidopsis. Additionally, these two plant species have extensive cDNA and expressed sequence tag (EST libraries. We employed the Program to Assemble Spliced Alignments (PASA to identify and analyze alternatively spliced isoforms in both species. Results A comprehensive analysis of alternative splicing was performed in rice that started with >1.1 million publicly available spliced ESTs and over 30,000 full length cDNAs in conjunction with the newly enhanced PASA software. A parallel analysis was performed with Arabidopsis to compare and ascertain potential differences between monocots and dicots. Alternative splicing is a widespread phenomenon (observed in greater than 30% of the loci with transcript support and we have described nine alternative splicing variations. While alternative splicing has the potential to create many RNA isoforms from a single locus, the majority of loci generate only two or three isoforms and transcript support indicates that these isoforms are generally not rare events. For the alternate donor (AD and acceptor (AA classes, the distance between the splice sites for the majority of events was found to be less than 50 basepairs (bp. In both species, the most frequent distance between AA is 3 bp, consistent with reports in mammalian systems. Conversely, the most frequent distance between AD is 4 bp in both plant species, as previously observed in mouse. Most alternative splicing variations are localized to the protein coding sequence and are predicted to significantly alter the coding sequence. Conclusion Alternative splicing is widespread in both rice and Arabidopsis and these species share many common features. Interestingly, alternative splicing may play a role beyond creating novel combinations of transcripts that expand the proteome. Many isoforms will presumably have negative

  16. A de novo transcriptome assembly of Lucilia sericata (Diptera: Calliphoridae) with predicted alternative splices, single nucleotide polymorphisms and transcript expression estimates.

    Science.gov (United States)

    Sze, S-H; Dunham, J P; Carey, B; Chang, P L; Li, F; Edman, R M; Fjeldsted, C; Scott, M J; Nuzhdin, S V; Tarone, Aaron M

    2012-04-01

    The blow fly Lucilia sericata (Diptera: Calliphoridae) (Meigen) is a nonmodel organism with no reference genome that is associated with numerous areas of research spanning the ecological, evolutionary, medical, veterinary and forensic sciences. To facilitate scientific discovery in this species, the transcriptome was assembled from more than six billion bases of Illumina and twenty-one million bases of 454 sequence derived from embryonic, larval, pupal, adult and larval salivary gland libraries. The assembly was carried out in a manner that enabled identification of putative single nucleotide polymorphisms (SNPs) and alternative splices, and that provided expression estimates for various life history stages and for salivary tissue. The assembled transcriptome was also used to identify transcribed transposable elements in L. sericata. The results of this study will enable blow fly biologists, dipterists and comparative genomicists to more rapidly develop and test molecular and genetic hypotheses, especially those regarding blow fly development and salivary gland biology. © 2012 The Authors. Insect Molecular Biology © 2012 The Royal Entomological Society.

  17. Co-assembly of two GluR6 kainate receptor splice variants within a functional protein complex.

    Science.gov (United States)

    Coussen, Françoise; Perrais, David; Jaskolski, Frédéric; Sachidhanandam, Shankar; Normand, Elisabeth; Bockaert, Joel; Marin, Philippe; Mulle, Christophe

    2005-08-18

    Kainate receptors (KAR) are composed of several distinct subunits and splice variants, but the functional relevance of this diversity remains largely unclear. Here we show that two splice variants of the GluR6 subunit, GluR6a and GluR6b, which differ in their C-terminal domains, do not show distinct functional properties, but coassemble as heteromers in vitro and in vivo. Using a proteomic approach combining affinity purification and MALDI-TOF mass spectrometry, we found that GluR6a and GluR6b interact with two distinct subsets of cytosolic proteins mainly involved in Ca(2+) regulation of channel function and intracellular trafficking. Guided by these results, we provide evidence that the regulation of native KAR function by NMDA receptors depends on the heteromerization of GluR6a and GluR6b and interaction of calcineurin with GluR6b. Thus, GluR6a and GluR6b bring in close proximity two separate subsets of interacting proteins that contribute to the fine regulation of KAR trafficking and function.

  18. Self-assembly of folic acid: a chiral-aligning medium for enantiodiscrimination of organic molecules in an aqueous environment.

    Science.gov (United States)

    Lokesh; Suryaprakash, N

    2012-09-10

    Weak orienting medium: Self-assembly of alkaline salt of folic acid yielded a weak liquid-crystalline phase in an aqueous environment. This medium has the ability to discriminate enantiomers. The mesophase exists over a broad range and has the physical parameter dependent tunability of degree of alignment (see scheme). Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Effect of molecular structure and packing density of an azo self-assembled monolayer on liquid crystal alignment.

    Science.gov (United States)

    Vengatesan, M R; Lee, Seung-Ho; Son, Jong-Ho; Lim, Jeong-Ku; Song, Jang Kun

    2013-10-01

    We studied the alignment of liquid crystals (LCs) on a photo-switchable azo-containing self-assembled monolayer (azo-SAM) with different packing densities and molecular structures. The packing density of the azo-SAM substrates was varied by changing the dipping time of the substrate in azosilane monomers solution (2mM in toluene). The thickness of the monolayer on the silicon substrate increased as the dipping time was increased. The relative surface packing density on the glass substrates was estimated from the surface energies of the azo-SAM. The photo-induced dynamics of liquid crystal alignment on the azo-SAM significantly varied according to the packing density of the azo-SAM and the structure of the azo-SAM molecules. The azo-SAM from long octyloxy chain-terminated azosilane (azo-S1) possessed stable homeotropic alignment even after photobuffing, while the azo-SAM from short methyl group-terminated azosilane monomer (azo-S2) showed photo-switchable homeotropic and planar alignments. However, when the packing density was increased to an excessive degree, even the azo-SAM from azo-S2 exhibited stable homeotropic alignment regardless of photobuffing. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Self-Assembled Formation of Well-Aligned Cu-Te Nano-Rods on Heavily Cu-Doped ZnTe Thin Films

    National Research Council Canada - National Science Library

    Liang, Jing; Cheng, Man Kit; Lai, Ying Hoi; Wei, Guanglu; Yang, Sean Derman; Wang, Gan; Ho, Sut Kam; Tam, Kam Weng; Sou, Iam Keong

    2016-01-01

    ... reported. We found an interesting self-assembled formation of crystalline well-aligned Cu-Te nano-rods near the surface of heavily Cu-doped ZnTe thin films grown via the molecular beam epitaxy technique...

  1. Doubly Spliced RNA of Hepatitis B Virus Suppresses Viral Transcription via TATA-Binding Protein and Induces Stress Granule Assembly.

    Science.gov (United States)

    Tsai, Kuen-Nan; Chong, Chin-Liew; Chou, Yu-Chi; Huang, Chien-Chiao; Wang, Yi-Ling; Wang, Shao-Win; Chen, Mong-Liang; Chen, Chun-Hong; Chang, Chungming

    2015-11-01

    The risk of liver cancer in patients infected with the hepatitis B virus (HBV) and their clinical response to interferon alpha therapy vary based on the HBV genotype. The mechanisms underlying these differences in HBV pathogenesis remain unclear. In HepG2 cells transfected with a mutant HBV(G2335A) expression plasmid that does not transcribe the 2.2-kb doubly spliced RNA (2.2DS-RNA) expressed by wild-type HBV genotype A, the level of HBV pregenomic RNA (pgRNA) was higher than that in cells transfected with an HBV genotype A expression plasmid. By using cotransfection with HBV genotype D and 2.2DS-RNA expression plasmids, we found that a reduction of pgRNA was observed in the cells even in the presence of small amounts of the 2.2DS-RNA plasmid. Moreover, ectopic expression of 2.2DS-RNA in the HBV-producing cell line 1.3ES2 reduced the expression of pgRNA. Further analysis showed that exogenously transcribed 2.2DS-RNA inhibited a reconstituted transcription in vitro. In Huh7 cells ectopically expressing 2.2DS-RNA, RNA immunoprecipitation revealed that 2.2DS-RNA interacted with the TATA-binding protein (TBP) and that nucleotides 432 to 832 of 2.2DS-RNA were required for efficient TBP binding. Immunofluorescence experiments showed that 2.2DS-RNA colocalized with cytoplasmic TBP and the stress granule components, G3BP and poly(A)-binding protein 1 (PABP1), in Huh7 cells. In conclusion, our study reveals that 2.2DS-RNA acts as a repressor of HBV transcription through an interaction with TBP that induces stress granule formation. The expression of 2.2DS-RNA may be one of the viral factors involved in viral replication, which may underlie differences in clinical outcomes of liver disease and responses to interferon alpha therapy between patients infected with different HBV genotypes. Patients infected with certain genotypes of HBV have a lower risk of hepatocellular carcinoma and exhibit a more favorable response to antiviral therapy than patients infected with other HBV

  2. Charge transport in vertically aligned, self-assembled peptide nanotube junctions.

    Science.gov (United States)

    Mizrahi, Mordechay; Zakrassov, Alexander; Lerner-Yardeni, Jenny; Ashkenasy, Nurit

    2012-01-21

    The self-assembly propensity of peptides has been extensively utilized in recent years for the formation of supramolecular nanostructures. In particular, the self-assembly of peptides into fibrils and nanotubes makes them promising building blocks for electronic and electro-optic applications. However, the mechanisms of charge transfer in these wire-like structures, especially in ambient conditions, are not yet fully understood. We describe here a layer-by-layer deposition methodology of short self-assembled cyclic peptide nanotubes, which results in vertically oriented nanotubes on gold substrates. Using this novel deposition methodology, we have fabricated molecular junctions with a conductive atomic force microscopy tip as a second electrode. Studies of the junctions' current-voltage characteristics as a function of the nanotube length revealed an efficient charge transfer in these supramolecular structures, with a low current attenuation constant of 0.1 Å(-1), which indicate that electron transfer is dominated by hopping. Moreover, the threshold voltage to field-emission dominated transport was found to increase with peptide length in a manner that depends on the nature of the contact with the electrodes. The flexibility in the design of the peptide monomers and the ability to control their sequential order over the nanotube by means of the layer-by-layer assembly process, which is demonstrated in this work, can be used to engineer the electronic properties of self-assembled peptide nanotubes toward device applications.

  3. Application of capillary forces and stiction for lateral displacement, alignment, suspension and locking of self-assembled microcantilevers

    Science.gov (United States)

    Rose, F.; Hattori, M.; Kobayashi, D.; Toshiyoshi, H.; Fujita, H.; Kawakatsu, H.

    2006-10-01

    We report the surface-tension-powered self-assembly (displacement, alignment, pulling down and locking) of microcantilevers. Capillary forces-assisted displacement is realized by compression of four arrays of springs linked to the opposite lateral sides of the cantilevers. After in-plane translation along the initial cantilever orientation, the microstructure is pulled down and locked on the substrate by stiction. Spring and capillary forces are described with a simple analytical model. Complete self-assembly occurs when the layout of the whole system (the cantilever with its traveling spring structure, the surrounding area and the local distribution of the liquid) is well designed. We show that the presence of a water meniscus trapped at a step edge in the vicinity of the tip end of a microcantilever could lead to stiction failure before traveling of the structure. Small beams (6 µm long) protruding over step edges were fabricated by adding a mechanically assisted displacement step (with a microneedle) to the self-assembly experiment.

  4. Alignment and Use of Self-Assembled Peptide Nanotubes as Dry-Etching Mask

    DEFF Research Database (Denmark)

    Andersen, Karsten Brandt; Castillo, Jaime; Bakmand, Tanya

    2012-01-01

    Self-assembled diphenylalanine peptide nanotubes provide a means of achieving nanostructured materials in a very simple and fast way. Recent discoveries have shown that this unique material, in addition to remaining stable under dry conditions, rapidly dissolves in water making it a promising can...

  5. Self-formation of highly aligned metallic, semiconducting and single chiral single-walled carbon nanotubes assemblies via a crystal template method

    Energy Technology Data Exchange (ETDEWEB)

    Kawai, Hideki; Hasegawa, Kai; Yanagi, Kazuhiro, E-mail: yanagi-kazuhiro@tmu.ac.jp [Department of Physics, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397 (Japan); Oyane, Ayako [Nanosystem Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8562 (Japan); Naitoh, Yasuhisa [Nanosystem Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8564 (Japan)

    2014-09-01

    The fabrication of an aligned array of single-walled carbon nanotubes (SWCNTs) with a single chiral state has been a significant challenge for SWCNT applications as well as for basic science research. Here, we developed a simple, unique technique to produce assemblies in which metallic, semiconducting, and single chiral state SWCNTs were densely and highly aligned. We utilized a crystal of surfactant as a template on which mono-dispersed SWCNTs in solution self-assembled. Micro-Raman measurements and scanning electron microscopy measurements clearly showed that the SWCNTs were highly and densely aligned parallel to the crystal axis, indicating that approximately 70% of the SWCNTs were within 7° of being parallel. Moreover, the assemblies exhibited good field effect transistor characteristics with an on/off ratio of 1.3 × 10{sup 5}.

  6. Direct comparison of performance of single nucleotide variant calling in human genome with alignment-based and assembly-based approaches.

    Science.gov (United States)

    Wu, Leihong; Yavas, Gokhan; Hong, Huixiao; Tong, Weida; Xiao, Wenming

    2017-09-08

    Complementary to reference-based variant detection, recent studies revealed that many novel variants could be detected with de novo assembled genomes. To evaluate the effect of reads coverage and the accuracy of assembly-based variant calling, we simulated short reads containing more than 3 million of single nucleotide variants (SNVs) from the whole human genome and compared the efficiency of SNV calling between the assembly-based and alignment-based calling approaches. We assessed the quality of the assembled contig and found that a minimum of 30X coverage of short reads was needed to ensure reliable SNV calling and to generate assembled contigs with a good coverage of genome and genes. In addition, we observed that the assembly-based approach had a much lower recall rate and precision comparing to the alignment-based approach that would recover 99% of imputed SNVs. We observed similar results with experimental reads for NA24385, an individual whose germline variants were well characterized. Although there are additional values for SNVs detection, the assembly-based approach would have great risk of false discovery of novel SNVs. Further improvement of de novo assembly algorithms are needed in order to warrant a good completeness of genome with haplotype resolved and high fidelity of assembled sequences.

  7. Self-assembly of vertically aligned quantum ring-dot structure by Multiple Droplet Epitaxy

    Science.gov (United States)

    Elborg, Martin; Noda, Takeshi; Mano, Takaaki; Kuroda, Takashi; Yao, Yuanzhao; Sakuma, Yoshiki; Sakoda, Kazuaki

    2017-11-01

    We successfully grow vertically aligned quantum ring-dot structures by Multiple Droplet Epitaxy technique. The growth is achieved by depositing GaAs quantum rings in a first droplet epitaxy process which are subsequently covered by a thin AlGaAs barrier. In a second droplet epitaxy process, Ga droplets preferentially position in the center indentation of the ring as well as attached to the edge of the ring in [ 1 1 bar 0 ] direction. By designing the ring geometry, full selectivity for the center position of the ring is achieved where we crystallize the droplets into quantum dots. The geometry of the ring and dot as well as barrier layer can be controlled in separate growth steps. This technique offers great potential for creating complex quantum molecules for novel quantum information technologies.

  8. Shaping the Arabidopsis Transcriptome through Alternative Splicing

    Directory of Open Access Journals (Sweden)

    Dorothee Staiger

    2015-01-01

    Full Text Available Alternative splicing is a molecular tool of the cell to generate more than one messenger RNA from the same gene. Through variable combinations of exons blueprints for different proteins are assembled from one and the same pre-messenger RNA, thus increasing the complexity of the proteome. Moreover, through alternative splicing different transcript variants with different stabilities and different regulatory motifs can be generated, leading to variation in the transcriptome. The importance of alternative splicing in plants has been increasingly recognized in the last decade. Alternative splicing has been found during abiotic and biotic stress and during development. Here, recent advancements in the understanding of alternative splicing in higher plants are presented. Mechanistic details and functional consequences of alternative splicing are discussed with a focus on the model plant Arabidopsis thaliana.

  9. Self-assembly of designed precursors: A route to crystallographically aligned new materials with controlled nanoarchitecture

    Energy Technology Data Exchange (ETDEWEB)

    Westover, Richard, E-mail: rwestove@uoregon.edu; Atkins, Ryan A.; Falmbigl, Matthias; Ditto, Jeffrey J.; Johnson, David C.

    2016-04-15

    Modulated elemental reactants is a method by which new and complex intergrowth compounds can be synthesized by the self-assembly of designed precursors prepared by physical vapor deposition. Careful calibration of the composition and thickness of the precursors ensures the formation of the desired product by precise control of local composition and diffusion lengths. Superstructures of increasing complexity can be realized using binary and ternary systems as starting points. The synthesis of systems based on three different binary compounds, either alloyed together or separated into distinct layers, expands the number of possible superstructures that can be formed using this technique, but provides analytical challenges. The synthesis of [(SnSe){sub 1.15}]{sub 1}([Ta{sub x}V{sub 1−x}]Se{sub 2}){sub 1}[(SnSe){sub 1.15}]{sub 1}([V{sub y}Ta{sub 1−y}]Se{sub 2}){sub 1} compound is used to illustrate the preparation of precursors and the challenges in both measuring and limiting the interdiffusion of layers during self-assembly. Systematic changes in the electrical properties of (SnSe){sub 1+δ}(Ta{sub x}V{sub 1−x})Se{sub 2} alloys are observed as x is varied. The electrical resistivity of [(SnSe){sub 1.15}]{sub 1}([Ta{sub x}V{sub 1−x}]Se{sub 2}){sub 1}[(SnSe){sub 1.15}]{sub 1}([V{sub y}Ta{sub 1−y}]Se{sub 2}){sub 1} can be modeled as the two constituent layers in parallel.

  10. Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle function

    Science.gov (United States)

    Gallagher, Thomas L.; Arribere, Joshua A.; Geurts, Paul A.; Exner, Cameron R. T.; McDonald, Kent L.; Dill, Kariena K.; Marr, Henry L.; Adkar, Shaunak S.; Garnett, Aaron T.; Amacher, Sharon L.; Conboy, John G.

    2012-01-01

    Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos was strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle function. PMID:21925157

  11. Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle functions.

    Science.gov (United States)

    Gallagher, Thomas L; Arribere, Joshua A; Geurts, Paul A; Exner, Cameron R T; McDonald, Kent L; Dill, Kariena K; Marr, Henry L; Adkar, Shaunak S; Garnett, Aaron T; Amacher, Sharon L; Conboy, John G

    2011-11-15

    Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos were strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle functions. Published by Elsevier Inc.

  12. Safely splicing glass optical fibers

    Science.gov (United States)

    Korbelak, K.

    1980-01-01

    Field-repair technique fuses glass fibers in flammable environment. Apparatus consists of v-groove vacuum chucks on manipulators, high-voltage dc power supply and tungsten electrodes, microscope to observe joint alignment and fusion, means of test transmission through joint. Apparatus is enclosed in gas tight bos filled with inert gas during fusion. About 2 feet of fiber end are necessary for splicing.

  13. ScanIndel: a hybrid framework for indel detection via gapped alignment, split reads and de novo assembly.

    Science.gov (United States)

    Yang, Rendong; Nelson, Andrew C; Henzler, Christine; Thyagarajan, Bharat; Silverstein, Kevin A T

    2015-12-07

    Comprehensive identification of insertions/deletions (indels) across the full size spectrum from second generation sequencing is challenging due to the relatively short read length inherent in the technology. Different indel calling methods exist but are limited in detection to specific sizes with varying accuracy and resolution. We present ScanIndel, an integrated framework for detecting indels with multiple heuristics including gapped alignment, split reads and de novo assembly. Using simulation data, we demonstrate ScanIndel's superior sensitivity and specificity relative to several state-of-the-art indel callers across various coverage levels and indel sizes. ScanIndel yields higher predictive accuracy with lower computational cost compared with existing tools for both targeted resequencing data from tumor specimens and high coverage whole-genome sequencing data from the human NIST standard NA12878. Thus, we anticipate ScanIndel will improve indel analysis in both clinical and research settings. ScanIndel is implemented in Python, and is freely available for academic use at https://github.com/cauyrd/ScanIndel.

  14. Survey of gene splicing algorithms based on reads.

    Science.gov (United States)

    Si, Xiuhua; Wang, Qian; Zhang, Lei; Wu, Ruo; Ma, Jiquan

    2017-09-05

    Gene splicing is the process of assembling a large number of unordered short sequence fragments to the original genome sequence as accurately as possible. Several popular splicing algorithms based on reads are reviewed in this article, including reference genome algorithms and de novo splicing algorithms (Greedy-extension, Overlap-Layout-Consensus graph, De Bruijn graph). We also discuss a new splicing method based on the MapReduce strategy and Hadoop. By comparing these algorithms, some conclusions are drawn and some suggestions on gene splicing research are made.

  15. Novel field installable optical fiber splice and connector for optical access networks

    Science.gov (United States)

    Kihara, Mitsuru; Koyama, Ryo; Abe, Yoshiteru; Son, Hitoshi; Kobayashi, Masaru; Tomita, Shigeru

    2013-08-01

    We have developed two types of field installable connection techniques. One is mechanical splicing, which is used to connect coated optical fibers without the need for stripping or cleaning procedures. The other is a field assembly connection technique, which employs a new type of field installable connector that makes it possible to realize a physical contact connection with chamfer grinding. Mechanical splicing is achieved by precisely aligning and directly connecting coated fibers with a capillary. The assembled splice is installed with 1.3-μm single-mode fibers that have an 80-μm cladding and a 125-μm coating and they exhibit good optical performance with a low average insertion loss of 0.2 dB. Connection is achieved with our developed field installable connector by using a chamfered fiber endface and the compression force of the buckled fiber. The assembled connectors achieve physical contact with the chamfered fiber endface, which provides good optical performance with a low insertion loss of 0.11 dB.

  16. Choice of reference sequence and assembler for alignment of Listeria monocytogenes short-read sequence data greatly influences rates of error in SNP analyses.

    Directory of Open Access Journals (Sweden)

    Arthur W Pightling

    Full Text Available The wide availability of whole-genome sequencing (WGS and an abundance of open-source software have made detection of single-nucleotide polymorphisms (SNPs in bacterial genomes an increasingly accessible and effective tool for comparative analyses. Thus, ensuring that real nucleotide differences between genomes (i.e., true SNPs are detected at high rates and that the influences of errors (such as false positive SNPs, ambiguously called sites, and gaps are mitigated is of utmost importance. The choices researchers make regarding the generation and analysis of WGS data can greatly influence the accuracy of short-read sequence alignments and, therefore, the efficacy of such experiments. We studied the effects of some of these choices, including: i depth of sequencing coverage, ii choice of reference-guided short-read sequence assembler, iii choice of reference genome, and iv whether to perform read-quality filtering and trimming, on our ability to detect true SNPs and on the frequencies of errors. We performed benchmarking experiments, during which we assembled simulated and real Listeria monocytogenes strain 08-5578 short-read sequence datasets of varying quality with four commonly used assemblers (BWA, MOSAIK, Novoalign, and SMALT, using reference genomes of varying genetic distances, and with or without read pre-processing (i.e., quality filtering and trimming. We found that assemblies of at least 50-fold coverage provided the most accurate results. In addition, MOSAIK yielded the fewest errors when reads were aligned to a nearly identical reference genome, while using SMALT to align reads against a reference sequence that is ∼0.82% distant from 08-5578 at the nucleotide level resulted in the detection of the greatest numbers of true SNPs and the fewest errors. Finally, we show that whether read pre-processing improves SNP detection depends upon the choice of reference sequence and assembler. In total, this study demonstrates that researchers

  17. Choice of reference sequence and assembler for alignment of Listeria monocytogenes short-read sequence data greatly influences rates of error in SNP analyses.

    Science.gov (United States)

    Pightling, Arthur W; Petronella, Nicholas; Pagotto, Franco

    2014-01-01

    The wide availability of whole-genome sequencing (WGS) and an abundance of open-source software have made detection of single-nucleotide polymorphisms (SNPs) in bacterial genomes an increasingly accessible and effective tool for comparative analyses. Thus, ensuring that real nucleotide differences between genomes (i.e., true SNPs) are detected at high rates and that the influences of errors (such as false positive SNPs, ambiguously called sites, and gaps) are mitigated is of utmost importance. The choices researchers make regarding the generation and analysis of WGS data can greatly influence the accuracy of short-read sequence alignments and, therefore, the efficacy of such experiments. We studied the effects of some of these choices, including: i) depth of sequencing coverage, ii) choice of reference-guided short-read sequence assembler, iii) choice of reference genome, and iv) whether to perform read-quality filtering and trimming, on our ability to detect true SNPs and on the frequencies of errors. We performed benchmarking experiments, during which we assembled simulated and real Listeria monocytogenes strain 08-5578 short-read sequence datasets of varying quality with four commonly used assemblers (BWA, MOSAIK, Novoalign, and SMALT), using reference genomes of varying genetic distances, and with or without read pre-processing (i.e., quality filtering and trimming). We found that assemblies of at least 50-fold coverage provided the most accurate results. In addition, MOSAIK yielded the fewest errors when reads were aligned to a nearly identical reference genome, while using SMALT to align reads against a reference sequence that is ∼0.82% distant from 08-5578 at the nucleotide level resulted in the detection of the greatest numbers of true SNPs and the fewest errors. Finally, we show that whether read pre-processing improves SNP detection depends upon the choice of reference sequence and assembler. In total, this study demonstrates that researchers should

  18. The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells.

    Directory of Open Access Journals (Sweden)

    Thomas C Whisenant

    Full Text Available Activation of CD4 T cells is a reaction to challenges such as microbial pathogens, cancer and toxins that defines adaptive immune responses. The roles of T cell receptor crosslinking, intracellular signaling, and transcription factor activation are well described, but the importance of post-transcriptional regulation by RNA-binding proteins (RBPs has not been considered in depth. We describe a new model expanding and activating primary human CD4 T cells and applied this to characterizing activation-induced assembly of splicing factors centered on U2AF2. We immunoprecipitated U2AF2 to identify what mRNA transcripts were bound as a function of activation by TCR crosslinking and costimulation. In parallel, mass spectrometry revealed the proteins incorporated into the U2AF2-centered RNA/protein interactome. Molecules that retained interaction with the U2AF2 complex after RNAse treatment were designated as "central" interactome members (CIMs. Mass spectrometry also identified a second class of activation-induced proteins, "peripheral" interactome members (PIMs, that bound to the same transcripts but were not in physical association with U2AF2 or its partners. siRNA knockdown of two CIMs and two PIMs caused changes in activation marker expression, cytokine secretion, and gene expression that were unique to each protein and mapped to pathways associated with key aspects of T cell activation. While knocking down the PIM, SYNCRIP, impacts a limited but immunologically important set of U2AF2-bound transcripts, knockdown of U2AF1 significantly impairs assembly of the majority of protein and mRNA components in the activation-induced interactome. These results demonstrated that CIMs and PIMs, either directly or indirectly through RNA, assembled into activation-induced U2AF2 complexes and play roles in post-transcriptional regulation of genes related to cytokine secretion. These data suggest an additional layer of regulation mediated by the activation

  19. Spray-assisted alignment of Layer-by-Layer assembled silver nanowires: a general approach for the preparation of highly anisotropic nano-composite films.

    Science.gov (United States)

    Hu, H; Pauly, M; Felix, O; Decher, G

    2017-01-19

    The present article focuses on the build-up and the properties of oriented silver nanowire monolayer films and Layer-by-Layer assembled multilayer films. We describe the template-free oriented spray-assisted assembly of silver nanowires at solid/air-interfaces using Grazing Incidence Spraying, a simple and versatile approach that allows the formation of highly oriented thin films with a tunable density and in-plane orientation. Depending on the spraying conditions the nematic order parameter, which describes the angular spread of misaligned nanowires, can be as high as 0.98 (a value of 1.00 corresponding to a perfectly parallel alignment). The combination with the Layer-by-Layer assembly allows building multilayer thin films possessing in-plane anisotropy. In order to demonstrate that the local alignment does not cancel out on the macroscopic scale but leads to direction-dependent properties, we use linearly polarized UV-Vis-NIR spectroscopy to probe the selective excitation of the transverse and longitudinal localized plasmon resonances of the nanowires. The polarization efficiency of the thin films increases strongly with the in-plane density, the degree of orientation, and the number of silver nanowire layers. Multilayer films containing 4 layers of nanowires oriented in the same direction reach a polarization efficiency of up to 97% in the near-infrared region.

  20. Self-Assembly of Chip-Size Components with Cavity Structures: High-Precision Alignment and Direct Bonding without Thermal Compression for Hetero Integration

    Directory of Open Access Journals (Sweden)

    Mitsumasa Koyanagi

    2011-02-01

    Full Text Available New surface mounting and packaging technologies, using self-assembly with chips having cavity structures, were investigated for three-dimensional (3D and hetero integration of complementary metal-oxide semiconductors (CMOS and microelectromechanical systems (MEMS. By the surface tension of small droplets of 0.5 wt% hydrogen fluoride (HF aqueous solution, the cavity chips, with a side length of 3 mm, were precisely aligned to hydrophilic bonding regions on the surface of plateaus formed on Si substrates. The plateaus have micro-channels to readily evaporate and fully remove the liquid from the cavities. The average alignment accuracy of the chips with a 1 mm square cavity was found to be 0.4 mm. The alignment accuracy depends, not only on the area of the bonding regions on the substrates and the length of chip periphery without the widths of channels in the plateaus, but also the area wetted by the liquid on the bonding regions. The precisely aligned chips were then directly bonded to the substrates at room temperature without thermal compression, resulting in a high shear bonding strength of more than 10 MPa.

  1. Alternative Splicing in CKD

    OpenAIRE

    Stevens, Megan; Oltean, Sebastian

    2016-01-01

    Alternative splicing (AS) has emerged in the postgenomic era as one of the main drivers of proteome diversity, with ���94% of multiexon genes alternatively spliced in humans. AS is therefore one of the main control mechanisms for cell phenotype, and is a process deregulated in disease. Numerous reports describe pathogenic mutations in splice factors, splice sites, or regulatory sequences. Additionally, compared with the physiologic state, disease often associates with an abnormal proportion o...

  2. Computational prediction of splicing regulatory elements shared by Tetrapoda organisms

    Science.gov (United States)

    Churbanov, Alexander; Vořechovský, Igor; Hicks, Chindo

    2009-01-01

    Background Auxiliary splicing sequences play an important role in ensuring accurate and efficient splicing by promoting or repressing recognition of authentic splice sites. These cis-acting motifs have been termed splicing enhancers and silencers and are located both in introns and exons. They co-evolved into an intricate splicing code together with additional functional constraints, such as tissue-specific and alternative splicing patterns. We used orthologous exons extracted from the University of California Santa Cruz multiple genome alignments of human and 22 Tetrapoda organisms to predict candidate enhancers and silencers that have reproducible and statistically significant bias towards annotated exonic boundaries. Results A total of 2,546 Tetrapoda enhancers and silencers were clustered into 15 putative core motifs based on their Markov properties. Most of these elements have been identified previously, but 118 putative silencers and 260 enhancers (~15%) were novel. Examination of previously published experimental data for the presence of predicted elements showed that their mutations in 21/23 (91.3%) cases altered the splicing pattern as expected. Predicted intronic motifs flanking 3' and 5' splice sites had higher evolutionary conservation than other sequences within intronic flanks and the intronic enhancers were markedly differed between 3' and 5' intronic flanks. Conclusion Difference in intronic enhancers supporting 5' and 3' splice sites suggests an independent splicing commitment for neighboring exons. Increased evolutionary conservation for ISEs/ISSs within intronic flanks and effect of modulation of predicted elements on splicing suggest functional significance of found elements in splicing regulation. Most of the elements identified were shown to have direct implications in human splicing and therefore could be useful for building computational splicing models in biomedical research. PMID:19889216

  3. Computational prediction of splicing regulatory elements shared by Tetrapoda organisms

    Directory of Open Access Journals (Sweden)

    Hicks Chindo

    2009-11-01

    Full Text Available Abstract Background Auxiliary splicing sequences play an important role in ensuring accurate and efficient splicing by promoting or repressing recognition of authentic splice sites. These cis-acting motifs have been termed splicing enhancers and silencers and are located both in introns and exons. They co-evolved into an intricate splicing code together with additional functional constraints, such as tissue-specific and alternative splicing patterns. We used orthologous exons extracted from the University of California Santa Cruz multiple genome alignments of human and 22 Tetrapoda organisms to predict candidate enhancers and silencers that have reproducible and statistically significant bias towards annotated exonic boundaries. Results A total of 2,546 Tetrapoda enhancers and silencers were clustered into 15 putative core motifs based on their Markov properties. Most of these elements have been identified previously, but 118 putative silencers and 260 enhancers (~15% were novel. Examination of previously published experimental data for the presence of predicted elements showed that their mutations in 21/23 (91.3% cases altered the splicing pattern as expected. Predicted intronic motifs flanking 3' and 5' splice sites had higher evolutionary conservation than other sequences within intronic flanks and the intronic enhancers were markedly differed between 3' and 5' intronic flanks. Conclusion Difference in intronic enhancers supporting 5' and 3' splice sites suggests an independent splicing commitment for neighboring exons. Increased evolutionary conservation for ISEs/ISSs within intronic flanks and effect of modulation of predicted elements on splicing suggest functional significance of found elements in splicing regulation. Most of the elements identified were shown to have direct implications in human splicing and therefore could be useful for building computational splicing models in biomedical research.

  4. Computational prediction of splicing regulatory elements shared by Tetrapoda organisms.

    Science.gov (United States)

    Churbanov, Alexander; Vorechovský, Igor; Hicks, Chindo

    2009-11-04

    Auxiliary splicing sequences play an important role in ensuring accurate and efficient splicing by promoting or repressing recognition of authentic splice sites. These cis-acting motifs have been termed splicing enhancers and silencers and are located both in introns and exons. They co-evolved into an intricate splicing code together with additional functional constraints, such as tissue-specific and alternative splicing patterns. We used orthologous exons extracted from the University of California Santa Cruz multiple genome alignments of human and 22 Tetrapoda organisms to predict candidate enhancers and silencers that have reproducible and statistically significant bias towards annotated exonic boundaries. A total of 2,546 Tetrapoda enhancers and silencers were clustered into 15 putative core motifs based on their Markov properties. Most of these elements have been identified previously, but 118 putative silencers and 260 enhancers (~15%) were novel. Examination of previously published experimental data for the presence of predicted elements showed that their mutations in 21/23 (91.3%) cases altered the splicing pattern as expected. Predicted intronic motifs flanking 3' and 5' splice sites had higher evolutionary conservation than other sequences within intronic flanks and the intronic enhancers were markedly differed between 3' and 5' intronic flanks. Difference in intronic enhancers supporting 5' and 3' splice sites suggests an independent splicing commitment for neighboring exons. Increased evolutionary conservation for ISEs/ISSs within intronic flanks and effect of modulation of predicted elements on splicing suggest functional significance of found elements in splicing regulation. Most of the elements identified were shown to have direct implications in human splicing and therefore could be useful for building computational splicing models in biomedical research.

  5. Improving the Accuracy of Fitted Atomic Models in Cryo-EM Density Maps of Protein Assemblies Using Evolutionary Information from Aligned Homologous Proteins.

    Science.gov (United States)

    Rakesh, Ramachandran; Srinivasan, Narayanaswamy

    2016-01-01

    Cryo-Electron Microscopy (cryo-EM) has become an important technique to obtain structural insights into large macromolecular assemblies. However the resolution of the density maps do not allow for its interpretation at atomic level. Hence they are combined with high resolution structures along with information from other experimental or bioinformatics techniques to obtain pseudo-atomic models. Here, we describe the use of evolutionary conservation of residues as obtained from protein structures and alignments of homologous proteins to detect errors in the fitting of atomic structures as well as improve accuracy of the protein-protein interfacial regions in the cryo-EM density maps.

  6. A gene-based radiation hybrid map of chicken microchromosome 14: Comparison to human and alignment to the assembled chicken sequence

    Directory of Open Access Journals (Sweden)

    Milan Denis

    2005-03-01

    Full Text Available Abstract We present a gene-based RH map of the chicken microchromosome GGA14, known to have synteny conservations with human chromosomal regions HSA16p13.3 and HSA17p11.2. Microsatellite markers from the genetic map were used to check the validity of the RH map and additional markers were developed from chicken EST data to yield comparative mapping data. A high rate of intra-chromosomal rearrangements was detected by comparison to the assembled human sequence. Finally, the alignment of the RH map to the assembled chicken sequence showed a small number of discordances, most of which involved the same region of the chromosome spanning between 40.5 and 75.9 cR6000 on the RH map.

  7. Neuron-specific splicing.

    Science.gov (United States)

    Hakim, Nor Hakimah Ab; Majlis, Burhanuddin Yeop; Suzuki, Hitoshi; Tsukahara, Toshifumi

    2017-03-22

    During pre-mRNA splicing events, introns are removed from the pre-mRNA, and the remaining exons are connected together to form a single continuous molecule. Alternative splicing is a common mechanism for the regulation of gene expression in eukaryotes. More than 90% of human genes are known to undergo alternative splicing. The most common type of alternative splicing is exon skipping, which is also known as cassette exon. Other known alternative splicing events include alternative 5' splice sites, alternative 3' splice sites, intron retention, and mutually exclusive exons. Alternative splicing events are controlled by regulatory proteins responsible for both positive and negative regulation. In this review, we focus on neuronal splicing regulators and discuss several notable regulators in depth. In addition, we have also included an example of splicing regulation mediated by the RBFox protein family. Lastly, as previous studies have shown that a number of splicing factors are associated with neuronal diseases such as Alzheime's disease (AD) and Autism spectrum disorder (ASD), here we consider their importance in neuronal diseases wherein the underlying mechanisms have yet to be elucidated.

  8. Alternative splicing in ascomycetes.

    Science.gov (United States)

    Kempken, Frank

    2013-05-01

    Alternative splicing is a complex and regulated process, which results in mRNA with different coding capacities from a single gene. Extend and types of alternative splicing vary greatly among eukaryotes. In this review, I focus on alternative splicing in ascomycetes, which in general have significant lower extend of alternative splicing than mammals. Yeast-like species have low numbers of introns and consequently alternative splicing is lower compared to filamentous fungi. Several examples from single studies as well as from genomic scale analysis are presented, including a survey of alternative splicing in Neurospora crassa. Another focus is regulation by riboswitch RNA and alternative splicing in a heterologous system, along with putative protein factors involved in regulation.

  9. Alternative Splicing in CKD.

    Science.gov (United States)

    Stevens, Megan; Oltean, Sebastian

    2016-06-01

    Alternative splicing (AS) has emerged in the postgenomic era as one of the main drivers of proteome diversity, with ≥94% of multiexon genes alternatively spliced in humans. AS is therefore one of the main control mechanisms for cell phenotype, and is a process deregulated in disease. Numerous reports describe pathogenic mutations in splice factors, splice sites, or regulatory sequences. Additionally, compared with the physiologic state, disease often associates with an abnormal proportion of splice isoforms (or novel isoforms), without an apparent driver mutation. It is therefore essential to study how AS is regulated in physiology, how it contributes to pathogenesis, and whether we can manipulate faulty splicing for therapeutic advantage. Although the disease most commonly linked to deregulation of AS in several genes is cancer, many reports detail pathogenic splice variants in diseases ranging from neuromuscular disorders to diabetes or cardiomyopathies. A plethora of splice variants have been implicated in CKDs as well. In this review, we describe examples of these CKD-associated splice variants and ideas on how to manipulate them for therapeutic benefit. Copyright © 2016 by the American Society of Nephrology.

  10. Conditional control of alternative splicing through light-triggered splice-switching oligonucleotides.

    Science.gov (United States)

    Hemphill, James; Liu, Qingyang; Uprety, Rajendra; Samanta, Subhas; Tsang, Michael; Juliano, Rudolph L; Deiters, Alexander

    2015-03-18

    The spliceosome machinery is composed of several proteins and multiple small RNA molecules that are involved in gene regulation through the removal of introns from pre-mRNAs in order to assemble exon-based mRNA containing protein-coding sequences. Splice-switching oligonucleotides (SSOs) are genetic control elements that can be used to specifically control the expression of genes through correction of aberrant splicing pathways. A current limitation with SSO methodologies is the inability to achieve conditional control of their function paired with high spatial and temporal resolution. We addressed this limitation through site-specific installation of light-removable nucleobase-caging groups as well as photocleavable backbone linkers into synthetic SSOs. This enables optochemical OFF → ON and ON → OFF switching of their activity and thus precise control of alternative splicing. The use of light as a regulatory element allows for tight spatial and temporal control of splice switching in mammalian cells and animals.

  11. Splicing Regulation in Neurologic Disease

    National Research Council Canada - National Science Library

    Licatalosi, Donny D; Darnell, Robert B

    2006-01-01

    .... It is becoming evident that alternative splicing plays a particularly important role in neurologic disease, which is perhaps not surprising given the important role splicing plays in generating...

  12. Low resistance splices for HTS devices and applications

    Science.gov (United States)

    Lalitha, S. L.

    2017-09-01

    This paper discusses the preparation methodology and performance evaluation of low resistance splices made of the second generation (2G) high-temperature superconductor (HTS). These splices are required in a broad spectrum of HTS devices including a large aperture, high-field solenoid built in the laboratory to demonstrate a superconducting magnetic energy storage (SMES) device. Several pancake coils are assembled in the form of a nested solenoid, and each coil requires a hundred meters or more of 2G (RE)BCO tape. However, commercial availability of this superconductor with a very uniform physical properties is currently limited to shorter piece lengths. This necessitates us having splices to inter-connect the tape pieces within a pancake coil, between adjacent pancake coils, and to attach HTS current leads to the magnet assembly. As a part of the optimization and qualification of splicing process, a systematic study was undertaken to analyze the electrical performance of splices in two different configurations suitable for this magnet assembly: lap joint and spiral joint. The electrical performance is quantified in terms of the resistance of splices estimated from the current-voltage characteristics. It has been demonstrated that a careful application of this splicing technique can generate lap joints with resistance less than 1 nΩ at 77 K.

  13. spliceR

    DEFF Research Database (Denmark)

    Vitting-Seerup, Kristoffer; Porse, Bo Torben; Sandelin, Albin

    2014-01-01

    RNA-seq data is currently underutilized, in part because it is difficult to predict the functional impact of alternate transcription events. Recent software improvements in full-length transcript deconvolution prompted us to develop spliceR, an R package for classification of alternative splicing...

  14. Efficient Photocatalytic Degradation of Rhodamine B Dye by Aligned Arrays of Self-Assembled Hydrogen Titanate Nanotubes

    Directory of Open Access Journals (Sweden)

    Sriparna Chatterjee

    2014-01-01

    Full Text Available We show that an aligned array of hydrothermally grown, multiwalled hydrogen titanate (H2Ti3O7 nanotubes—anchored to both faces of a metallic Ti foil—acts as an efficient photocatalyst. We studied the degradation of rhodamine B dye in the presence of the nanostructured photocatalyst under UV irradiation, by monitoring the optical absorption of the dye. Rhodamine B was chosen as a representative—and particularly harmful—industrial pollutant dye. The inner and outer diameters of the H2Ti3O7 nanotubes were 5 nm and 10 nm, respectively. The nanotube array catalyst is recyclable and structurally stable. Most importantly, it shows comparable or higher photodecomposition rate constant than those of both H2Ti3O7 nanotube powder and P-25 (Degussa. The enhanced photocatalytic performance may be ascribed to the nanotube array having a superhydrophilic surface with a high accessible surface area.

  15. Genome-wide analysis of alternative splicing in Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Thomas Julie

    2010-02-01

    Full Text Available Abstract Background Genome-wide computational analysis of alternative splicing (AS in several flowering plants has revealed that pre-mRNAs from about 30% of genes undergo AS. Chlamydomonas, a simple unicellular green alga, is part of the lineage that includes land plants. However, it diverged from land plants about one billion years ago. Hence, it serves as a good model system to study alternative splicing in early photosynthetic eukaryotes, to obtain insights into the evolution of this process in plants, and to compare splicing in simple unicellular photosynthetic and non-photosynthetic eukaryotes. We performed a global analysis of alternative splicing in Chlamydomonas reinhardtii using its recently completed genome sequence and all available ESTs and cDNAs. Results Our analysis of AS using BLAT and a modified version of the Sircah tool revealed AS of 498 transcriptional units with 611 events, representing about 3% of the total number of genes. As in land plants, intron retention is the most prevalent form of AS. Retained introns and skipped exons tend to be shorter than their counterparts in constitutively spliced genes. The splice site signals in all types of AS events are weaker than those in constitutively spliced genes. Furthermore, in alternatively spliced genes, the prevalent splice form has a stronger splice site signal than the non-prevalent form. Analysis of constitutively spliced introns revealed an over-abundance of motifs with simple repetitive elements in comparison to introns involved in intron retention. In almost all cases, AS results in a truncated ORF, leading to a coding sequence that is around 50% shorter than the prevalent splice form. Using RT-PCR we verified AS of two genes and show that they produce more isoforms than indicated by EST data. All cDNA/EST alignments and splice graphs are provided in a website at http://combi.cs.colostate.edu/as/chlamy. Conclusions The extent of AS in Chlamydomonas that we observed is much

  16. Optical Fiber Fusion Splicing

    CERN Document Server

    Yablon, Andrew D

    2005-01-01

    This book is an up-to-date treatment of optical fiber fusion splicing incorporating all the recent innovations in the field. It provides a toolbox of general strategies and specific techniques that the reader can apply when optimizing fusion splices between novel fibers. It specifically addresses considerations important for fusion splicing of contemporary specialty fibers including dispersion compensating fiber, erbium-doped gain fiber, polarization maintaining fiber, and microstructured fiber. Finally, it discusses the future of optical fiber fusion splicing including silica and non-silica based optical fibers as well as the trend toward increasing automation. Whilst serving as a self-contained reference work, abundant citations from the technical literature will enable readers to readily locate primary sources.

  17. Chromatin and alternative splicing.

    Science.gov (United States)

    Alló, M; Schor, I E; Muñoz, M J; de la Mata, M; Agirre, E; Valcárcel, J; Eyras, E; Kornblihtt, A R

    2010-01-01

    Alternative splicing affects more than 90% of human genes. Coupling between transcription and splicing has become crucial in the complex network underlying alternative splicing regulation. Because chromatin is the real template for nuclear transcription, changes in its structure, but also in the "reading" and "writing" of the histone code, could modulate splicing choices. Here, we discuss the evidence supporting these ideas, from the first proposal of chromatin affecting alternative splicing, performed 20 years ago, to the latest findings including genome-wide evidence that nucleosomes are preferentially positioned in exons. We focus on two recent reports from our laboratories that add new evidence to this field. The first report shows that a physiological stimulus such as neuron depolarization promotes intragenic histone acetylation (H3K9ac) and chromatin relaxation, causing the skipping of exon 18 of the neural cell adhesion molecule gene. In the second report, we show how specific histone modifications can be created at targeted gene regions as a way to affect alternative splicing: Using small interfering RNAs (siRNAs), we increased the levels of H3K9me2 and H3K27me3 in the proximity of alternative exon 33 of the human fibronectin gene, favoring its inclusion into mature messenger RNA (mRNA) through a mechanism that recalls RNA-mediated transcriptional gene silencing.

  18. Self-Assembled Formation of Well-Aligned Cu-Te Nano-Rods on Heavily Cu-Doped ZnTe Thin Films

    Science.gov (United States)

    Liang, Jing; Cheng, Man Kit; Lai, Ying Hoi; Wei, Guanglu; Yang, Sean Derman; Wang, Gan; Ho, Sut Kam; Tam, Kam Weng; Sou, Iam Keong

    2016-11-01

    Cu doping of ZnTe, which is an important semiconductor for various optoelectronic applications, has been successfully achieved previously by several techniques. However, besides its electrical transport characteristics, other physical and chemical properties of heavily Cu-doped ZnTe have not been reported. We found an interesting self-assembled formation of crystalline well-aligned Cu-Te nano-rods near the surface of heavily Cu-doped ZnTe thin films grown via the molecular beam epitaxy technique. A phenomenological growth model is presented based on the observed crystallographic morphology and measured chemical composition of the nano-rods using various imaging and chemical analysis techniques. When substitutional doping reaches its limit, the extra Cu atoms favor an up-migration toward the surface, leading to a one-dimensional surface modulation and formation of Cu-Te nano-rods, which explain unusual observations on the reflection high energy electron diffraction patterns and apparent resistivity of these thin films. This study provides an insight into some unexpected chemical reactions involved in the heavily Cu-doped ZnTe thin films, which may be applied to other material systems that contain a dopant having strong reactivity with the host matrix.

  19. Herboxidiene triggers splicing repression and abiotic stress responses in plants

    KAUST Repository

    Alshareef, Sahar

    2017-03-27

    Background Constitutive and alternative splicing of pre-mRNAs from multiexonic genes controls the diversity of the proteome; these precisely regulated processes also fine-tune responses to cues related to growth, development, and stresses. Small-molecule inhibitors that perturb splicing provide invaluable tools for use as chemical probes to uncover the molecular underpinnings of splicing regulation and as potential anticancer compounds. Results Here, we show that herboxidiene (GEX1A) inhibits both constitutive and alternative splicing. Moreover, GEX1A activates genome-wide transcriptional patterns involved in abiotic stress responses in plants. GEX1A treatment -activated ABA-inducible promoters, and led to stomatal closure. Interestingly, GEX1A and pladienolide B (PB) elicited similar cellular changes, including alterations in the patterns of transcription and splicing, suggesting that these compounds might target the same spliceosome complex in plant cells. Conclusions Our study establishes GEX1A as a potent splicing inhibitor in plants that can be used to probe the assembly, dynamics, and molecular functions of the spliceosome and to study the interplay between splicing stress and abiotic stresses, as well as having potential biotechnological applications.

  20. Transcriptome-wide modulation of splicing by the exon junction complex.

    Science.gov (United States)

    Wang, Zhen; Murigneux, Valentine; Le Hir, Hervé

    2014-01-01

    The exon junction complex (EJC) is a dynamic multi-protein complex deposited onto nuclear spliced mRNAs upstream of exon-exon junctions. The four core proteins, eIF4A3, Magoh, Y14 and MLN51, are stably bound to mRNAs during their lifecycle, serving as a binding platform for other nuclear and cytoplasmic proteins. Recent evidence has shown that the EJC is involved in the splicing regulation of some specific events in both Drosophila and mammalian cells. Here, we show that knockdown of EJC core proteins causes widespread alternative splicing changes in mammalian cells. These splicing changes are specific to EJC core proteins, as knockdown of eIF4A3, Y14 and MLN51 shows similar splicing changes, and are different from knockdown of other splicing factors. The splicing changes can be rescued by a siRNA-resistant form of eIF4A3, indicating an involvement of EJC core proteins in regulating alternative splicing. Finally, we find that the splicing changes are linked with RNA polymerase II elongation rates. Taken together, this study reveals that the coupling between EJC proteins and splicing is broader than previously suspected, and that a possible link exists between mRNP assembly and splice site recognition.

  1. A 5' splice site enhances the recruitment of basal transcription initiation factors in vivo

    DEFF Research Database (Denmark)

    Damgaard, Christian Kroun; Kahns, Søren; Lykke-Andersen, Søren

    2008-01-01

    Transcription and pre-mRNA splicing are interdependent events. Although mechanisms governing the effects of transcription on splicing are becoming increasingly clear, the means by which splicing affects transcription remain elusive. Using cell lines stably expressing HIV-1 or β-globin mRNAs, harb...... a promoter-proximal 5′ splice site via its U1 snRNA interaction can feed back to stimulate transcription initiation by enhancing preinitiation complex assembly.......Transcription and pre-mRNA splicing are interdependent events. Although mechanisms governing the effects of transcription on splicing are becoming increasingly clear, the means by which splicing affects transcription remain elusive. Using cell lines stably expressing HIV-1 or β-globin mRNAs......, harboring wild-type or various 5′ splice site mutations, we demonstrate a strong positive correlation between splicing efficiency and transcription activity. Interestingly, a 5′ splice site can stimulate transcription even in the absence of splicing. Chromatin immunoprecipitation experiments show enhanced...

  2. Expressiveness of basic Splice

    NARCIS (Netherlands)

    J.C. van de Pol (Jaco)

    2000-01-01

    textabstractWe study a simple software architecture, in which application processes are coordinated by writing into and reading from a global set. This architecture underlies Splice, which is developed and used at the company Hollandse Signaalapparaten. Our approach is distinguished by viewing the

  3. Towards a Consolidation of LHC Superconducting Splices for 7 TeV Operation

    CERN Document Server

    Bertinelli, F; Fessia, P; Garion, C; Mathot, S; Perin, A; Scheuerlein, C; Sgobba, S; ten Kat, H; Tock, J P; Verweij, A; Willering, G

    2010-01-01

    Following the analysis of the September 2008 LHC incident, the assembly process and the quality assurance of the main 13 kA interconnection splices were improved, with new measurement and diagnostics methods introduced. During the 2008-2009 shutdown ~5% of these 10 000 splices were newly assembled with these improvements implemented, but essentially maintaining the original design. It is known today that a limiting factor towards 7 TeV operation is the normal conducting resistance of ~15% of the original main 13 kA interconnection splices, associated to the electrical continuity of the copper stabiliser. A “Splices Task Force” has been set up at CERN to evaluate the need for, develop and test design improvements and prepare the implementation of a consolidation campaign. Important issues of splice design, process choice, resources and time requirements are considered.

  4. The second RNA-binding domain of the human splicing factor ASF/SF2 is the critical domain controlling adenovirus E1A alternative 5'-splice site selection.

    Science.gov (United States)

    Dauksaite, Vita; Akusjärvi, Göran

    2004-07-15

    The human splicing factor ASF/SF2 (alternative splicing factor/splicing factor 2) is modular in structure with two RNA-binding domains (RBD1 and RBD2) and a C-terminal domain rich in arginine-serine dipeptide repeats. ASF/SF2 is an essential splicing factor that also functions as an important regulator of alternative splicing. In adenovirus E1A (early region 1A) alternative pre-mRNA splicing, ASF/SF2 functions as a strong inducer of proximal 5'-splice-site selection, both in vitro and in vivo. In the present study, we tested the functional role of individual domains of ASF/SF2 in alternative splicing in vitro. We show that ASF/SF2-RBD2 is the critical domain controlling E1A alternative splicing. In fact, RBD2 alone is sufficient to mimic the activity of the full-length ASF/SF2 protein as an inducer of proximal 5'-splice-site selection in vitro. The RBD2 domain induces a switch to E1A-proximal 5'-splice-site usage by repressing distal 12 S splicing and simultaneously stimulates proximal 13 S splicing. In contrast, the ASF/SF2-RBD1 domain has a more general splicing enhancer phenotype and appears to stimulate preferentially cap-proximal 5'-splice-site selection. Furthermore, the SWQDLKD motif, which is conserved in all SR proteins (serine/arginine-rich proteins) containing two RBDs, and the ribonucleoprotein-1-type RNA recognition motif were both found to be necessary for the alternative splice-site-switching activity of ASF/SF2. The RNP-1 motif was necessary for efficient RNA binding, whereas the SWQDLKD motif most probably contributes by functioning as a surface-mediating critical protein-protein contact during spliceosome assembly.

  5. Transcriptome sequencing revealed significant alteration of cortical promoter usage and splicing in schizophrenia.

    Directory of Open Access Journals (Sweden)

    Jing Qin Wu

    Full Text Available While hybridization based analysis of the cortical transcriptome has provided important insight into the neuropathology of schizophrenia, it represents a restricted view of disease-associated gene activity based on predetermined probes. By contrast, sequencing technology can provide un-biased analysis of transcription at nucleotide resolution. Here we use this approach to investigate schizophrenia-associated cortical gene expression.The data was generated from 76 bp reads of RNA-Seq, aligned to the reference genome and assembled into transcripts for quantification of exons, splice variants and alternative promoters in postmortem superior temporal gyrus (STG/BA22 from 9 male subjects with schizophrenia and 9 matched non-psychiatric controls. Differentially expressed genes were then subjected to further sequence and functional group analysis. The output, amounting to more than 38 Gb of sequence, revealed significant alteration of gene expression including many previously shown to be associated with schizophrenia. Gene ontology enrichment analysis followed by functional map construction identified three functional clusters highly relevant to schizophrenia including neurotransmission related functions, synaptic vesicle trafficking, and neural development. Significantly, more than 2000 genes displayed schizophrenia-associated alternative promoter usage and more than 1000 genes showed differential splicing (FDR<0.05. Both types of transcriptional isoforms were exemplified by reads aligned to the neurodevelopmentally significant doublecortin-like kinase 1 (DCLK1 gene.This study provided the first deep and un-biased analysis of schizophrenia-associated transcriptional diversity within the STG, and revealed variants with important implications for the complex pathophysiology of schizophrenia.

  6. Biochemical identification of new proteins involved in splicing repression at the Drosophila P-element exonic splicing silencer

    Science.gov (United States)

    Horan, Lucas; Yasuhara, Jiro C.; Kohlstaedt, Lori A.; Rio, Donald C.

    2015-01-01

    Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5′ exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5′ exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites. PMID:26545814

  7. Genome-wide analysis of alternative splicing events in Hordeum vulgare: Highlighting retention of intron-based splicing and its possible function through network analysis.

    Science.gov (United States)

    Panahi, Bahman; Mohammadi, Seyed Abolghasem; Ebrahimi Khaksefidi, Reyhaneh; Fallah Mehrabadi, Jalil; Ebrahimie, Esmaeil

    2015-11-30

    In this study, using homology mapping of assembled expressed sequence tags against the genomic data, we identified alternative splicing events in barley. Results demonstrated that intron retention is frequently associated with specific abiotic stresses. Network analysis resulted in discovery of some specific sub-networks between miRNAs and transcription factors in genes with high number of alternative splicing, such as cross talk between SPL2, SPL10 and SPL11 regulated by miR156 and miR157 families. To confirm the alternative splicing events, elongation factor protein (MLOC_3412) was selected followed by experimental verification of the predicted splice variants by Semi quantitative Reverse Transcription PCR (qRT-PCR). Our novel integrative approach opens a new avenue for functional annotation of alternative splicing through regulatory-based network discovery. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  8. Method of predicting Splice Sites based on signal interactions

    Directory of Open Access Journals (Sweden)

    Deogun Jitender S

    2006-04-01

    Full Text Available Abstract Background Predicting and proper ranking of canonical splice sites (SSs is a challenging problem in bioinformatics and machine learning communities. Any progress in SSs recognition will lead to better understanding of splicing mechanism. We introduce several new approaches of combining a priori knowledge for improved SS detection. First, we design our new Bayesian SS sensor based on oligonucleotide counting. To further enhance prediction quality, we applied our new de novo motif detection tool MHMMotif to intronic ends and exons. We combine elements found with sensor information using Naive Bayesian Network, as implemented in our new tool SpliceScan. Results According to our tests, the Bayesian sensor outperforms the contemporary Maximum Entropy sensor for 5' SS detection. We report a number of putative Exonic (ESE and Intronic (ISE Splicing Enhancers found by MHMMotif tool. T-test statistics on mouse/rat intronic alignments indicates, that detected elements are on average more conserved as compared to other oligos, which supports our assumption of their functional importance. The tool has been shown to outperform the SpliceView, GeneSplicer, NNSplice, Genio and NetUTR tools for the test set of human genes. SpliceScan outperforms all contemporary ab initio gene structural prediction tools on the set of 5' UTR gene fragments. Conclusion Designed methods have many attractive properties, compared to existing approaches. Bayesian sensor, MHMMotif program and SpliceScan tools are freely available on our web site. Reviewers This article was reviewed by Manyuan Long, Arcady Mushegian and Mikhail Gelfand.

  9. Mutual interdependence of splicing and transcription elongation.

    Science.gov (United States)

    Brzyżek, Grzegorz; Świeżewski, Szymon

    2015-01-01

    Transcription and splicing are intrinsically linked, as splicing needs a pre-mRNA substrate to commence. The more nuanced view is that the rate of transcription contributes to splicing regulation. On the other hand there is accumulating evidence that splicing has an active role in controlling transcription elongation by DNA-dependent RNA polymerase II (RNAP II). We briefly review those mechanisms and propose a unifying model where splicing controls transcription elongation to provide an optimal timing for successive rounds of splicing.

  10. Development of a single vector system that enhances trans-splicing of SMN2 transcripts.

    Directory of Open Access Journals (Sweden)

    Tristan H Coady

    Full Text Available RNA modalities are developing as a powerful means to re-direct pathogenic pre-mRNA splicing events. Improving the efficiency of these molecules in vivo is critical as they move towards clinical applications. Spinal muscular atrophy (SMA is caused by loss of SMN1. A nearly identical copy gene called SMN2 produces low levels of functional protein due to alternative splicing. We previously reported a trans-splicing RNA (tsRNA that re-directed SMN2 splicing. Now we show that reducing the competition between endogenous splices sites enhanced the efficiency of trans-splicing. A single vector system was developed that expressed the SMN tsRNA and a splice-site blocking antisense (ASO-tsRNA. The ASO-tsRNA vector significantly elevated SMN levels in primary SMA patient fibroblasts, within the central nervous system of SMA mice and increased SMN-dependent in vitro snRNP assembly. These results demonstrate that the ASO-tsRNA strategy provides insight into the trans-splicing mechanism and a means of significantly enhancing trans-splicing activity in vivo.

  11. Mutations in the small subunit of the Drosophila U2AF splicing factor cause lethality and developmental defects

    NARCIS (Netherlands)

    D.Z. Rudner (David); R. Kanaar (Roland); K.S. Breger (Kevin); D.C. Rio (Donald)

    1996-01-01

    textabstractThe essential eukaryotic pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein auxiliary factor) is required to specify the 3' splice site at an early step in spliceosome assembly. U2AF binds site-specifically to the intron polypyrimidine tract and recruits U2 small nuclear

  12. MolAlign: an algorithm for aligning multiple small molecules.

    Science.gov (United States)

    Chan, Shek Ling

    2017-06-01

    In small molecule drug discovery projects, the receptor structure is not always available. In such cases it is enormously useful to be able to align known ligands in the way they bind in the receptor. Here we shall present an algorithm for the alignment of multiple small molecule ligands. This algorithm takes pre-generated conformers as input, and proposes aligned assemblies of the ligands. The algorithm consists of two stages: the first stage is to perform alignments for each pair of ligands, the second stage makes use of the results from the first stage to build up multiple ligand alignment assemblies using a novel iterative procedure. The scoring functions are improved versions of the one mentioned in our previous work. We have compared our results with some recent publications. While an exact comparison is impossible, it is clear that our algorithm is fast and produces very competitive results.

  13. The strength of intron donor splice sites in human genes displays a bell-shaped pattern

    DEFF Research Database (Denmark)

    Wang, Kai; Wernersson, Rasmus; Brunak, Søren

    2011-01-01

    during spliceosome assembly are fused and in tandem are transcribed and spliced into a single mature mRNA sequence. In their splice site patterns, these genes individually seem to deviate from the convex pattern, offering a possible rationale behind their fusion into a single transcript.......MOTIVATION: The gene concept has recently changed from the classical one protein notion into a much more diverse picture, where overlapping or fused transcripts, alternative transcription initiation, and genes within genes, add to the complexity generated by alternative splicing. Increased...... understanding of the mechanisms controlling pre-mRNA splicing is thus important for a wide range of aspects relating to gene expression. RESULTS: We have discovered a convex gene delineating pattern in the strength of 5' intron splice sites. When comparing the strengths of >18 000 intron containing Human genes...

  14. Analysis of a splice array experiment elucidates roles of chromatin elongation factor Spt4-5 in splicing.

    Directory of Open Access Journals (Sweden)

    Yuanyuan Xiao

    2005-09-01

    Full Text Available Splicing is an important process for regulation of gene expression in eukaryotes, and it has important functional links to other steps of gene expression. Two examples of these linkages include Ceg1, a component of the mRNA capping enzyme, and the chromatin elongation factors Spt4-5, both of which have recently been shown to play a role in the normal splicing of several genes in the yeast Saccharomyces cerevisiae. Using a genomic approach to characterize the roles of Spt4-5 in splicing, we used splicing-sensitive DNA microarrays to identify specific sets of genes that are mis-spliced in ceg1, spt4, and spt5 mutants. In the context of a complex, nested, experimental design featuring 22 dye-swap array hybridizations, comprising both biological and technical replicates, we applied five appropriate statistical models for assessing differential expression between wild-type and the mutants. To refine selection of differential expression genes, we then used a robust model-synthesizing approach, Differential Expression via Distance Synthesis, to integrate all five models. The resultant list of differentially expressed genes was then further analyzed with regard to select attributes: we found that highly transcribed genes with long introns were most sensitive to spt mutations. QPCR confirmation of differential expression was established for the limited number of genes evaluated. In this paper, we showcase splicing array technology, as well as powerful, yet general, statistical methodology for assessing differential expression, in the context of a real, complex experimental design. Our results suggest that the Spt4-Spt5 complex may help coordinate splicing with transcription under conditions that present kinetic challenges to spliceosome assembly or function.

  15. Intronic Alus influence alternative splicing.

    Directory of Open Access Journals (Sweden)

    Galit Lev-Maor

    2008-09-01

    Full Text Available Examination of the human transcriptome reveals higher levels of RNA editing than in any other organism tested to date. This is indicative of extensive double-stranded RNA (dsRNA formation within the human transcriptome. Most of the editing sites are located in the primate-specific retrotransposed element called Alu. A large fraction of Alus are found in intronic sequences, implying extensive Alu-Alu dsRNA formation in mRNA precursors. Yet, the effect of these intronic Alus on splicing of the flanking exons is largely unknown. Here, we show that more Alus flank alternatively spliced exons than constitutively spliced ones; this is especially notable for those exons that have changed their mode of splicing from constitutive to alternative during human evolution. This implies that Alu insertions may change the mode of splicing of the flanking exons. Indeed, we demonstrate experimentally that two Alu elements that were inserted into an intron in opposite orientation undergo base-pairing, as evident by RNA editing, and affect the splicing patterns of a downstream exon, shifting it from constitutive to alternative. Our results indicate the importance of intronic Alus in influencing the splicing of flanking exons, further emphasizing the role of Alus in shaping of the human transcriptome.

  16. MUON DETECTORS: ALIGNMENT

    CERN Multimedia

    S. Szillasi

    2013-01-01

    The CMS detector has been gradually opened and whenever a wheel became exposed the first operation was the removal of the MABs, the sensor structures of the Hardware Barrel Alignment System. By the last days of June all 36 MABs have arrived at the Alignment Lab at the ISR where, as part of the Alignment Upgrade Project, they are refurbished with new Survey target holders. Their electronic checkout is on the way and finally they will be recalibrated. During LS1 the alignment system will be upgraded in order to allow more precise reconstruction of the MB4 chambers in Sector 10 and Sector 4. This requires new sensor components, so called MiniMABs (pictured below), that have already been assembled and calibrated. Image 6: Calibrated MiniMABs are ready for installation For the track-based alignment, the systematic uncertainties of the algorithm are under scrutiny: this study will enable the production of an improved Monte Carlo misalignment scenario and to update alignment position errors eventually, crucial...

  17. Splicing Activation by Rbfox Requires Self-Aggregation through Its Tyrosine-Rich Domain.

    Science.gov (United States)

    Ying, Yi; Wang, Xiao-Jun; Vuong, Celine K; Lin, Chia-Ho; Damianov, Andrey; Black, Douglas L

    2017-07-13

    Proteins of the Rbfox family act with a complex of proteins called the Large Assembly of Splicing Regulators (LASR). We find that Rbfox interacts with LASR via its C-terminal domain (CTD), and this domain is essential for its splicing activity. In addition to LASR recruitment, a low-complexity (LC) sequence within the CTD contains repeated tyrosines that mediate higher-order assembly of Rbfox/LASR and are required for splicing activation by Rbfox. This sequence spontaneously aggregates in solution to form fibrous structures and hydrogels, suggesting an assembly similar to the insoluble cellular inclusions formed by FUS and other proteins in neurologic disease. Unlike the pathological aggregates, we find that assembly of the Rbfox CTD plays an essential role in its normal splicing function. Rather than simple recruitment of individual regulators to a target exon, alternative splicing choices also depend on the higher-order assembly of these regulators within the nucleus. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Handbook of knotting and splicing

    CERN Document Server

    Hasluck, Paul N

    2005-01-01

    Clearly written and amply illustrated with 208 figures, this classic guide ranges from simple and useful knots to complex varieties. Additional topics include rope splicing, working cordage, hammock making, more.

  19. 3′ Splice Site Sequences of Spinal Muscular Atrophy Related SMN2 Pre-mRNA Include Enhancers for Nearby Exons

    Directory of Open Access Journals (Sweden)

    Sunghee Cho

    2014-01-01

    Full Text Available Spinal muscular atrophy (SMA is a human genetic disease which occurs because of the deletion or mutation of SMN1 gene. SMN1 gene encodes the SMN protein which plays a key role in spliceosome assembly. Although human patients contain SMN2, a duplicate of SMN1, splicing of SMN2 produces predominantly exon 7 skipped isoform. In order to understand the functions of splice site sequences on exon 7 and 8, we analyzed the effects of conserved splice site sequences on exon 7 skipping of SMN2 and SMN1 pre-mRNA. We show here that conserved 5′ splice site sequence of exon 7 promoted splicing of nearby exons and subsequently reduced splicing of distant exons. However, to our surprise, conserved 3′ splice site sequence of exon 7 and 8 did not promote splicing of nearby exons. By contrast, the mutation inhibited splicing of nearby exons and subsequently promoted splicing of distant exons. Our study shows that 3′ splice sites of exon 7 and 8 contain enhancer for their splice site selection, in addition to providing cleavage sites.

  20. Solid State NMR Structure Analysis of the Antimicrobial Peptide Gramicidin S in Lipid Membranes: Concentration-Dependent Re-alignment and Self-Assembly as a β-Barrel

    Science.gov (United States)

    Afonin, Sergii; Dürr, Ulrich H. N.; Wadhwani, Parvesh; Salgado, Jesus; Ulrich, Anne S.

    Antimicrobial peptides can kill bacteria by permeabilizing their cell membrane, as these amphiphilic molecules interact favourably with lipid bilayers. This mechanism of action is attributed either to the formation of a peptide “carpet” on the membrane surface, or to a transmembrane pore. However, the structure of such a pore has not yet been resolved under relevant conditions. Gramicidin S is a symmetrical cyclic β-sheet decapeptide, which has been previously shown by solid state NMR to lie flat on the membrane surface at low peptide:lipid ratios (≤ 1:80). Using highly sensitive 19F-NMR, supported by 15N-labelling, we found that gramicidin S can flip into an upright transmembrane alignment at high peptide:lipid ratios (≥ 1:40). Orientational NMR constraints suggest that the peptide may self-assemble as an oligomeric β-barrel pore, which is stabilized by intermolecular hydrogen bonds. Comparison of different model membranes shows that the observed re-alignment is favoured in thin bilayers with short-chain lipids, especially near the chain melting temperature, whereas long-chain lipids suppress pore formation. Based on the oligomeric structural model and the conditions of pore formation, guidelines may now be derived for rationally designing peptide analogues as antibiotics with improved selectivity and reduced side effects.

  1. Design of RNA-Binding Proteins: Manipulate Alternative Splicing in Human Cells with Artificial Splicing Factors.

    Science.gov (United States)

    Wang, Yang; Wang, Zefeng

    2016-01-01

    The majority of human genes undergo alternative splicing to produce multiple isoforms with distinct functions. The dysregulations of alternative splicing have been found to be closely associated with various human diseases; thus new approaches to modulate disease-associated splicing events will provide great therapeutic potentials. Here we report protocols for constructing novel artificial splicing factors that can be designed to specifically modulate alternative splicing of target genes. By following the method outlined in this protocol, it is possible to design and generate artificial splicing factors with diverse activities in regulating different types of alternative splicing. The artificial splicing factors can be used to change splicing of either minigenes or endogenous genes in cultured human cells, providing a new strategy to study the regulation of alternative splicing and function of alternatively spliced products.

  2. Binary Mixtures of SH- and CH3-Terminated Self-Assembled Monolayers to Control the Average Spacing Between Aligned Gold Nanoparticles

    Directory of Open Access Journals (Sweden)

    Pavelka Laura

    2009-01-01

    Full Text Available Abstract This paper presents a method to control the average spacing between organometallic chemical vapor deposition (OMCVD grown gold nanoparticles (Au NPs in a line. Focused ion beam patterned CH3-terminated self-assembled monolayers are refilled systematically with different mixtures of SH- and CH3-terminated silanes. The average spacing between OMCVD Au NPs is demonstrated systematically to decrease by increasing the v/v% ratio of the thiols in the binary silane mixtures with SH- and CH3-terminated groups.

  3. Pairagon: a highly accurate, HMM-based cDNA-to-genome aligner

    DEFF Research Database (Denmark)

    Lu, David V; Brown, Randall H; Arumugam, Manimozhiyan

    2009-01-01

    MOTIVATION: The most accurate way to determine the intron-exon structures in a genome is to align spliced cDNA sequences to the genome. Thus, cDNA-to-genome alignment programs are a key component of most annotation pipelines. The scoring system used to choose the best alignment is a primary...... heuristics. RESULTS: We present Pairagon, a pair hidden Markov model based cDNA-to-genome alignment program, as the most accurate aligner for sequences with high- and low-identity levels. We conducted a series of experiments testing alignment accuracy with varying sequence identity. We first created 'perfect......' simulated cDNA sequences by splicing the sequences of exons in the reference genome sequences of fly and human. The complete reference genome sequences were then mutated to various degrees using a realistic mutation simulator and the perfect cDNAs were aligned to them using Pairagon and 12 other aligners...

  4. Beyond Alignment

    DEFF Research Database (Denmark)

    Beyond Alignment: Applying Systems Thinking to Architecting Enterprises is a comprehensive reader about how enterprises can apply systems thinking in their enterprise architecture practice, for business transformation and for strategic execution. The book's contributors find that systems thinking...

  5. Supplementary Material for: Herboxidiene triggers splicing repression and abiotic stress responses in plants

    KAUST Repository

    Alshareef, Sahar

    2017-01-01

    Abstract Background Constitutive and alternative splicing of pre-mRNAs from multiexonic genes controls the diversity of the proteome; these precisely regulated processes also fine-tune responses to cues related to growth, development, and stresses. Small-molecule inhibitors that perturb splicing provide invaluable tools for use as chemical probes to uncover the molecular underpinnings of splicing regulation and as potential anticancer compounds. Results Here, we show that herboxidiene (GEX1A) inhibits both constitutive and alternative splicing. Moreover, GEX1A activates genome-wide transcriptional patterns involved in abiotic stress responses in plants. GEX1A treatment -activated ABA-inducible promoters, and led to stomatal closure. Interestingly, GEX1A and pladienolide B (PB) elicited similar cellular changes, including alterations in the patterns of transcription and splicing, suggesting that these compounds might target the same spliceosome complex in plant cells. Conclusions Our study establishes GEX1A as a potent splicing inhibitor in plants that can be used to probe the assembly, dynamics, and molecular functions of the spliceosome and to study the interplay between splicing stress and abiotic stresses, as well as having potential biotechnological applications.

  6. Splice testing for LHC quadrupole magnets

    CERN Document Server

    Barzi, E; Fehér, S; Kashikhin, V V; Kerby, J S; Lamm, M J; Orris, D; Ray, G; Tartaglia, M; Zlobin, A V

    2003-01-01

    Electrical splices between NbTi Rutherford type cables need to be made for the LHC IR inner triplet quadrupoles. Splices between magnets as well as internal to the magnets are necessary. Various splice configurations, solders, and fluxes have been considered. Testing of these splices at cryogenic temperatures and at various currents has been completed. The results were satisfactory; Fermilab is capable of making excellent low resistance (<1n Omega ) solder joints for the LHC project. (4 refs).

  7. Extending reference assembly models

    DEFF Research Database (Denmark)

    Church, Deanna M.; Schneider, Valerie A.; Steinberg, Karyn Meltz

    2015-01-01

    The human genome reference assembly is crucial for aligning and analyzing sequence data, and for genome annotation, among other roles. However, the models and analysis assumptions that underlie the current assembly need revising to fully represent human sequence diversity. Improved analysis tools...... and updated data reporting formats are also required....

  8. Influenza A Virus Utilizes Suboptimal Splicing to Coordinate the Timing of Infection

    Directory of Open Access Journals (Sweden)

    Mark A. Chua

    2013-01-01

    Full Text Available Influenza A virus is unique as an RNA virus in that it replicates in the nucleus and undergoes splicing. With only ten major proteins, the virus must gain nuclear access, replicate, assemble progeny virions in the cytoplasm, and then egress. In an effort to elucidate the coordination of these events, we manipulated the transcript levels from the bicistronic nonstructural segment that encodes the spliced virus product responsible for genomic nuclear export. We find that utilization of an erroneous splice site ensures the slow accumulation of the viral nuclear export protein (NEP while generating excessive levels of an antagonist that inhibits the cellular response to infection. Modulation of this simple transcriptional event results in improperly timed export and loss of virus infection. Together, these data demonstrate that coordination of the influenza A virus life cycle is set by a “molecular timer” that operates on the inefficient splicing of a virus transcript.

  9. The Integrity of ACSR Full Tension Single-Stage Splice Connector at Higher Operation Temperature

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jy-An John [ORNL; Lara-Curzio, Edgar [ORNL; King Jr, Thomas J [ORNL

    2008-10-01

    Due to increases in power demand and limited investment in new infrastructure, existing overhead power transmission lines often need to operate at temperatures higher than those used for the original design criteria. This has led to the accelerated aging and degradation of splice connectors. It is manifested by the formation of hot-spots that have been revealed by infrared imaging during inspection. The implications of connector aging is two-fold: (1) significant increases in resistivity of the splice connector (i.e., less efficient transmission of electricity) and (2) significant reductions in the connector clamping strength, which could ultimately result in separation of the power transmission line at the joint. Therefore, the splice connector appears to be the weakest link in electric power transmission lines. This report presents a protocol for integrating analytical and experimental approaches to evaluate the integrity of full tension single-stage splice connector assemblies and the associated effective lifetime at high operating temperature.

  10. Bos taurus genome assembly.

    Science.gov (United States)

    Liu, Yue; Qin, Xiang; Song, Xing-Zhi Henry; Jiang, Huaiyang; Shen, Yufeng; Durbin, K James; Lien, Sigbjørn; Kent, Matthew Peter; Sodeland, Marte; Ren, Yanru; Zhang, Lan; Sodergren, Erica; Havlak, Paul; Worley, Kim C; Weinstock, George M; Gibbs, Richard A

    2009-04-24

    We present here the assembly of the bovine genome. The assembly method combines the BAC plus WGS local assembly used for the rat and sea urchin with the whole genome shotgun (WGS) only assembly used for many other animal genomes including the rhesus macaque. The assembly process consisted of multiple phases: First, BACs were assembled with BAC generated sequence, then subsequently in combination with the individual overlapping WGS reads. Different assembly parameters were tested to separately optimize the performance for each BAC assembly of the BAC and WGS reads. In parallel, a second assembly was produced using only the WGS sequences and a global whole genome assembly method. The two assemblies were combined to create a more complete genome representation that retained the high quality BAC-based local assembly information, but with gaps between BACs filled in with the WGS-only assembly. Finally, the entire assembly was placed on chromosomes using the available map information.Over 90% of the assembly is now placed on chromosomes. The estimated genome size is 2.87 Gb which represents a high degree of completeness, with 95% of the available EST sequences found in assembled contigs. The quality of the assembly was evaluated by comparison to 73 finished BACs, where the draft assembly covers between 92.5 and 100% (average 98.5%) of the finished BACs. The assembly contigs and scaffolds align linearly to the finished BACs, suggesting that misassemblies are rare. Genotyping and genetic mapping of 17,482 SNPs revealed that more than 99.2% were correctly positioned within the Btau_4.0 assembly, confirming the accuracy of the assembly. The biological analysis of this bovine genome assembly is being published, and the sequence data is available to support future bovine research.

  11. Bos taurus genome assembly

    Directory of Open Access Journals (Sweden)

    Sodergren Erica

    2009-04-01

    Full Text Available Abstract Background We present here the assembly of the bovine genome. The assembly method combines the BAC plus WGS local assembly used for the rat and sea urchin with the whole genome shotgun (WGS only assembly used for many other animal genomes including the rhesus macaque. Results The assembly process consisted of multiple phases: First, BACs were assembled with BAC generated sequence, then subsequently in combination with the individual overlapping WGS reads. Different assembly parameters were tested to separately optimize the performance for each BAC assembly of the BAC and WGS reads. In parallel, a second assembly was produced using only the WGS sequences and a global whole genome assembly method. The two assemblies were combined to create a more complete genome representation that retained the high quality BAC-based local assembly information, but with gaps between BACs filled in with the WGS-only assembly. Finally, the entire assembly was placed on chromosomes using the available map information. Over 90% of the assembly is now placed on chromosomes. The estimated genome size is 2.87 Gb which represents a high degree of completeness, with 95% of the available EST sequences found in assembled contigs. The quality of the assembly was evaluated by comparison to 73 finished BACs, where the draft assembly covers between 92.5 and 100% (average 98.5% of the finished BACs. The assembly contigs and scaffolds align linearly to the finished BACs, suggesting that misassemblies are rare. Genotyping and genetic mapping of 17,482 SNPs revealed that more than 99.2% were correctly positioned within the Btau_4.0 assembly, confirming the accuracy of the assembly. Conclusion The biological analysis of this bovine genome assembly is being published, and the sequence data is available to support future bovine research.

  12. GC content around splice sites affects splicing through pre-mRNA secondary structures

    Directory of Open Access Journals (Sweden)

    Chen Liang

    2011-01-01

    Full Text Available Abstract Background Alternative splicing increases protein diversity by generating multiple transcript isoforms from a single gene through different combinations of exons or through different selections of splice sites. It has been reported that RNA secondary structures are involved in alternative splicing. Here we perform a genomic study of RNA secondary structures around splice sites in humans (Homo sapiens, mice (Mus musculus, fruit flies (Drosophila melanogaster, and nematodes (Caenorhabditis elegans to further investigate this phenomenon. Results We observe that GC content around splice sites is closely associated with the splice site usage in multiple species. RNA secondary structure is the possible explanation, because the structural stability difference among alternative splice sites, constitutive splice sites, and skipped splice sites can be explained by the GC content difference. Alternative splice sites tend to be GC-enriched and exhibit more stable RNA secondary structures in all of the considered species. In humans and mice, splice sites of first exons and long exons tend to be GC-enriched and hence form more stable structures, indicating the special role of RNA secondary structures in promoter proximal splicing events and the splicing of long exons. In addition, GC-enriched exon-intron junctions tend to be overrepresented in tissue-specific alternative splice sites, indicating the functional consequence of the GC effect. Compared with regions far from splice sites and decoy splice sites, real splice sites are GC-enriched. We also found that the GC-content effect is much stronger than the nucleotide-order effect to form stable secondary structures. Conclusion All of these results indicate that GC content is related to splice site usage and it may mediate the splicing process through RNA secondary structures.

  13. Titin Diversity—Alternative Splicing Gone Wild

    Directory of Open Access Journals (Sweden)

    Wei Guo

    2010-01-01

    Full Text Available Titin is an extremely large protein found in highest concentrations in heart and skeletal muscle. The single mammalian gene is expressed in multiple isoforms as a result of alternative splicing. Although titin isoform expression is controlled developmentally and in a tissue specific manner, the vast number of potential splicing pathways far exceeds those described in any other alternatively spliced gene. Over 1 million human splice pathways for a single individual can be potentially derived from the PEVK region alone. A new splicing pattern for the human cardiac N2BA isoform type has been found in which the PEVK region includes only the N2B type exons. The alterations in splicing and titin isoform expression in human heart disease provide impetus for future detailed study of the splicing mechanisms for this giant protein.

  14. Production and Quality Assurance of Main Busbar Interconnection Splices during the LHC 2008-2009 Shutdown.

    CERN Document Server

    Bertinelli, F; Dalin, J-M; Fessia, P; Flora, R H; Heck, S; Pfeffer, H; Prin, H; Scheuerlein, C; Thonet, P; Tock, J-P; Williams, L

    2011-01-01

    The main busbar interconnection splices of the Large Hadron Collider are assembled by inductive soldering of the Rutherford type cables and the copper profiles of the stabilizer. Following the September 2008 incident, the assembly process and the quality assurance have been improved, with new measurement and diagnostics methods introduced. In the 2008-2009 shutdown the resistance both in the superconducting and in the normal conducting states have been the focus for improvements. The introduction of gamma radiography has allowed the visualization of voids between cable and stabilizer. It is now known that during the standard soldering heating cycle solder is lost from the busbar extremities adjacent to the splice profiles, leaving parts of the cable in poor contact with the stabilizer. A room temperature resistance measurement has been introduced as a simple, non-destructive test to measure the electrical continuity of the splice in its normal conducting state. An ultrasonic test has been performed systematic...

  15. Splicing and alternative splicing in rice and humans

    Directory of Open Access Journals (Sweden)

    Zhiguo E

    2013-09-01

    Full Text Available Rice is a monocot gramineous crop, and one of the mostimportant staple foods. Rice is considered a model species formost gramineous crops. Extensive research on rice hasprovided critical guidance for other crops, such as maize andwheat. In recent years, climate change and exacerbated soildegradation have resulted in a variety of abiotic stresses, suchas greenhouse effects, lower temperatures, drought, floods,soil salinization and heavy metal pollution. As such, there isan extremely high demand for additional research, in order toaddress these negative factors. Studies have shown that thealternative splicing of many genes in rice is affected by stressconditions, suggesting that manipulation of the alternativesplicing of specific genes may be an effective approach for riceto adapt to abiotic stress. With the advancement ofmicroarrays, and more recently, next generation sequencingtechnology, several studies have shown that more than half ofthe genes in the rice genome undergo alternative splicing. Thismini-review summarizes the latest progress in the research ofsplicing and alternative splicing in rice, compared to splicingin humans. Furthermore, we discuss how additional studiesmay change the landscape of investigation of rice functionalgenomics and genetically improved rice. [BMB Reports 2013;46(9: 439-447

  16. Self-aligning biaxial load frame

    Science.gov (United States)

    Ward, M.B.; Epstein, J.S.; Lloyd, W.R.

    1994-01-18

    An self-aligning biaxial loading apparatus for use in testing the strength of specimens while maintaining a constant specimen centroid during the loading operation. The self-aligning biaxial loading apparatus consists of a load frame and two load assemblies for imparting two independent perpendicular forces upon a test specimen. The constant test specimen centroid is maintained by providing elements for linear motion of the load frame relative to a fixed cross head, and by alignment and linear motion elements of one load assembly relative to the load frame. 3 figures.

  17. SplicePlot: a utility for visualizing splicing quantitative trait loci.

    Science.gov (United States)

    Wu, Eric; Nance, Tracy; Montgomery, Stephen B

    2014-04-01

    RNA sequencing has provided unprecedented resolution of alternative splicing and splicing quantitative trait loci (sQTL). However, there are few tools available for visualizing the genotype-dependent effects of splicing at a population level. SplicePlot is a simple command line utility that produces intuitive visualization of sQTLs and their effects. SplicePlot takes mapped RNA sequencing reads in BAM format and genotype data in VCF format as input and outputs publication-quality Sashimi plots, hive plots and structure plots, enabling better investigation and understanding of the role of genetics on alternative splicing and transcript structure. Source code and detailed documentation are available at http://montgomerylab.stanford.edu/spliceplot/index.html under Resources and at Github. SplicePlot is implemented in Python and is supported on Linux and Mac OS. A VirtualBox virtual machine running Ubuntu with SplicePlot already installed is also available.

  18. The In Vivo Kinetics of RNA Polymerase II Elongation during Co-Transcriptional Splicing

    Science.gov (United States)

    Brody, Yehuda; Neufeld, Noa; Bieberstein, Nicole; Causse, Sebastien Z.; Böhnlein, Eva-Maria; Neugebauer, Karla M.; Darzacq, Xavier; Shav-Tal, Yaron

    2011-01-01

    RNA processing events that take place on the transcribed pre-mRNA include capping, splicing, editing, 3′ processing, and polyadenylation. Most of these processes occur co-transcriptionally while the RNA polymerase II (Pol II) enzyme is engaged in transcriptional elongation. How Pol II elongation rates are influenced by splicing is not well understood. We generated a family of inducible gene constructs containing increasing numbers of introns and exons, which were stably integrated in human cells to serve as actively transcribing gene loci. By monitoring the association of the transcription and splicing machineries on these genes in vivo, we showed that only U1 snRNP localized to the intronless gene, consistent with a splicing-independent role for U1 snRNP in transcription. In contrast, all snRNPs accumulated on intron-containing genes, and increasing the number of introns increased the amount of spliceosome components recruited. This indicates that nascent RNA can assemble multiple spliceosomes simultaneously. Kinetic measurements of Pol II elongation in vivo, Pol II ChIP, as well as use of Spliceostatin and Meayamycin splicing inhibitors showed that polymerase elongation rates were uncoupled from ongoing splicing. This study shows that transcription elongation kinetics proceed independently of splicing at the model genes studied here. Surprisingly, retention of polyadenylated mRNA was detected at the transcription site after transcription termination. This suggests that the polymerase is released from chromatin prior to the completion of splicing, and the pre-mRNA is post-transcriptionally processed while still tethered to chromatin near the gene end. PMID:21264352

  19. DIDA: Distributed Indexing Dispatched Alignment.

    Directory of Open Access Journals (Sweden)

    Hamid Mohamadi

    Full Text Available One essential application in bioinformatics that is affected by the high-throughput sequencing data deluge is the sequence alignment problem, where nucleotide or amino acid sequences are queried against targets to find regions of close similarity. When queries are too many and/or targets are too large, the alignment process becomes computationally challenging. This is usually addressed by preprocessing techniques, where the queries and/or targets are indexed for easy access while searching for matches. When the target is static, such as in an established reference genome, the cost of indexing is amortized by reusing the generated index. However, when the targets are non-static, such as contigs in the intermediate steps of a de novo assembly process, a new index must be computed for each run. To address such scalability problems, we present DIDA, a novel framework that distributes the indexing and alignment tasks into smaller subtasks over a cluster of compute nodes. It provides a workflow beyond the common practice of embarrassingly parallel implementations. DIDA is a cost-effective, scalable and modular framework for the sequence alignment problem in terms of memory usage and runtime. It can be employed in large-scale alignments to draft genomes and intermediate stages of de novo assembly runs. The DIDA source code, sample files and user manual are available through http://www.bcgsc.ca/platform/bioinfo/software/dida. The software is released under the British Columbia Cancer Agency License (BCCA, and is free for academic use.

  20. Vane segment support and alignment device

    Energy Technology Data Exchange (ETDEWEB)

    McLaurin, Leroy Dixon (Winter Springs, FL); Sizemore, John Derek (Orlando, FL)

    1999-01-01

    A support and alignment assembly for supporting and aligning a vane segment is provided. The support and alignment assembly comprises a torque plate which defines an opening for receiving an eccentric pin and a locking end member for receiving a lock socket member. An eccentric pin adjustably supported by the torque plate opening for supporting and aligning a vane segment is provided. A lock socket member adapted to securely receive the eccentric pin and rotated therewith, and adjustably engage the torque plate locking end is provided. The lock socket member receives the eccentric pin, such that when the eccentric pin is adjusted to align the vane segment, the lock socket member engages the torque plate locking end to secure the vane segment in the desired position.

  1. Vane segment support and alignment device

    Science.gov (United States)

    McLaurin, L.D.; Sizemore, J.D.

    1999-07-13

    A support and alignment assembly for supporting and aligning a vane segment is provided. The support and alignment assembly comprises a torque plate which defines an opening for receiving an eccentric pin and a locking end member for receiving a lock socket member. An eccentric pin adjustably supported by the torque plate opening for supporting and aligning a vane segment is provided. A lock socket member adapted to securely receive the eccentric pin and rotated therewith, and adjustably engage the torque plate locking end is provided. The lock socket member receives the eccentric pin, such that when the eccentric pin is adjusted to align the vane segment, the lock socket member engages the torque plate locking end to secure the vane segment in the desired position. 5 figs.

  2. Computational prediction of splicing regulatory elements shared by Tetrapoda organisms

    OpenAIRE

    Churbanov, Alexander; Vo?echovsk?, Igor; Hicks, Chindo

    2009-01-01

    Abstract Background Auxiliary splicing sequences play an important role in ensuring accurate and efficient splicing by promoting or repressing recognition of authentic splice sites. These cis-acting motifs have been termed splicing enhancers and silencers and are located both in introns and exons. They co-evolved into an intricate splicing code together with additional functional constraints, such as tissue-specific and alternative splicing patterns. We used orthologous exons extracted from t...

  3. Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes.

    Science.gov (United States)

    Sveen, A; Kilpinen, S; Ruusulehto, A; Lothe, R A; Skotheim, R I

    2016-05-12

    Alternative splicing is a widespread process contributing to structural transcript variation and proteome diversity. In cancer, the splicing process is commonly disrupted, resulting in both functional and non-functional end-products. Cancer-specific splicing events are known to contribute to disease progression; however, the dysregulated splicing patterns found on a genome-wide scale have until recently been less well-studied. In this review, we provide an overview of aberrant RNA splicing and its regulation in cancer. We then focus on the executors of the splicing process. Based on a comprehensive catalog of splicing factor encoding genes and analyses of available gene expression and somatic mutation data, we identify cancer-associated patterns of dysregulation. Splicing factor genes are shown to be significantly differentially expressed between cancer and corresponding normal samples, and to have reduced inter-individual expression variation in cancer. Furthermore, we identify enrichment of predicted cancer-critical genes among the splicing factors. In addition to previously described oncogenic splicing factor genes, we propose 24 novel cancer-critical splicing factors predicted from somatic mutations.

  4. Preparation of Splicing Competent Nuclear Extract from Mammalian Cells and In Vitro Pre-mRNA Splicing Assay.

    Science.gov (United States)

    Movassat, Maliheh; Shenasa, Hossein; Hertel, Klemens J

    2017-01-01

    The ability to perform in vitro splicing assays has paved the way for in-depth studies of the mechanisms and machinery involved in the process of splicing. The in vitro splicing assay is a valuable experimental approach that combines the complexity of the spliceosome and regulatory systems with the flexibility of performing endless splicing and alternative splicing reactions. Through the use of crude nuclear extract and radiolabeled pre-mRNA, spliced mRNAs can be visualized using autoradiography for downstream analysis. This chapter describes the necessary steps to perform an in vitro splicing reaction, including the generation of the key components necessary for the splicing reaction; nuclear extract.

  5. Spliced

    DEFF Research Database (Denmark)

    Addison, Courtney Page

    2017-01-01

    been initiated. In this article I present a brief history of HGT, showing how the ethical and practical viability of the field was achieved by key scientific and regulatory actors. These parties carefully articulated gene therapy’s scope, limiting it to therapeutic interventions on somatic cells......, and cultivated alliances and divisions that bolstered the field’s legitimacy. At times these measures faltered, and then practitioners and sometimes patients would invoke an ethical imperative, posing gene therapy as the best solution to life and death problems. I suggest that we consider how boundary...

  6. Alternate splicing of transcripts shape macrophage response to Mycobacterium tuberculosis infection.

    Directory of Open Access Journals (Sweden)

    Haroon Kalam

    2017-03-01

    Full Text Available Transcriptional reprogramming of macrophages upon Mycobacterium tuberculosis (Mtb infection is widely studied; however, the significance of alternate splicing (AS in shaping cellular responses to mycobacterial infections is not yet appreciated. Alternate splicing can influence transcript stability or structure, function and localization of corresponding proteins thereby altering protein stoichiometry and physiological consequences. Using comprehensive analysis of a time-series RNA-seq data obtained from human macrophages infected with virulent or avirulent strains of Mtb, we show extensive remodeling of alternate splicing in macrophage transcriptome. The global nature of this regulation was evident since genes belonging to functional classes like trafficking, immune response, autophagy, redox and metabolism showed marked departure in the pattern of splicing in the infected macrophages. The systemic perturbation of splicing machinery in the infected macrophages was apparent as genes involved at different stages of spliceosome assembly were also regulated at the splicing level. Curiously there was a considerable increase in the expression of truncated/non-translatable variants of several genes, specifically upon virulent infections. Increased expression of truncated transcripts correlated with a decline in the corresponding protein levels. We verified the physiological relevance for one such candidate gene RAB8B; whose truncated variant gets enriched in H37Rv infected cells. Upon tweaking relative abundance of longer or shorter variants of RAB8B transcripts by specialized transduction, mycobacterial targeting to lysosomes could be promoted or blocked respectively, which also resulted in corresponding changes in the bacterial survival. Our results show RAB8B recruitment to the mycobacterial phagosomes is required for phagosome maturation. Thus the abundance of truncated RAB8B variant helps virulent Mtb survival by limiting the RAB8B levels in the

  7. An SXF Extension for Alignment

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, W. [Brookhaven National Lab. (BNL), Upton, NY (United States); Pilat, F. [Brookhaven National Lab. (BNL), Upton, NY (United States)

    1998-11-13

    The accelerator description in the standard exchange format SXF is now used for RHIC and the LHC. Parsers exist to a growing number of simulation programs. SXF, however, is a fiat ordered list of elements and does not support the simultaneous misalignment of adjacent elements. The introduction of an element hierarchy is needed to align several elements by the same amount. This is of interest for two reasons: First, magnets are often modeled by more than one element and all these elements should be misaligned by the same amount. For example, a magnet model may consist of a body and two end kicks; and the body itself may be modeled by several kicks. Second, sometimes there are several magnets assembled in one cryostat. It is then desirable to misalign the whole cryostat while the individual magnets within are misaligned relative to the cryostat. The proper description of alignment errors is of great importance to the US-LHC col­laboration in which the US will deliver assembled cryostat-s of interaction region magnets to CERN. In the design phase an estimate of tolerable alignment errors is needed. Once magnets are assembled in a cryostat, the best position of the cryostat must be found in order to minimize the harmful effects of field errors. We developed a description that allows the simultaneous misalignment of adjacent magnets. The description is closely related to the SXF format. A filter program can be used to process and merge the misalignment information into a flat SXF file.

  8. Sensor Alignment by Tracks

    CERN Document Server

    Karimäki, V; Lampen, T; Lindén, T

    2003-01-01

    Good geometrical calibration is essential in the use of high resolution detectors. The individual sensors in the detector have to be calibrated with an accuracy better than the intrinsic resolution, which typically is of the order of 10 um. We present an effective method to perform fine calibration of sensor positions in a detector assembly consisting of a large number of pixel and strip sensors. Up to six geometric parameters, three for location and three for orientation, can be computed for each sensor on a basis of particle trajectories traversing the detector system. The performance of the method is demonstrated with both simulated tracks and tracks reconstructed from experimental data. We also present a brief review of other alignment methods reported in the literature.

  9. Comparative transcriptomics uncovers alternative splicing and molecular marker development in radish (Raphanus sativus L.).

    Science.gov (United States)

    Luo, Xiaobo; Xu, Liang; Liang, Dongyi; Wang, Yan; Zhang, Wei; Zhu, Xianwen; Zhu, Yuelin; Jiang, Haiyan; Tang, Mingjia; Liu, Liwang

    2017-07-03

    Alternative splicing (AS) plays important roles in gene expression and proteome diversity. Single nucleotide polymorphism (SNP) and insertion/deletion (InDel) are abundant polymorphisms and co-dominant inheritance markers, which have been widely used in germplasm identification, genetic mapping and marker-assisted selection in plants. So far, however, little information is available on utilization of AS events and development of SNP and InDel markers from transcriptome in radish. In this study, three radish transcriptome datasets were collected and aligned to the reference radish genome. A total of 56,530 AS events were identified from three radish genotypes with intron retention (IR) being the most frequent AS type, which accounted for 59.4% of the total expressed genes in radish. In all, 22,412 SNPs and 9436 InDels were identified with an average frequency of 1 SNP/17.9 kb and 1 InDel/42.5 kb, respectively. A total of 43,680 potential SSRs were identified in 31,604 assembled unigenes with a density of 1 SSR/2.5 kb. The ratio of SNPs with nonsynonymous/synonymous mutations was 1.05:1. Moreover, 35 SNPs and 200 InDels were randomly selected and validated by Sanger sequencing, 83.9% of the SNPs and 70% of the InDels exhibited polymorphism among these three genotypes. In addition, the 15 SNPs and 125 InDels were found to be unevenly distributed on 9 linkage groups. Furthermore, 40 informative InDel markers were successfully used for the genetic diversity analysis on 32 radish accessions. These results would not only provide new insights into transcriptome complexity and AS regulation, but also furnish large amount of molecular marker resources for germplasm identification, genetic mapping and further genetic improvement of radish in breeding programs.

  10. An Interspecific Plant Hybrid Shows Novel Changes in Parental Splice Forms of Genes for Splicing Factors

    Science.gov (United States)

    Scascitelli, Moira; Cognet, Marie; Adams, Keith L.

    2010-01-01

    Interspecific hybridization plays an important role in plant adaptive evolution and speciation, and the process often results in phenotypic novelty. Hybrids can show changes in genome structure and gene expression compared with their parents including chromosomal rearrangments, changes in cytosine methylation, up- and downregulation of gene expression, and gene silencing. Alternative splicing (AS) is a fundamental aspect of the expression of many genes. However alternative splicing patterns have not been examined in multiple genes in an interspecific plant hybrid compared with its parents. Here we studied alternative splicing patterns in an interspecific Populus hybrid and its parents by assaying 40 genes using reverse transcription PCR. Most of the genes showed identical alternative splicing patterns between the parents and the hybrid. We found new alternative splicing variants present in the hybrid in two SR genes involved in the regulation of splicing and alternative splicing. The novel alternative splicing patterns included changes in donor and acceptor sites to create a new exon in one allele of PtRSZ22 in the hybrid and retention of an intron in both alleles of PtSR34a.1 in the hybrid, with effects on the function of the corresponding truncated proteins, if present. Our results suggest that novel alternative splicing patterns are present in a small percentage of genes in hybrids, but they could make a considerable impact on the expression of some genes. Changes in alternative splicing are likely to be an important component of the genetic changes that occur upon interspecific hybridization. PMID:20100939

  11. Aberrant Splicing in Cancer: Mediators of Malignant Progression through an Imperfect Splice Program Shift.

    Science.gov (United States)

    Luz, Felipe Andrés Cordero; Brígido, Paula Cristina; Moraes, Alberto Silva; Silva, Marcelo José Barbosa

    2017-01-01

    Although the efforts to understand the genetic basis of cancer allowed advances in diagnosis and therapy, little is known about other molecular bases. Splicing is a key event in gene expression, controlling the excision of introns decoded inside genes and being responsible for 80% of the proteome amplification through events of alternative splicing. Growing data from the last decade point to deregulation of splicing events as crucial in carcinogenesis and tumor progression. Several alterations in splicing events were observed in cancer, caused by either missexpression of or detrimental mutations in some splicing factors, and appear to be critical in carcinogenesis and key events during tumor progression. Notwithstanding, it is difficult to determine whether it is a cause or consequence of cancer and/or tumorigenesis. Most reviews focus on the generated isoforms of deregulated splicing pattern, while others mainly summarize deregulated splicing factors observed in cancer. In this review, events associated with carcinogenesis and tumor progression mainly, and epithelial-to-mesenchymal transition, which is also implicated in alternative splicing regulation, will be progressively discussed in the light of a new perspective, suggesting that splicing deregulation mediates cell reprogramming in tumor progression by an imperfect shift of the splice program. © 2016 S. Karger AG, Basel.

  12. Microbial and Natural Metabolites That Inhibit Splicing: A Powerful Alternative for Cancer Treatment

    Science.gov (United States)

    Rosas-Murrieta, Nora Hilda; Martínez-Montiel, Mónica; Gaspariano-Cholula, Mayra Patricia

    2016-01-01

    In eukaryotes, genes are frequently interrupted with noncoding sequences named introns. Alternative splicing is a nuclear mechanism by which these introns are removed and flanking coding regions named exons are joined together to generate a message that will be translated in the cytoplasm. This mechanism is catalyzed by a complex machinery known as the spliceosome, which is conformed by more than 300 proteins and ribonucleoproteins that activate and regulate the precision of gene expression when assembled. It has been proposed that several genetic diseases are related to defects in the splicing process, including cancer. For this reason, natural products that show the ability to regulate splicing have attracted enormous attention due to its potential use for cancer treatment. Some microbial metabolites have shown the ability to inhibit gene splicing and the molecular mechanism responsible for this inhibition is being studied for future applications. Here, we summarize the main types of natural products that have been characterized as splicing inhibitors, the recent advances regarding molecular and cellular effects related to these molecules, and the applications reported so far in cancer therapeutics. PMID:27610372

  13. Single Molecule Cluster Analysis Identifies Signature Dynamic Conformations along the Splicing Pathway

    Science.gov (United States)

    Blanco, Mario R.; Martin, Joshua S.; Kahlscheuer, Matthew L.; Krishnan, Ramya; Abelson, John; Laederach, Alain; Walter, Nils G.

    2016-01-01

    The spliceosome is the dynamic RNA-protein machine responsible for faithfully splicing introns from precursor messenger RNAs (pre-mRNAs). Many of the dynamic processes required for the proper assembly, catalytic activation, and disassembly of the spliceosome as it acts on its pre-mRNA substrate remain poorly understood, a challenge that persists for many biomolecular machines. Here, we developed a fluorescence-based Single Molecule Cluster Analysis (SiMCAn) tool to dissect the manifold conformational dynamics of a pre-mRNA through the splicing cycle. By clustering common dynamic behaviors derived from selectively blocked splicing reactions, SiMCAn was able to identify signature conformations and dynamic behaviors of multiple ATP-dependent intermediates. In addition, it identified a conformation adopted late in splicing by a 3′ splice site mutant, invoking a mechanism for substrate proofreading. SiMCAn presents a novel framework for interpreting complex single molecule behaviors that should prove widely useful for the comprehensive analysis of a plethora of dynamic cellular machines. PMID:26414013

  14. Microbial and Natural Metabolites That Inhibit Splicing: A Powerful Alternative for Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Nancy Martínez-Montiel

    2016-01-01

    Full Text Available In eukaryotes, genes are frequently interrupted with noncoding sequences named introns. Alternative splicing is a nuclear mechanism by which these introns are removed and flanking coding regions named exons are joined together to generate a message that will be translated in the cytoplasm. This mechanism is catalyzed by a complex machinery known as the spliceosome, which is conformed by more than 300 proteins and ribonucleoproteins that activate and regulate the precision of gene expression when assembled. It has been proposed that several genetic diseases are related to defects in the splicing process, including cancer. For this reason, natural products that show the ability to regulate splicing have attracted enormous attention due to its potential use for cancer treatment. Some microbial metabolites have shown the ability to inhibit gene splicing and the molecular mechanism responsible for this inhibition is being studied for future applications. Here, we summarize the main types of natural products that have been characterized as splicing inhibitors, the recent advances regarding molecular and cellular effects related to these molecules, and the applications reported so far in cancer therapeutics.

  15. SU-E-I-113: Alignment and Assembly of Small Field of View Pre-Clinical Images Taken with a Micro-Solid State X-Ray Imaging Detector.

    Science.gov (United States)

    Loughran, B; Singh, V; Bednarek, D; Rudin, S

    2012-06-01

    We have developed a Micro-Solid State X-ray Imaging Intensifier (μSSXII) detector with an electron multiplying (EM)CCD chip which allows for an ultra-high pixel resolution of 8 × 8 microns. However, the EMCCD chip area of 1004 × 1002 pixels results in an 8.032 × 8.016 mm imaging area and restricts the ability of the user to orient and properly image larger pre-clinical objects such as rat-kidney-vasculature casts. We propose a method to align and assemble images of such larger structures while preserving the ultra-high spatial resolution of the μSSXII. An imaging platform of negligible attenuation was attached to a stepper motor giving the platform free movement in the plane normal to the fluoroscopic x-ray beam. By alternating the detector's image acquisition and the stepper movement, many adjacent overlapping views of the object are acquired. The user then identifies the two pixel coordinates in adjacent images which represent the same point in space. A custom Matlab computer code uses the pixel coordinates to aggregate and average the input images which results in a larger field of view consisting of many ultra-high resolution images. The images were able to be successfully combined to form a large image while preserving the ultra-high resolution of the detector. In experimental tests, multiple portions of a mammography test object were imaged and virtually no spatial resolution degradation was found in the combined image. Additionally, when imaging a resin cast of rat-kidney-vasculature, vessels of less than 50 μm could be viewed in the combined image. The setup and method were found to preserve the ultra-high resolution inherent to the μSSXII while allowing pre-clinical imaging of objects larger than the detector's field of view. The large field of view was effective in orienting the user towards specific areas of interest in the objects imaged. Supported in part by: NIH Grants R01-EB008425, R01-EB002873, and an equipment grant from Toshiba Medical

  16. Depolarization-mediated regulation of alternative splicing

    Directory of Open Access Journals (Sweden)

    Alok eSharma

    2011-12-01

    Full Text Available Alternative splicing in eukaryotes plays an important role in regulating gene expression by selectively including alternative exons. A wealth of information has been accumulated that explains how alternative exons are selected in a developmental stage- or tissue-specific fashion. However, our knowledge of how cells respond to environmental changes to alter alternative splicing is very limited. For example, although a number of alternative exons have been shown to be regulated by calcium level alterations, the underlying mechanisms are not well understood. As calcium signaling in neurons plays a crucial role in essential neuronal functions such as learning and memory formation, it is important to understand how this process is regulated at every level in gene expression. The significance of the dynamic control of alternative splicing in response to changes of calcium levels has been largely unappreciated. In this communication, we will summarize the recent advances in calcium signaling-mediated alternative splicing that have provided some insights into the important regulatory mechanisms. In addition to describing the cis-acting RNA elements on the pre-mRNA molecules that respond to changes of intracellular calcium levels, we will summarize how splicing regulators change and affect alternative splicing in this process. We will also discuss a novel mode of calcium-mediated splicing regulation at the level of chromatin structure and transcription.

  17. Rail-RNA: scalable analysis of RNA-seq splicing and coverage.

    Science.gov (United States)

    Nellore, Abhinav; Collado-Torres, Leonardo; Jaffe, Andrew E; Alquicira-Hernández, José; Wilks, Christopher; Pritt, Jacob; Morton, James; Leek, Jeffrey T; Langmead, Ben

    2017-12-15

    RNA sequencing (RNA-seq) experiments now span hundreds to thousands of samples. Current spliced alignment software is designed to analyze each sample separately. Consequently, no information is gained from analyzing multiple samples together, and it requires extra work to obtain analysis products that incorporate data from across samples. We describe Rail-RNA, a cloud-enabled spliced aligner that analyzes many samples at once. Rail-RNA eliminates redundant work across samples, making it more efficient as samples are added. For many samples, Rail-RNA is more accurate than annotation-assisted aligners. We use Rail-RNA to align 667 RNA-seq samples from the GEUVADIS project on Amazon Web Services in under 16 h for US$0.91 per sample. Rail-RNA outputs alignments in SAM/BAM format; but it also outputs (i) base-level coverage bigWigs for each sample; (ii) coverage bigWigs encoding normalized mean and median coverages at each base across samples analyzed; and (iii) exon-exon splice junctions and indels (features) in columnar formats that juxtapose coverages in samples in which a given feature is found. Supplementary outputs are ready for use with downstream packages for reproducible statistical analysis. We use Rail-RNA to identify expressed regions in the GEUVADIS samples and show that both annotated and unannotated (novel) expressed regions exhibit consistent patterns of variation across populations and with respect to known confounding variables. Rail-RNA is open-source software available at http://rail.bio. anellore@gmail.com or langmea@cs.jhu.edu. Supplementary data are available at Bioinformatics online.

  18. Splicing pattern - ASTRA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us ASTRA Splicing pattern Data detail Data name Splicing pattern DOI 10.18908/lsdba.nbdc00371-0...04 Description of data contents The patterns of alternative splicing/transcriptional initiation Data file Fi...le name: astra_splicing_pattern.zip File URL: ftp://ftp.biosciencedbc.jp/archive/astra/LATEST/astra_splicing_pattern...ogodb/view/astra_splicing_pattern#en Data acquisition method For the five organisms (H. sapiens, M. musculus...apping data into bit arrays, detection of splicing patterns and distribution to t

  19. Regular languages, regular grammars and automata in splicing systems

    Science.gov (United States)

    Mohamad Jan, Nurhidaya; Fong, Wan Heng; Sarmin, Nor Haniza

    2013-04-01

    Splicing system is known as a mathematical model that initiates the connection between the study of DNA molecules and formal language theory. In splicing systems, languages called splicing languages refer to the set of double-stranded DNA molecules that may arise from an initial set of DNA molecules in the presence of restriction enzymes and ligase. In this paper, some splicing languages resulted from their respective splicing systems are shown. Since all splicing languages are regular, languages which result from the splicing systems can be further investigated using grammars and automata in the field of formal language theory. The splicing language can be written in the form of regular languages generated by grammar. Besides that, splicing systems can be accepted by automata. In this research, two restriction enzymes are used in splicing systems namely BfuCI and NcoI.

  20. RNA-Binding Proteins: Splicing Factors and Disease

    Directory of Open Access Journals (Sweden)

    Alger M. Fredericks

    2015-05-01

    Full Text Available Pre-mRNA splicing is mediated by interactions of the Core Spliceosome and an array of accessory RNA binding proteins with cis-sequence elements. Splicing is a major regulatory component in higher eukaryotes. Disruptions in splicing are a major contributor to human disease. One in three hereditary disease alleles are believed to cause aberrant splicing. Hereditary disease alleles can alter splicing by disrupting a splicing element, creating a toxic RNA, or affecting splicing factors. One of the challenges of medical genetics is identifying causal variants from the thousands of possibilities discovered in a clinical sequencing experiment. Here we review the basic biochemistry of splicing, the mechanisms of splicing mutations, the methods for identifying splicing mutants, and the potential of therapeutic interventions.

  1. Heat shock activates splicing at latent alternative 5' splice sites in nematodes.

    Science.gov (United States)

    Nevo, Yuval; Sperling, Joseph; Sperling, Ruth

    2015-01-01

    Pre-mRNA splicing is essential for the regulation of gene expression in eukaryotes and is fundamental in development and cancer, and involves the selection of a consensus sequence that defines the 5' splice site (5'SS). Human introns harbor multiple sequences that conform to the 5'SS consensus, which are not used under normal growth conditions. Under heat shock conditions, splicing at such intronic latent 5'SSs occurred in thousands of human transcripts, resulting in pre-maturely terminated aberrant proteins. Here we performed a survey of the C. elegans genome, showing that worm's introns contain latent 5'SSs, whose use for splicing would have resulted in pre-maturely terminated mRNAs. Splicing at these latent 5'SSs could not be detected under normal growth conditions, while heat shock activated latent splicing in a number of tested C. elegans transcripts. Two scenarios could account for the lack of latent splicing under normal growth conditions (i) Splicing at latent 5'SSs do occur, but the nonsense mRNAs thus formed are rapidly and efficiently degraded (e.g. by NMD); and (ii) Splicing events at intronic latent 5'SSs are suppressed. Here we support the second scenario, because, nematode smg mutants that are devoid of NMD-essential factors, did not show latent splicing under normal growth conditions. Hence, these experiments together with our previous experiments in mammalian cells, indicate the existence of a nuclear quality control mechanism, termed Suppression Of Splicing (SOS), which discriminates between latent and authentic 5'SSs in an open reading frame dependent manner, and allows splicing only at the latter. Our results show that SOS is an evolutionary conserved mechanism, probably shared by most eukaryotes.

  2. SpliceAid-F: a database of human splicing factors and their RNA-binding sites.

    Science.gov (United States)

    Giulietti, Matteo; Piva, Francesco; D'Antonio, Mattia; D'Onorio De Meo, Paolo; Paoletti, Daniele; Castrignanò, Tiziana; D'Erchia, Anna Maria; Picardi, Ernesto; Zambelli, Federico; Principato, Giovanni; Pavesi, Giulio; Pesole, Graziano

    2013-01-01

    A comprehensive knowledge of all the factors involved in splicing, both proteins and RNAs, and of their interaction network is crucial for reaching a better understanding of this process and its functions. A large part of relevant information is buried in the literature or collected in various different databases. By hand-curated screenings of literature and databases, we retrieved experimentally validated data on 71 human RNA-binding splicing regulatory proteins and organized them into a database called 'SpliceAid-F' (http://www.caspur.it/SpliceAidF/). For each splicing factor (SF), the database reports its functional domains, its protein and chemical interactors and its expression data. Furthermore, we collected experimentally validated RNA-SF interactions, including relevant information on the RNA-binding sites, such as the genes where these sites lie, their genomic coordinates, the splicing effects, the experimental procedures used, as well as the corresponding bibliographic references. We also collected information from experiments showing no RNA-SF binding, at least in the assayed conditions. In total, SpliceAid-F contains 4227 interactions, 2590 RNA-binding sites and 1141 'no-binding' sites, including information on cellular contexts and conditions where binding was tested. The data collected in SpliceAid-F can provide significant information to explain an observed splicing pattern as well as the effect of mutations in functional regulatory elements.

  3. Protein splicing and its evolution in eukaryotes

    Directory of Open Access Journals (Sweden)

    Starokadomskyy P. L.

    2010-02-01

    Full Text Available Inteins, or protein introns, are parts of protein sequences that are post-translationally excised, their flanking regions (exteins being spliced together. This process was called protein splicing. Originally inteins were found in prokaryotic or unicellular eukaryotic organisms. But the general principles of post-translation protein rearrangement are evolving yielding different post-translation modification of proteins in multicellular organisms. For clarity, these non-intein mediated events call either protein rearrangements or protein editing. The most intriguing example of protein editing is proteasome-mediated splicing of antigens in vertebrates that may play important role in antigen presentation. Other examples of protein rearrangements are maturation of Hg-proteins (critical receptors in embryogenesis as well as maturation of several metabolic enzymes. Despite a lack of experimental data we try to analyze some intriguing examples of protein splicing evolution.

  4. Strategies and tools for whole genome alignments

    Energy Technology Data Exchange (ETDEWEB)

    Couronne, Olivier; Poliakov, Alexander; Bray, Nicolas; Ishkhanov,Tigran; Ryaboy, Dmitriy; Rubin, Edward; Pachter, Lior; Dubchak, Inna

    2002-11-25

    The availability of the assembled mouse genome makespossible, for the first time, an alignment and comparison of two largevertebrate genomes. We have investigated different strategies ofalignment for the subsequent analysis of conservation of genomes that areeffective for different quality assemblies. These strategies were appliedto the comparison of the working draft of the human genome with the MouseGenome Sequencing Consortium assembly, as well as other intermediatemouse assemblies. Our methods are fast and the resulting alignmentsexhibit a high degree of sensitivity, covering more than 90 percent ofknown coding exons in the human genome. We have obtained such coveragewhile preserving specificity. With a view towards the end user, we havedeveloped a suite of tools and websites for automatically aligning, andsubsequently browsing and working with whole genome comparisons. Wedescribe the use of these tools to identify conserved non-coding regionsbetween the human and mouse genomes, some of which have not beenidentified by other methods.

  5. Thermopriming Triggers Splicing Memory in Arabidopsis

    KAUST Repository

    Ling, Yu

    2018-02-20

    Abiotic and biotic stresses limit crop productivity. Exposure to a non-lethal stress, referred to as priming, can allow plants to survive subsequent and otherwise lethal conditions; the priming effect persists even after a prolonged stress-free period. However, the molecular mechanisms underlying priming are not fully understood. Here, we investigated the molecular basis of heat shock memory and the role of priming in Arabidopsisthaliana. Comprehensive analysis of transcriptome-wide changes in gene expression and alternative splicing in primed and non-primed plants revealed that alternative splicing functions as a novel component of heat shock memory. We show that priming of plants with a non-lethal heat stress results in de-repression of splicing after a second exposure to heat stress. By contrast, non-primed plants showed significant repression of splicing. These observations link ‘splicing memory’ to the ability of plants to survive subsequent and otherwise lethal heat stress. This newly discovered priming-induced splicing memory may represent a general feature of heat stress responses in plants and other organisms as many of the key components of heat shock responses are conserved among eukaryotes. Furthermore, this finding could facilitate the development of novel approaches to improve plant survival under extreme heat stress.

  6. Spliced leader trapping reveals widespread alternative splicing patterns in the highly dynamic transcriptome of Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Daniel Nilsson

    2010-08-01

    Full Text Available Trans-splicing of leader sequences onto the 5'ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5'splice sites and analyze the corresponding gene expression profile, that we term spliced leader trapping (SLT. The method can be applied to any organism with a sequenced genome and trans-splicing of a conserved leader sequence. We analyzed the expression profiles and splicing patterns of bloodstream and insect forms of the parasite Trypanosoma brucei. We detected the 5' splice sites of 85% of the annotated protein-coding genes and, contrary to previous reports, found up to 40% of transcripts to be differentially expressed. Furthermore, we discovered more than 2500 alternative splicing events, many of which appear to be stage-regulated. Based on our findings we hypothesize that alternatively spliced transcripts present a new means of regulating gene expression and could potentially contribute to protein diversity in the parasite. The entire dataset can be accessed online at TriTrypDB or through: http://splicer.unibe.ch/.

  7. Differential splicing using whole-transcript microarrays

    Directory of Open Access Journals (Sweden)

    Robinson Mark D

    2009-05-01

    Full Text Available Abstract Background The latest generation of Affymetrix microarrays are designed to interrogate expression over the entire length of every locus, thus giving the opportunity to study alternative splicing genome-wide. The Exon 1.0 ST (sense target platform, with versions for Human, Mouse and Rat, is designed primarily to probe every known or predicted exon. The smaller Gene 1.0 ST array is designed as an expression microarray but still interrogates expression with probes along the full length of each well-characterized transcript. We explore the possibility of using the Gene 1.0 ST platform to identify differential splicing events. Results We propose a strategy to score differential splicing by using the auxiliary information from fitting the statistical model, RMA (robust multichip analysis. RMA partitions the probe-level data into probe effects and expression levels, operating robustly so that if a small number of probes behave differently than the rest, they are downweighted in the fitting step. We argue that adjacent poorly fitting probes for a given sample can be evidence of differential splicing and have designed a statistic to search for this behaviour. Using a public tissue panel dataset, we show many examples of tissue-specific alternative splicing. Furthermore, we show that evidence for putative alternative splicing has a strong correspondence between the Gene 1.0 ST and Exon 1.0 ST platforms. Conclusion We propose a new approach, FIRMAGene, to search for differentially spliced genes using the Gene 1.0 ST platform. Such an analysis complements the search for differential expression. We validate the method by illustrating several known examples and we note some of the challenges in interpreting the probe-level data. Software implementing our methods is freely available as an R package.

  8. A random-permutations-based approach to fast read alignment.

    Science.gov (United States)

    Lederman, Roy

    2013-01-01

    Read alignment is a computational bottleneck in some sequencing projects. Most of the existing software packages for read alignment are based on two algorithmic approaches: prefix-trees and hash-tables. We propose a new approach to read alignment using random permutations of strings. We present a prototype implementation and experiments performed with simulated and real reads of human DNA. Our experiments indicate that this permutations-based prototype is several times faster than comparable programs for fast read alignment and that it aligns more reads correctly. This approach may lead to improved speed, sensitivity, and accuracy in read alignment. The algorithm can also be used for specialized alignment applications and it can be extended to other related problems, such as assembly.More information: http://alignment.commons.yale.edu.

  9. Purification of RNA-Protein Splicing Complexes Using a Tagged Protein from In Vitro Splicing Reaction Mixture.

    Science.gov (United States)

    Kataoka, Naoyuki

    2016-01-01

    In eukaryotes, pre-mRNA splicing is an essential step for gene expression. Splicing reactions have been well investigated by using in vitro splicing reactions with extracts prepared from cultured cells. Here, we describe protocols for the preparation of splicing-competent extracts from cells expressing a tagged spliceosomal protein. The whole-cell extracts are able to splice exogenously added pre-mRNA and the RNA-protein complex formed in the in vitro splicing reaction can be purified by immunoprecipitation using antibodies against the peptide tag on the splicing protein. The method described here to prepare splicing-active extracts from whole cells is particularly useful when studying pre-mRNA splicing in various cell types, and the expression of a tagged spliceosomal protein allows one to purify and analyze the RNA-protein complexes by simple immunoprecipitation.

  10. Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals

    Directory of Open Access Journals (Sweden)

    Elela Sherif

    2006-01-01

    Full Text Available Abstract Background We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. Results We show that an oligonucleotide with a 5' tail containing the human β-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. Conclusion Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.

  11. Silicon Alignment Pins: An Easy Way to Realize a Wafer-To-Wafer Alignment

    Science.gov (United States)

    Jung-Kubiak, Cecile (Inventor); Reck, Theodore (Inventor); Thomas, Bertrand (Inventor); Lin, Robert H. (Inventor); Peralta, Alejandro (Inventor); Gill, John J. (Inventor); Lee, Choonsup (Inventor); Siles, Jose V. (Inventor); Toda, Risaku (Inventor); Chattopadhyay, Goutam (Inventor)

    2016-01-01

    A silicon alignment pin is used to align successive layers of components made in semiconductor chips and/or metallic components to make easier the assembly of devices having a layered structure. The pin is made as a compressible structure which can be squeezed to reduce its outer diameter, have one end fit into a corresponding alignment pocket or cavity defined in a layer of material to be assembled into a layered structure, and then allowed to expand to produce an interference fit with the cavity. The other end can then be inserted into a corresponding cavity defined in a surface of a second layer of material that mates with the first layer. The two layers are in registry when the pin is mated to both. Multiple layers can be assembled to create a multilayer structure. Examples of such devices are presented.

  12. Fusion splicing: a novel approach to fiber connections for the Dark Energy Spectroscopic Instrument

    Science.gov (United States)

    Fagrelius, Parker; Poppett, Claire; Edelstein, Jerry

    2016-08-01

    The Dark Energy Spectroscopic Instrument (DESI) is under construction to measure the expansion history of the universe using the Baryon Acoustic Oscillation (BAO) technique and the growth of structure using redshift-space distortions (RSD). The spectra of 40 million galaxies over 14,000 square degrees will be measured during the life of the experiment. A new prime focus corrector for the KPNO Mayall telescope will deliver light to 5000 fiber optic positioners. The fibers will run 50 meters from the focal plane to the coudé room where they feed ten broadband spectrographs. The focal plane assembly will be integrated separately from the spectrograph slits and long fiber cables in order to ease integration flow, and the two subsystems will be connected before final integration on the telescope. In order to retain maximum throughput and minimize the focal ratio degradation (FRD) when connecting the fiber system, we are employing fusion splicing as opposed to mechanical connectorization. For the best splice performance, the optical fibers are stripped of their polyimide coating, precision cleaved, and then fused with a heating filament. We report results from the splicing process, measuring a collimated FRD increase of less than 0.5 degrees for a f/3.9 input beam compared to >1 degree increase for mechanical connectors. We also show that the near field performance is minimally degraded after splicing. These results represent the first of their kind for a fiber-fed astronomical instrument.

  13. A high quality Arabidopsis transcriptome for accurate transcript-level analysis of alternative splicing

    KAUST Repository

    Zhang, Runxuan

    2017-04-05

    Alternative splicing generates multiple transcript and protein isoforms from the same gene and thus is important in gene expression regulation. To date, RNA-sequencing (RNA-seq) is the standard method for quantifying changes in alternative splicing on a genome-wide scale. Understanding the current limitations of RNA-seq is crucial for reliable analysis and the lack of high quality, comprehensive transcriptomes for most species, including model organisms such as Arabidopsis, is a major constraint in accurate quantification of transcript isoforms. To address this, we designed a novel pipeline with stringent filters and assembled a comprehensive Reference Transcript Dataset for Arabidopsis (AtRTD2) containing 82,190 non-redundant transcripts from 34 212 genes. Extensive experimental validation showed that AtRTD2 and its modified version, AtRTD2-QUASI, for use in Quantification of Alternatively Spliced Isoforms, outperform other available transcriptomes in RNA-seq analysis. This strategy can be implemented in other species to build a pipeline for transcript-level expression and alternative splicing analyses.

  14. Rbfox Proteins Regulate Splicing as Part of a Large Multiprotein Complex LASR.

    Science.gov (United States)

    Damianov, Andrey; Ying, Yi; Lin, Chia-Ho; Lee, Ji-Ann; Tran, Diana; Vashisht, Ajay A; Bahrami-Samani, Emad; Xing, Yi; Martin, Kelsey C; Wohlschlegel, James A; Black, Douglas L

    2016-04-21

    Rbfox proteins control alternative splicing and posttranscriptional regulation in mammalian brain and are implicated in neurological disease. These proteins recognize the RNA sequence (U)GCAUG, but their structures and diverse roles imply a variety of protein-protein interactions. We find that nuclear Rbfox proteins are bound within a large assembly of splicing regulators (LASR), a multimeric complex containing the proteins hnRNP M, hnRNP H, hnRNP C, Matrin3, NF110/NFAR-2, NF45, and DDX5, all approximately equimolar to Rbfox. We show that splicing repression mediated by hnRNP M is stimulated by Rbfox. Virtually all the intron-bound Rbfox is associated with LASR, and hnRNP M motifs are enriched adjacent to Rbfox crosslinking sites in vivo. These findings demonstrate that Rbfox proteins bind RNA with a defined set of cofactors and affect a broader set of exons than previously recognized. The function of this multimeric LASR complex has implications for deciphering the regulatory codes controlling splicing networks. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Desktop aligner for fabrication of multilayer microfluidic devices

    Science.gov (United States)

    Li, Xiang; Yu, Zeta Tak For; Geraldo, Dalton; Weng, Shinuo; Alve, Nitesh; Dun, Wu; Kini, Akshay; Patel, Karan; Shu, Roberto; Zhang, Feng; Li, Gang; Jin, Qinghui; Fu, Jianping

    2015-07-01

    Multilayer assembly is a commonly used technique to construct multilayer polydimethylsiloxane (PDMS)-based microfluidic devices with complex 3D architecture and connectivity for large-scale microfluidic integration. Accurate alignment of structure features on different PDMS layers before their permanent bonding is critical in determining the yield and quality of assembled multilayer microfluidic devices. Herein, we report a custom-built desktop aligner capable of both local and global alignments of PDMS layers covering a broad size range. Two digital microscopes were incorporated into the aligner design to allow accurate global alignment of PDMS structures up to 4 in. in diameter. Both local and global alignment accuracies of the desktop aligner were determined to be about 20 μm cm-1. To demonstrate its utility for fabrication of integrated multilayer PDMS microfluidic devices, we applied the desktop aligner to achieve accurate alignment of different functional PDMS layers in multilayer microfluidics including an organs-on-chips device as well as a microfluidic device integrated with vertical passages connecting channels located in different PDMS layers. Owing to its convenient operation, high accuracy, low cost, light weight, and portability, the desktop aligner is useful for microfluidic researchers to achieve rapid and accurate alignment for generating multilayer PDMS microfluidic devices.

  16. Human-specific protein isoforms produced by novel splice sites in the human genome after the human-chimpanzee divergence

    Directory of Open Access Journals (Sweden)

    Kim Dong Seon

    2012-11-01

    Full Text Available Abstract Background Evolution of splice sites is a well-known phenomenon that results in transcript diversity during human evolution. Many novel splice sites are derived from repetitive elements and may not contribute to protein products. Here, we analyzed annotated human protein-coding exons and identified human-specific splice sites that arose after the human-chimpanzee divergence. Results We analyzed multiple alignments of the annotated human protein-coding exons and their respective orthologous mammalian genome sequences to identify 85 novel splice sites (50 splice acceptors and 35 donors in the human genome. The novel protein-coding exons, which are expressed either constitutively or alternatively, produce novel protein isoforms by insertion, deletion, or frameshift. We found three cases in which the human-specific isoform conferred novel molecular function in the human cells: the human-specific IMUP protein isoform induces apoptosis of the trophoblast and is implicated in pre-eclampsia; the intronization of a part of SMOX gene exon produces inactive spermine oxidase; the human-specific NUB1 isoform shows reduced interaction with ubiquitin-like proteins, possibly affecting ubiquitin pathways. Conclusions Although the generation of novel protein isoforms does not equate to adaptive evolution, we propose that these cases are useful candidates for a molecular functional study to identify proteomic changes that might bring about novel phenotypes during human evolution.

  17. Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF.

    Science.gov (United States)

    Chen, Zhiqi; Ma, Xuezhong; Zhang, Jianhua; Hu, Jim; Gorczynski, Reginald M

    2010-10-01

    CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200(tr)) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200.

  18. Splicing variants of porcine synphilin-1

    DEFF Research Database (Denmark)

    Larsen, Knud Erik; Madsen, Lone Bruhn; Farajzadeh, Leila

    2015-01-01

    %) and to mouse (84%) synphilin-1. Three shorter transcript variants of the synphilin-1 gene were identified, all lacking one or more exons. SNCAIP transcripts were detected in most examined organs and tissues and the highest expression was found in brain tissues and lung. Conserved splicing variants and a novel......RNA was investigated by RNAseq. The presented work reports the molecular cloning and characterization of the porcine (Sus scrofa) synphilin-1 cDNA (SNCAIP) and three splice variants hereof. The porcine SNCAIP cDNA codes for a protein (synphilin-1) of 919 amino acids which shows a high similarity to human (90...... splice form of synhilin-1 were found in this study. All synphilin-1 isoforms encoded by the identified transcript variants lack functional domains important for protein degradation....

  19. Capillary Electrophoresis Analysis of Conventional Splicing Assays

    DEFF Research Database (Denmark)

    de Garibay, Gorka Ruiz; Acedo, Alberto; García-Casado, Zaida

    2014-01-01

    Rare sequence variants in "high-risk" disease genes, often referred as unclassified variants (UVs), pose a serious challenge to genetic testing. However, UVs resulting in splicing alterations can be readily assessed by in vitro assays. Unfortunately, analytical and clinical interpretation...... of these assays is often challenging. Here, we explore this issue by conducting splicing assays in 31 BRCA2 genetic variants. All variants were assessed by RT-PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear-cut outputs (Class-2 or Class-5 according to analytical...... International Agency for Research on Cancer guidelines), we performed qPCR and/or minigene assays. The latter were performed with a new splicing vector (pSAD) developed by authors of the present manuscript (patent #P201231427 CSIC). We have identified three clinically relevant Class-5 variants (c.682-2A>G, c...

  20. Genomics of alternative splicing: evolution, development and pathophysiology.

    Science.gov (United States)

    Gamazon, Eric R; Stranger, Barbara E

    2014-06-01

    Alternative splicing is a major cellular mechanism in metazoans for generating proteomic diversity. A large proportion of protein-coding genes in multicellular organisms undergo alternative splicing, and in humans, it has been estimated that nearly 90 % of protein-coding genes-much larger than expected-are subject to alternative splicing. Genomic analyses of alternative splicing have illuminated its universal role in shaping the evolution of genomes, in the control of developmental processes, and in the dynamic regulation of the transcriptome to influence phenotype. Disruption of the splicing machinery has been found to drive pathophysiology, and indeed reprogramming of aberrant splicing can provide novel approaches to the development of molecular therapy. This review focuses on the recent progress in our understanding of alternative splicing brought about by the unprecedented explosive growth of genomic data and highlights the relevance of human splicing variation on disease and therapy.

  1. Tissue-specific splicing factor gene expression signatures

    NARCIS (Netherlands)

    A.R. Grosso; A.Q. Gomes (Anita); N.L. Barbosa-Morais (Nuno); S. Caldeira (Sandra); N.P. Thorne (Natalie); G. Grech (Godfrey); M.M. von Lindern (Marieke); M. Carmo-Fonseca (Maria)

    2008-01-01

    textabstractThe alternative splicing code that controls and coordinates the transcriptome in complex multicellular organisms remains poorly understood. It has long been argued that regulation of alternative splicing relies on combinatorial interactions between multiple proteins, and that

  2. Tissue-specific splicing factor gene expression signatures

    NARCIS (Netherlands)

    Grosso, Ana Rita; Gomes, Anita Q.; Barbosa-Morais, Nuno L.; Caldeira, Sandra; Thorne, Natalie P.; Grech, Godfrey; von Lindern, Marieke; Carmo-Fonseca, Maria

    2008-01-01

    The alternative splicing code that controls and coordinates the transcriptome in complex multicellular organisms remains poorly understood. It has long been argued that regulation of alternative splicing relies on combinatorial interactions between multiple proteins, and that tissue-specific

  3. MUON DETECTORS: ALIGNMENT

    CERN Multimedia

    G.Gomez.

    Since June of 2009, the muon alignment group has focused on providing new alignment constants and on finalizing the hardware alignment reconstruction. Alignment constants for DTs and CSCs were provided for CRAFT09 data reprocessing. For DT chambers, the track-based alignment was repeated using CRAFT09 cosmic ray muons and validated using segment extrapolation and split cosmic tools. One difference with respect to the previous alignment is that only five degrees of freedom were aligned, leaving the rotation around the local x-axis to be better determined by the hardware system. Similarly, DT chambers poorly aligned by tracks (due to limited statistics) were aligned by a combination of photogrammetry and hardware-based alignment. For the CSC chambers, the hardware system provided alignment in global z and rotations about local x. Entire muon endcap rings were further corrected in the transverse plane (global x and y) by the track-based alignment. Single chamber track-based alignment suffers from poor statistic...

  4. Alternative Pre-mRNA Splicing in Mammals and Teleost Fish: A Effective Strategy for the Regulation of Immune Responses Against Pathogen Infection.

    Science.gov (United States)

    Chang, Ming Xian; Zhang, Jie

    2017-07-15

    Pre-mRNA splicing is the process by which introns are removed and the protein coding elements assembled into mature mRNAs. Alternative pre-mRNA splicing provides an important source of transcriptome and proteome complexity through selectively joining different coding elements to form mRNAs, which encode proteins with similar or distinct functions. In mammals, previous studies have shown the role of alternative splicing in regulating the function of the immune system, especially in the regulation of T-cell activation and function. As lower vertebrates, teleost fish mainly rely on a large family of pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) from various invading pathogens. In this review, we summarize recent advances in our understanding of alternative splicing of piscine PRRs including peptidoglycan recognition proteins (PGRPs), nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) and their downstream signaling molecules, compared to splicing in mammals. We also discuss what is known and unknown about the function of splicing isoforms in the innate immune responses against pathogens infection in mammals and teleost fish. Finally, we highlight the consequences of alternative splicing in the innate immune system and give our view of important directions for future studies.

  5. Comparison of splice sites in mammals and chicken

    OpenAIRE

    Abril Ferrando, Josep Francesc; Castelo Valdueza, Robert; Guigó Serra, Roderic

    2005-01-01

    We have carried out an initial analysis of the dynamics of the recent evolution of the splice-sites sequences on a large collection of human, rodent (mouse and rat), and chicken introns. Our results indicate that the sequences of splice sites are largely homogeneous within tetrapoda. We have also found that orthologous splice signals between human and rodents and within rodents are more conserved than unrelated splice sites, but the additional conservation can be explained mostly by backgroun...

  6. MUON DETECTORS: ALIGNMENT

    CERN Multimedia

    G. Gomez and J. Pivarski

    2011-01-01

    Alignment efforts in the first few months of 2011 have shifted away from providing alignment constants (now a well established procedure) and focussed on some critical remaining issues. The single most important task left was to understand the systematic differences observed between the track-based (TB) and hardware-based (HW) barrel alignments: a systematic difference in r-φ and in z, which grew as a function of z, and which amounted to ~4-5 mm differences going from one end of the barrel to the other. This difference is now understood to be caused by the tracker alignment. The systematic differences disappear when the track-based barrel alignment is performed using the new “twist-free” tracker alignment. This removes the largest remaining source of systematic uncertainty. Since the barrel alignment is based on hardware, it does not suffer from the tracker twist. However, untwisting the tracker causes endcap disks (which are aligned ...

  7. Functional and evolutionary analysis of alternatively spliced genes is consistent with an early eukaryotic origin of alternative splicing

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Penny, David

    2007-01-01

    , and may therefore predate multicellularity, is still unknown. To better understand the origin and evolution of alternative splicing and its usage in diverse organisms, we studied alternative splicing in 12 eukaryotic species, comparing rates of alternative splicing across genes of different functional...... classes, cellular locations, intron/exon structures and evolutionary origins. RESULTS: For each species, we find that genes from most functional categories are alternatively spliced. Ancient genes (shared between animals, fungi and plants) show high levels of alternative splicing. Genes with products...... expressed in the nucleus or plasma membrane are generally more alternatively spliced while those expressed in extracellular location show less alternative splicing. We find a clear correspondence between incidence of alternative splicing and intron number per gene both within and between genomes. In general...

  8. 30 CFR 77.504 - Electrical connections or splices; suitability.

    Science.gov (United States)

    2010-07-01

    ... UNDERGROUND COAL MINES Electrical Equipment-General § 77.504 Electrical connections or splices; suitability. Electrical connections or splices in electric conductors shall be mechanically and electrically efficient... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Electrical connections or splices; suitability...

  9. The implications of alternative splicing in the ENCODE protein complement

    DEFF Research Database (Denmark)

    Tress, Michael L.; Martelli, Pier Luigi; Frankish, Adam

    2007-01-01

    Alternative premessenger RNA splicing enables genes to generate more than one gene product. Splicing events that occur within protein coding regions have the potential to alter the biological function of the expressed protein and even to create new protein functions. Alternative splicing has been...

  10. Auxiliary splice factor U2AF26 and transcription factor Gfi1 cooperate directly in regulating CD45 alternative splicing.

    NARCIS (Netherlands)

    Heyd, F.; Dam, G.B. ten; Moroy, T.

    2006-01-01

    By alternative splicing, different isoforms of the transmembrane tyrosine phosphatase CD45 are generated that either enhance or limit T cell receptor signaling. We report here that CD45 alternative splicing is regulated by cooperative action of the splice factor U2AF26 and the transcription factor

  11. MUON DETECTORS: ALIGNMENT

    CERN Multimedia

    G.Gomez

    2010-01-01

    The main developments in muon alignment since March 2010 have been the production, approval and deployment of alignment constants for the ICHEP data reprocessing. In the barrel, a new geometry, combining information from both hardware and track-based alignment systems, has been developed for the first time. The hardware alignment provides an initial DT geometry, which is then anchored as a rigid solid, using the link alignment system, to a reference frame common to the tracker. The “GlobalPositionRecords” for both the Tracker and Muon systems are being used for the first time, and the initial tracker-muon relative positioning, based on the link alignment, yields good results within the photogrammetry uncertainties of the Tracker and alignment ring positions. For the first time, the optical and track-based alignments show good agreement between them; the optical alignment being refined by the track-based alignment. The resulting geometry is the most complete to date, aligning all 250 DTs, ...

  12. Girder Alignment Plan

    Energy Technology Data Exchange (ETDEWEB)

    Wolf, Zackary; Ruland, Robert; LeCocq, Catherine; Lundahl, Eric; Levashov, Yurii; Reese, Ed; Rago, Carl; Poling, Ben; Schafer, Donald; Nuhn, Heinz-Dieter; Wienands, Uli; /SLAC

    2010-11-18

    The girders for the LCLS undulator system contain components which must be aligned with high accuracy relative to each other. The alignment is one of the last steps before the girders go into the tunnel, so the alignment must be done efficiently, on a tight schedule. This note documents the alignment plan which includes efficiency and high accuracy. The motivation for girder alignment involves the following considerations. Using beam based alignment, the girder position will be adjusted until the beam goes through the center of the quadrupole and beam finder wire. For the machine to work properly, the undulator axis must be on this line and the center of the undulator beam pipe must be on this line. The physics reasons for the undulator axis and undulator beam pipe axis to be centered on the beam are different, but the alignment tolerance for both are similar. In addition, the beam position monitor must be centered on the beam to preserve its calibration. Thus, the undulator, undulator beam pipe, quadrupole, beam finder wire, and beam position monitor axes must all be aligned to a common line. All relative alignments are equally important, not just, for example, between quadrupole and undulator. We begin by making the common axis the nominal beam axis in the girder coordinate system. All components will be initially aligned to this axis. A more accurate alignment will then position the components relative to each other, without incorporating the girder itself.

  13. On the Possibility of an Early Evolutionary Origin for the Spliced Leader Trans-Splicing.

    Science.gov (United States)

    Krchňáková, Zuzana; Krajčovič, Juraj; Vesteg, Matej

    2017-08-01

    Trans-splicing is a process by which 5'- and 3'-ends of two pre-RNA molecules transcribed from different sites of the genome can be joined together to form a single RNA molecule. The spliced leader (SL) trans-splicing is mediated by the spliceosome and it allows the replacement of 5'-end of pre-mRNA by 5'(SL)-end of SL-RNA. This form of splicing has been observed in many phylogenetically unrelated eukaryotes. Either the SL trans-splicing (SLTS) originated in the last eukaryotic common ancestor (LECA) (or even earlier) and it was lost in most eukaryotic lineages, or this mechanism of RNA processing evolved several times independently in various unrelated eukaryotic taxa. The bioinformatic comparisons of SL-RNAs from various eukaryotic taxonomic groups have revealed the similarities of secondary structures of most SL-RNAs and a relative conservation of their splice sites (SSs) and Sm-binding sites (SmBSs). We propose that such structural and functional similarities of SL-RNAs are unlikely to have evolved repeatedly many times. Hence, we favor the scenario of an early evolutionary origin for the SLTS and multiple losses of SL-RNAs in various eukaryotic lineages.

  14. MUON DETECTORS: ALIGNMENT

    CERN Multimedia

    Z. Szillasi and G. Gomez.

    2013-01-01

    When CMS is opened up, major components of the Link and Barrel Alignment systems will be removed. This operation, besides allowing for maintenance of the detector underneath, is needed for making interventions that will reinforce the alignment measurements and make the operation of the alignment system more reliable. For that purpose and also for their general maintenance and recalibration, the alignment components will be transferred to the Alignment Lab situated in the ISR area. For the track-based alignment, attention is focused on the determination of systematic uncertainties, which have become dominant, since now there is a large statistics of muon tracks. This will allow for an improved Monte Carlo misalignment scenario and updated alignment position errors, crucial for high-momentum muon analysis such as Z′ searches.

  15. GAP: A computer program for gene assembly

    Energy Technology Data Exchange (ETDEWEB)

    Eisnstein, J.R.; Uberbacher, E.C.; Guan, X.; Mural, R.J.; Mann, R.C.

    1991-09-01

    A computer program, GAP (Gene Assembly Program), has been written to assemble and score hypothetical genes, given a DNA sequence containing the gene, and the outputs of several other programs which analyze the sequence. These programs include the codign-recognition and splice-junction-recognition modules developed in this laboratory. GAP is a prototype of a planned system in which it will be integrated with an expert system and rule base. Initial tests of GAP have been carried out with four sequences, the exons of which have been determined by biochemcial methods. The highest-scoring hypothetical genes for each of the four sequences had percent correct splice junctions ranging from 50 to 100% (average 81%) and percent correct bases ranging from 92 to 100% (average 96%). 9 refs., 1 tab.

  16. Discovery of a Splicing Regulator Required for Cell Cycle Progression

    Energy Technology Data Exchange (ETDEWEB)

    Suvorova, Elena S.; Croken, Matthew; Kratzer, Stella; Ting, Li-Min; Conde de Felipe, Magnolia; Balu, Bharath; Markillie, Lye Meng; Weiss, Louis M.; Kim, Kami; White, Michael W.

    2013-02-01

    In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms.

  17. Integrated magnetic transformer assembly

    DEFF Research Database (Denmark)

    2014-01-01

    The present invention relates to an integrated magnetics transformer assembly comprising a first magnetically permeable core forming a first substantially closed magnetic flux path and a second magnetically permeable core forming a second substantially closed magnetic flux path. A first input...... inductor winding is wound around a first predetermined segment of the first magnetically permeable core and a second input inductor winding is wound around a first predetermined segment of the second magnetically permeable core. The integrated magnetics transformer assembly further comprises a first output......-winding of the first output inductor winding and the first half-winding of the second output inductor winding are configured to produce aligned, i.e. in the same direction, magnetic fluxes through the first substantially closed magnetic flux path. The integrated magnetics transformer assembly is well- suited for use...

  18. Cauliflower mosaic virus Transcriptome Reveals a Complex Alternative Splicing Pattern.

    Directory of Open Access Journals (Sweden)

    Clément Bouton

    Full Text Available The plant pararetrovirus Cauliflower mosaic virus (CaMV uses alternative splicing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5' region and suggested that the main role of CaMV splicing is to downregulate expression of open reading frames (ORFs I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA.

  19. Mapping Quantitative Trait Loci (QTL in sheep. III. QTL for carcass composition traits derived from CT scans and aligned with a meta-assembly for sheep and cattle carcass QTL

    Directory of Open Access Journals (Sweden)

    Thomson Peter C

    2010-09-01

    Full Text Available Abstract An (Awassi × Merino × Merino single-sire backcross family with 165 male offspring was used to map quantitative trait loci (QTL for body composition traits on a framework map of 189 microsatellite loci across all autosomes. Two cohorts were created from the experimental progeny to represent alternative maturity classes for body composition assessment. Animals were raised under paddock conditions prior to entering the feedlot for a 90-day fattening phase. Body composition traits were derived in vivo at the end of the experiment prior to slaughter at 2 (cohort 1 and 3.5 (cohort 2 years of age, using computed tomography. Image analysis was used to gain accurate predictions for 13 traits describing major fat depots, lean muscle, bone, body proportions and body weight which were used for single- and two-QTL mapping analysis. Using a maximum-likelihood approach, three highly significant (LOD ≥ 3, 15 significant (LOD ≥ 2, and 11 suggestive QTL (1.7 ≤ LOD P P A meta-assembly of ovine QTL for carcass traits from this study and public domain sources was performed and compared with a corresponding bovine meta-assembly. The assembly demonstrated QTL with effects on carcass composition in homologous regions on OAR1, 2, 6 and 21.

  20. Novel bioinformatics method for identification of genome-wide non-canonical spliced regions using RNA-Seq data.

    Directory of Open Access Journals (Sweden)

    Yongsheng Bai

    Full Text Available During endoplasmic reticulum (ER stress, the endoribonuclease (RNase Ire1α initiates removal of a 26 nt region from the mRNA encoding the transcription factor Xbp1 via an unconventional mechanism (atypically within the cytosol. This causes an open reading frame-shift that leads to altered transcriptional regulation of numerous downstream genes in response to ER stress as part of the unfolded protein response (UPR. Strikingly, other examples of targeted, unconventional splicing of short mRNA regions have yet to be reported.Our goal was to develop an approach to identify non-canonical, possibly very short, splicing regions using RNA-Seq data and apply it to ER stress-induced Ire1α heterozygous and knockout mouse embryonic fibroblast (MEF cell lines to identify additional Ire1α targets.We developed a bioinformatics approach called the Read-Split-Walk (RSW pipeline, and evaluated it using two Ire1α heterozygous and two Ire1α-null samples. The 26 nt non-canonical splice site in Xbp1 was detected as the top hit by our RSW pipeline in heterozygous samples but not in the negative control Ire1α knockout samples. We compared the Xbp1 results from our approach with results using the alignment program BWA, Bowtie2, STAR, Exonerate and the Unix "grep" command. We then applied our RSW pipeline to RNA-Seq data from the SKBR3 human breast cancer cell line. RSW reported a large number of non-canonical spliced regions for 108 genes in chromosome 17, which were identified by an independent study.We conclude that our RSW pipeline is a practical approach for identifying non-canonical splice junction sites on a genome-wide level. We demonstrate that our pipeline can detect novel splice sites in RNA-Seq data generated under similar conditions for multiple species, in our case mouse and human.

  1. Development of a novel splice array platform and its application in the identification of alternative splice variants in lung cancer

    Directory of Open Access Journals (Sweden)

    Gomez-Roman Javier

    2010-06-01

    Full Text Available Abstract Background Microarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array approach was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events. Results The array consisted of exon probes and thermodynamically balanced junction probes. Suboptimal probes were tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was validated with artificial constructs for CDC6, VEGF, and PCBP4 isoforms. The platform was then applied to the analysis of differential splice forms in lung cancer samples compared to matched normal lung tissue. Overexpression of splice isoforms was identified for genes encoding CEACAM1, FHL-1, MLPH, and SUSD2. None of these splicing isoforms had been previously associated with lung cancer. Conclusions This methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies.

  2. Global genome splicing analysis reveals an increased number of alternatively spliced genes with aging.

    Science.gov (United States)

    Rodríguez, Sofía A; Grochová, Diana; McKenna, Tomás; Borate, Bhavesh; Trivedi, Niraj S; Erdos, Michael R; Eriksson, Maria

    2016-04-01

    Alternative splicing (AS) is a key regulatory mechanism for the development of different tissues; however, not much is known about changes to alternative splicing during aging. Splicing events may become more frequent and widespread genome-wide as tissues age and the splicing machinery stringency decreases. Using skin, skeletal muscle, bone, thymus, and white adipose tissue from wild-type C57BL6/J male mice (4 and 18 months old), we examined the effect of age on splicing by AS analysis of the differential exon usage of the genome. The results identified a considerable number of AS genes in skeletal muscle, thymus, bone, and white adipose tissue between the different age groups (ranging from 27 to 246 AS genes corresponding to 0.3-3.2% of the total number of genes analyzed). For skin, skeletal muscle, and bone, we included a later age group (28 months old) that showed that the number of alternatively spliced genes increased with age in all three tissues (P aging disease Hutchinson-Gilford progeria syndrome was performed. The results show that expression of the mutant protein, progerin, is associated with an impaired developmental splicing. As progerin accumulates, the number of genes with AS increases compared to in wild-type skin. Our results indicate the existence of a mechanism for increased AS during aging in several tissues, emphasizing that AS has a more important role in the aging process than previously known. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  3. Deciphering the plant splicing code: Experimental and computational approaches for predicting alternative splicing and splicing regulatory elements

    Directory of Open Access Journals (Sweden)

    Anireddy S.N. Reddy

    2012-02-01

    Full Text Available Extensive alternative splicing (AS of precursor mRNAs (pre-mRNAs in multicellular eukaryotes increases the protein-coding capacity of a genome and allows novel ways to regulate gene expression. In fowering plants, up to 48% of intron-containing genes exhibit AS. However, the full extent of AS in plants is not yet known, as only a few high throughput RNA-Seq studies have been performed. As the cost of obtaining RNA-Seq reads continues to fall, it is anticipated that huge amounts of plant sequence data will accumulate and help in obtaining a more complete picture of AS in plants. Although it is not an onerous task to obtain hundreds of millions of reads using high throughput sequencing technologies, computational tools to accurately predict and visualize AS are still being developed and refined. This review will discuss the tools to predict and visualize transcriptome-wide AS in plants using short reads and highlight their limitations. Comparative studies of AS events between plants and animals have revealed that there are major differences in the most prevalent types of AS events, suggesting that plants and animals differ in the way they recognize exons and introns. Extensive studies have been performed in animals to identify cis-elements involved in regulating AS, especially in exon skipping. However, such studies are in their infancy in plants. Here, we review the current state of research on splicing regulatory elements (SREs and briefly discuss emerging experimental and computational tools to identify cis-elements involved in regulation of AS in plants. The availability of curated alternative splice forms in plants makes it possible to use computational tools to predict SREs involved in AS regulation, which can then be verified experimentally. Such studies will permit identification of plant-specific features involved in AS regulation and contribute to deciphering the splicing code in plants.

  4. MUON DETECTORS: ALIGNMENT

    CERN Multimedia

    G.Gomez

    2010-01-01

    Most of the work in muon alignment since December 2009 has focused on the geometry reconstruction from the optical systems and improvements in the internal alignment of the DT chambers. The barrel optical alignment system has progressively evolved from reconstruction of single active planes to super-planes (December 09) to a new, full barrel reconstruction. Initial validation studies comparing this full barrel alignment at 0T with photogrammetry provide promising results. In addition, the method has been applied to CRAFT09 data, and the resulting alignment at 3.8T yields residuals from tracks (extrapolated from the tracker) which look smooth, suggesting a good internal barrel alignment with a small overall offset with respect to the tracker. This is a significant improvement, which should allow the optical system to provide a start-up alignment for 2010. The end-cap optical alignment has made considerable progress in the analysis of transfer line data. The next set of alignment constants for CSCs will there...

  5. Tidal alignment of galaxies

    Energy Technology Data Exchange (ETDEWEB)

    Blazek, Jonathan; Vlah, Zvonimir; Seljak, Uroš

    2015-08-01

    We develop an analytic model for galaxy intrinsic alignments (IA) based on the theory of tidal alignment. We calculate all relevant nonlinear corrections at one-loop order, including effects from nonlinear density evolution, galaxy biasing, and source density weighting. Contributions from density weighting are found to be particularly important and lead to bias dependence of the IA amplitude, even on large scales. This effect may be responsible for much of the luminosity dependence in IA observations. The increase in IA amplitude for more highly biased galaxies reflects their locations in regions with large tidal fields. We also consider the impact of smoothing the tidal field on halo scales. We compare the performance of this consistent nonlinear model in describing the observed alignment of luminous red galaxies with the linear model as well as the frequently used "nonlinear alignment model," finding a significant improvement on small and intermediate scales. We also show that the cross-correlation between density and IA (the "GI" term) can be effectively separated into source alignment and source clustering, and we accurately model the observed alignment down to the one-halo regime using the tidal field from the fully nonlinear halo-matter cross correlation. Inside the one-halo regime, the average alignment of galaxies with density tracers no longer follows the tidal alignment prediction, likely reflecting nonlinear processes that must be considered when modeling IA on these scales. Finally, we discuss tidal alignment in the context of cosmic shear measurements.

  6. Genomic HEXploring allows landscaping of novel potential splicing regulatory elements.

    Science.gov (United States)

    Erkelenz, Steffen; Theiss, Stephan; Otte, Marianne; Widera, Marek; Peter, Jan Otto; Schaal, Heiner

    2014-01-01

    Effective splice site selection is critically controlled by flanking splicing regulatory elements (SREs) that can enhance or repress splice site use. Although several computational algorithms currently identify a multitude of potential SRE motifs, their predictive power with respect to mutation effects is limited. Following a RESCUE-type approach, we defined a hexamer-based 'HEXplorer score' as average Z-score of all six hexamers overlapping with a given nucleotide in an arbitrary genomic sequence. Plotted along genomic regions, HEXplorer score profiles varied slowly in the vicinity of splice sites. They reflected the respective splice enhancing and silencing properties of splice site neighborhoods beyond the identification of single dedicated SRE motifs. In particular, HEXplorer score differences between mutant and reference sequences faithfully represented exonic mutation effects on splice site usage. Using the HIV-1 pre-mRNA as a model system highly dependent on SREs, we found an excellent correlation in 29 mutations between splicing activity and HEXplorer score. We successfully predicted and confirmed five novel SREs and optimized mutations inactivating a known silencer. The HEXplorer score allowed landscaping of splicing regulatory regions, provided a quantitative measure of mutation effects on splice enhancing and silencing properties and permitted calculation of the mutationally most effective nucleotide. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Alternative Splicing and Subfunctionalization Generates Functional Diversity in Fungal Proteomes

    Science.gov (United States)

    Jiménez-López, Claudia; Lorenz, Michael C.; van Hoof, Ambro

    2013-01-01

    Alternative splicing is commonly used by the Metazoa to generate more than one protein from a gene. However, such diversification of the proteome by alternative splicing is much rarer in fungi. We describe here an ancient fungal alternative splicing event in which these two proteins are generated from a single alternatively spliced ancestral SKI7/HBS1 gene retained in many species in both the Ascomycota and Basidiomycota. While the ability to express two proteins from a single SKI7/HBS1 gene is conserved in many fungi, the exact mechanism by which they achieve this varies. The alternative splicing was lost in Saccharomyces cerevisiae following the whole-genome duplication event as these two genes subfunctionalized into the present functionally distinct HBS1 and SKI7 genes. When expressed in yeast, the single gene from Lachancea kluyveri generates two functionally distinct proteins. Expression of one of these proteins complements hbs1, but not ski7 mutations, while the other protein complements ski7, but not hbs1. This is the first known case of subfunctionalization by loss of alternative splicing in yeast. By coincidence, the ancestral alternatively spliced gene was also duplicated in Schizosaccharomyces pombe with subsequent subfunctionalization and loss of splicing. Similar subfunctionalization by loss of alternative splicing in fungi also explains the presence of two PTC7 genes in the budding yeast Tetrapisispora blattae, suggesting that this is a common mechanism to preserve duplicate alternatively spliced genes. PMID:23516382

  8. Spliced leader RNA-mediated trans-splicing in phylum Rotifera.

    Science.gov (United States)

    Pouchkina-Stantcheva, Natalia N; Tunnacliffe, Alan

    2005-06-01

    In kinetoplastids, Euglena, and four metazoan phyla, trans-splicing has been described as a mechanism for the generation of mature messenger RNAs (mRNAs): 5'-ends of precursor mRNAs are replaced by a short spliced leader (SL) exon from a small SL RNA. Although the full phylogenetic range is unknown, trans-splicing has not been found in vertebrates, insects, plants, or yeast. In animal groups where it does occur, i.e., nematodes, cnidarians, platyhelminths, and primitive chordates, SL RNAs do not show sequence relatedness across phyla. The apparently sporadic phylogenetic distribution and the lack of SL RNA homology have led to opposing hypotheses on its evolution, involving either an ancient origin followed by loss in multiple lineages or independent acquisition in several taxa. Here we present evidence for the occurrence of trans-splicing in bdelloid rotifers (Bdelloidea, Rotifera). A common 23-nt sequence, representing the SL exon-diagnostic of SL RNA-mediated trans-splicing-was found at the 5'-end of at least 50%-65% of mRNAs from Adineta ricciae and Philodina sp. The trans-splicing pattern in bdelloid rotifers can be unusually complex, as observed in transcripts from a heat shock protein gene, hsp82-1, where the SL exon was spliced to three alternative positions. Bdelloid rotifer SL RNAs were found to be 105 or 106 nt long and comprised the SL sequence, a conserved splice donor site and an intron containing a putative spliceosome-binding motif. Intriguingly, some similarity of rotifer SL RNA sequence and predicted secondary structure was seen to that of the predominant SL1 RNA of nematodes, although it is unlikely that this demonstrates homology. In addition, sequence corresponding to the rotifer SL exon was found at the 5'-end of a number of full-length complementary DNA (cDNA) clones in a rice (Oryza sativa) database. None of these cDNAs gave a close match with homologous plant genes, suggesting that a small but significant portion of the rice expressed

  9. DNA splicing by directed ligation (SDL).

    Science.gov (United States)

    Berlin, Y A

    1999-01-01

    Splicing by directed ligation (SDL) is a method of in-phase joining of PCR-generated DNA fragments that is based on a pre-designed combination of class IIS restriction endonuclease recognition and cleavage sites. Since these enzymes cleave outside of their recognition sites, the resulting sticky end can have any desired sequence, and the site itself can be removed and does not appear in the final spliced DNA product. SDL is based on the addition of class IIS recognition sites onto primers used to amplify DNA sequences. Cleavage of the PCR products results in elimination of the recognition site-containing flanking sequences and leaves the DNA fragments crowned with protruding ends. With careful design of the sticky ends, several segments can be ligated together in a predetermined order in a single reaction. SDL requires fewer rounds of amplification than overlap extension methods, and is particularly useful for creating a series of recombinants that differ in one segment.

  10. Intravitreal Injection of Splice-switching Oligonucleotides to Manipulate Splicing in Retinal Cells

    Directory of Open Access Journals (Sweden)

    Xavier Gérard

    2015-01-01

    Full Text Available Leber congenital amaurosis is a severe hereditary retinal dystrophy responsible for neonatal blindness. The most common disease-causing mutation (c.2991+1655A>G; 10–15% creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. Recently, we reported that splice-switching oligonucleotides (SSO allow skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients, supporting the feasibility of a SSO-mediated exon skipping strategy to correct the aberrant splicing. Here, we present data in the wild-type mouse, which demonstrate that intravitreal administration of 2’-OMePS-SSO allows selective alteration of Cep290 splicing in retinal cells, including photoreceptors as shown by successful alteration of Abca4 splicing using the same approach. We show that both SSOs and Cep290 skipped mRNA were detectable for at least 1 month and that intravitreal administration of oligonucleotides did not provoke any serious adverse event. These data suggest that intravitreal injections of SSO should be considered to bypass protein truncation resulting from the c.2991+1655A>G mutation as well as other truncating mutations in genes which like CEP290 or ABCA4 have a mRNA size that exceed cargo capacities of US Food and Drug Administration (FDA-approved adeno-associated virus (AAV-vectors, thus hampering gene augmentation therapy.

  11. Resolving deconvolution ambiguity in gene alternative splicing

    Directory of Open Access Journals (Sweden)

    Hubbell Earl

    2009-08-01

    Full Text Available Abstract Background For many gene structures it is impossible to resolve intensity data uniquely to establish abundances of splice variants. This was empirically noted by Wang et al. in which it was called a "degeneracy problem". The ambiguity results from an ill-posed problem where additional information is needed in order to obtain an unique answer in splice variant deconvolution. Results In this paper, we analyze the situations under which the problem occurs and perform a rigorous mathematical study which gives necessary and sufficient conditions on how many and what type of constraints are needed to resolve all ambiguity. This analysis is generally applicable to matrix models of splice variants. We explore the proposal that probe sequence information may provide sufficient additional constraints to resolve real-world instances. However, probe behavior cannot be predicted with sufficient accuracy by any existing probe sequence model, and so we present a Bayesian framework for estimating variant abundances by incorporating the prediction uncertainty from the micro-model of probe responsiveness into the macro-model of probe intensities. Conclusion The matrix analysis of constraints provides a tool for detecting real-world instances in which additional constraints may be necessary to resolve splice variants. While purely mathematical constraints can be stated without error, real-world constraints may themselves be poorly resolved. Our Bayesian framework provides a generic solution to the problem of uniquely estimating transcript abundances given additional constraints that themselves may be uncertain, such as regression fit to probe sequence models. We demonstrate the efficacy of it by extensive simulations as well as various biological data.

  12. Stochastic principles governing alternative splicing of RNA

    OpenAIRE

    Jianfei Hu; Eli Boritz; William Wylie; Douek, Daniel C.

    2017-01-01

    Author summary Alternative RNA splicing within eukaryotic cells enables each gene to generate multiple different mature transcripts which further encode proteins with distinct or even opposing functions. The relative frequencies of the transcript isoforms generated by a particular gene are essential to the maintenance of normal cellular physiology; however, the underlying mechanisms and principles that govern these frequencies are unknown. We analyzed the frequency distribution of all transcr...

  13. Track based Alignment of the Inner Detector of ATLAS

    CERN Document Server

    Lacuesta, V; The ATLAS collaboration

    2011-01-01

    ATLAS is a multipurpose experiment that records the LHC collisions. In order to reconstruct trajectories of charged particle, ATLAS is equipped with a tracking system built using different technologies, silicon planar sensors (pixel and microstrips) and drift‐tubes. which is embedded in a 2 T solenoidal field. The ATLAS tracking system requires to determine accurately its almost 700,000 degrees of freedom. The demanded precision for the alignment of the silicon sensors is below 10 micrometers. The implementation of the track based alignment within the ATLAS software framework unifies different alignment approaches and allows the alignment of all tracking subsystems together. Primary vertexing and beam spot constraints have also been implemented, as well as constraints from on the particle momentum as measured by the Muon System. Finally the assembly survey data can be used as constraint to the alignment corrections. As alignment algorithms are based on minimization of the track‐hit residuals, one needs to...

  14. Evidence that C9ORF72 Dipeptide Repeat Proteins Associate with U2 snRNP to Cause Mis-splicing in ALS/FTD Patients

    Directory of Open Access Journals (Sweden)

    Shanye Yin

    2017-06-01

    Full Text Available Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR proteins that may disrupt pre-mRNA splicing in amyotrophic lateral sclerosis (ALS and frontotemporal dementia (FTD patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR and glycine-arginine (GR toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing. Proteomic and biochemical analyses identified the U2 small nuclear ribonucleoprotein particle (snRNP as a major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC-derived motor neurons and in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as ∼44% of the mis-spliced cassette exons in C9ORF72 patient brains.

  15. ASF/SF2-like maize pre-mRNA splicing factors affect splice site utilization and their transcripts are alternatively spliced.

    Science.gov (United States)

    Gao, Huirong; Gordon-Kamm, William J; Lyznik, L Alexander

    2004-09-15

    Three ASF/SF2-like alternative splicing genes from maize were identified, cloned, and analyzed. Each of these genes (zmSRp30, zmSRp31, and zmSRp32) contains two RNA binding domains, a signature sequence SWQDLKD, and a characteristic serine/ariginine-rich domain. There is a strong structural similarity to the human ASF/SF2 splicing factor and to the Arabidopsis atSRp34/p30 proteins. Similar to ASF/SF2-like genes in other organisms, the maize pre-mRNA messages are alternatively spliced. They are differentially expressed in maize tissues with relatively uniform levels of zmSRp30 and zmSRp31 messages being observed throughout the plant, while zmSRp32 messages preferentially accumulated in the meristematic regions. Overexpression of zmSRp32 in maize cells leads to the enhanced selection of weak 5' intron splice sites during the processing of pre-mRNA molecules. Overexpression of the zmSRp31 or zmSRp32 gene affects regulation of wheat dwarf virus rep gene pre-mRNA splicing, presumably by interacting with the weak 5' splice site, CCGU. Our results suggest that the described genes are functional homologues of the human ASF/SF2 alternative splicing factor and they indicate a diversity of the ASF/SF2-like alternative splicing factors in monocot plant cells.

  16. RF/Optical Demonstration: Focal Plane Assembly

    Science.gov (United States)

    Hoppe, D. J.; Chung, S.; Kovalik, J.; Gama, E.; Fernandez, M. M.

    2016-11-01

    In this article, we describe the second-generation focal plane optical assembly employed in the RF/optical demonstration at DSS-13. This assembly receives reflected light from the two mirror segments mounted on the RF primary. The focal plane assembly contains a fast steering mirror (FSM) to stabilize the focal plane spot, a pupil camera to aid in aligning the two segments, and several additional cameras for receiving the optical signal prior to as well as after the FSM loop.

  17. MUON DETECTORS: ALIGNMENT

    CERN Multimedia

    G.Gomez

    2011-01-01

    The Muon Alignment work now focuses on producing a new track-based alignment with higher track statistics, making systematic studies between the results of the hardware and track-based alignment methods and aligning the barrel using standalone muon tracks. Currently, the muon track reconstruction software uses a hardware-based alignment in the barrel (DT) and a track-based alignment in the endcaps (CSC). An important task is to assess the muon momentum resolution that can be achieved using the current muon alignment, especially for highly energetic muons. For this purpose, cosmic ray muons are used, since the rate of high-energy muons from collisions is very low and the event statistics are still limited. Cosmics have the advantage of higher statistics in the pT region above 100 GeV/c, but they have the disadvantage of having a mostly vertical topology, resulting in a very few global endcap muons. Only the barrel alignment has therefore been tested so far. Cosmic muons traversing CMS from top to bottom are s...

  18. MUON DETECTORS: ALIGNMENT

    CERN Multimedia

    Gervasio Gomez

    The main progress of the muon alignment group since March has been in the refinement of both the track-based alignment for the DTs and the hardware-based alignment for the CSCs. For DT track-based alignment, there has been significant improvement in the internal alignment of the superlayers inside the DTs. In particular, the distance between superlayers is now corrected, eliminating the residual dependence on track impact angles, and good agreement is found between survey and track-based corrections. The new internal geometry has been approved to be included in the forthcoming reprocessing of CRAFT samples. The alignment of DTs with respect to the tracker using global tracks has also improved significantly, since the algorithms use the latest B-field mapping, better run selection criteria, optimized momentum cuts, and an alignment is now obtained for all six degrees of freedom (three spatial coordinates and three rotations) of the aligned DTs. This work is ongoing and at a stage where we are trying to unders...

  19. MUON DETECTORS: ALIGNMENT

    CERN Multimedia

    G. Gomez

    Since December, the muon alignment community has focused on analyzing the data recorded so far in order to produce new DT and CSC Alignment Records for the second reprocessing of CRAFT data. Two independent algorithms were developed which align the DT chambers using global tracks, thus providing, for the first time, a relative alignment of the barrel with respect to the tracker. These results are an important ingredient for the second CRAFT reprocessing and allow, for example, a more detailed study of any possible mis-modelling of the magnetic field in the muon spectrometer. Both algorithms are constructed in such a way that the resulting alignment constants are not affected, to first order, by any such mis-modelling. The CSC chambers have not yet been included in this global track-based alignment due to a lack of statistics, since only a few cosmics go through the tracker and the CSCs. A strategy exists to align the CSCs using the barrel as a reference until collision tracks become available. Aligning the ...

  20. Evolution of alternative splicing regulation: changes in predicted exonic splicing regulators are not associated with changes in alternative splicing levels in primates

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob Lewin; Roy, Scott William

    2009-01-01

    of interspecific differences in these elements on the evolution of alternative splicing levels has not yet been investigated at genomic level. Here we study the effect of interspecific differences in predicted exonic splicing regulators (ESRs) on exon inclusion levels in human and chimpanzee. For this purpose, we...... compiled and studied comprehensive datasets of predicted ESRs, identified by several computational and experimental approaches, as well as microarray data for changes in alternative splicing levels between human and chimpanzee. Surprisingly, we found no association between changes in predicted ESRs...... and changes in alternative splicing levels. This observation holds across different ESR exon positions, exon lengths, and 5' splice site strengths. We suggest that this lack of association is mainly due to the great importance of context for ESR functionality: many ESR-like motifs in primates may have little...

  1. Accumulation of GC donor splice signals in mammals

    Directory of Open Access Journals (Sweden)

    Koonin Eugene V

    2008-07-01

    Full Text Available Abstract The GT dinucleotide in the first two intron positions is the most conserved element of the U2 donor splice signals. However, in a small fraction of donor sites, GT is replaced by GC. A substantial enrichment of GC in donor sites of alternatively spliced genes has been observed previously in human, nematode and Arabidopsis, suggesting that GC signals are important for regulation of alternative splicing. We used parsimony analysis to reconstruct evolution of donor splice sites and inferred 298 GT > GC conversion events compared to 40 GC > GT conversion events in primate and rodent genomes. Thus, there was substantive accumulation of GC donor splice sites during the evolution of mammals. Accumulation of GC sites might have been driven by selection for alternative splicing. Reviewers This article was reviewed by Jerzy Jurka and Anton Nekrutenko. For the full reviews, please go to the Reviewers' Reports section.

  2. Test and Analysis of Spliced DI-BSCCO HTS Tapes

    Science.gov (United States)

    Fetisov, S. S.; Sotnikov, D. V.; Radchenko, I. P.; Vysotsky, V. S.; Osabe, G.; Kinoshita, K.; Fujikami, J.; Kobayashi, S.; Yamazaki, K.

    For some applications, short unit lengths of HTS wires should be spliced if longer lengths are necessary and short unit lengths of HTS wires should be utilize by applying the splice technology to reduce the total wire cost in the application. The splice technology has been developed for DI-BSCCO Type HT-CA tapes by Sumitomo Electric and spliced tapes were tested in Russian Cable Institute. The test program included: measurements of splice's resistance, critical current anisotropy, thermo cycling tolerance, mechanical properties, overload tests and magnetization measurements. In the paper the results of tests are presented and discussed. The test results demonstrated that splices can be used for cable production if twisting and bending limitations are taken into account.

  3. Splicing modulation therapy in the treatment of genetic diseases

    Directory of Open Access Journals (Sweden)

    Arechavala-Gomeza V

    2014-12-01

    Full Text Available Virginia Arechavala-Gomeza,1 Bernard Khoo,2 Annemieke Aartsma-Rus3 1Neuromuscular Disorders Group, BioCruces Health Research Institute, Barakaldo, Bizkaia, Spain; 2Endocrinology, Division of Medicine, University College London, London, UK; 3Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands All authors contributed equally to this manuscript Abstract: Antisense-mediated splicing modulation is a tool that can be exploited in several ways to provide a potential therapy for rare genetic diseases. This approach is currently being tested in clinical trials for Duchenne muscular dystrophy and spinal muscular atrophy. The present review outlines the versatility of the approach to correct cryptic splicing, modulate alternative splicing, restore the open reading frame, and induce protein knockdown, providing examples of each. Finally, we outline a possible path forward toward the clinical application of this approach for a wide variety of inherited rare diseases. Keywords: splicing, therapy, antisense oligonucleotides, cryptic splicing, alternative splicing

  4. The emerging role of alternative splicing in senescence and aging.

    Science.gov (United States)

    Deschênes, Mathieu; Chabot, Benoit

    2017-10-01

    Deregulation of precursor mRNA splicing is associated with many illnesses and has been linked to age-related chronic diseases. Here we review recent progress documenting how defects in the machinery that performs intron removal and controls splice site selection contribute to cellular senescence and organismal aging. We discuss the functional association linking p53, IGF-1, SIRT1, and ING-1 splice variants with senescence and aging, and review a selection of splicing defects occurring in accelerated aging (progeria), vascular aging, and Alzheimer's disease. Overall, it is becoming increasingly clear that changes in the activity of splicing factors and in the production of key splice variants can impact cellular senescence and the aging phenotype. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  5. Splicing factor SUP-12 and the molecular complexity of apparent cooperativity.

    Science.gov (United States)

    Mackereth, Cameron D

    2014-01-01

    The splicing factor SUP-12 from C. elegans, in combination with either ASD-1 or FOX-1 from the Fox-1 (RBFOX) family, is required for generating a muscle-specific isoform of the fibroblast growth factor receptor EGL-15. Biophysical techniques have revealed the sequence preference for the RNA Recognition Motif (RRM) domain from SUP-12 as well as the structural details of the RNA-bound complex. Detailed genetics have identified a requisite need for the presence of both SUP-12 and ASD-1/FOX-1 to regulate the alternative splicing event, prompting speculation of a cooperative mechanism between these proteins on binding RNA. In contrast, the interplay between SUP-12 and ASD-1 suggests that although the RRM domains from each protein are in direct contact on the egl-15 pre-mRNA, there is no simple contribution of binding cooperativity. Evidence for an independent binding mechanism by SUP-12 and ASD-1 will be discussed, including a model in which both positive and negative contributions are balanced during complex assembly. The ability to monitor tissue-specific alternative splicing in live nematodes will continue to provide a powerful method to test in vivo mechanistic models derived from atomic-level investigation.

  6. MUON DETECTORS: ALIGNMENT

    CERN Multimedia

    G. Gomez

    2011-01-01

    A new set of muon alignment constants was approved in August. The relative position between muon chambers is essentially unchanged, indicating good detector stability. The main changes concern the global positioning of the barrel and of the endcap rings to match the new Tracker geometry. Detailed studies of the differences between track-based and optical alignment of DTs have proven to be a valuable tool for constraining Tracker alignment weak modes, and this information is now being used as part of the alignment procedure. In addition to the “split-cosmic” analysis used to investigate the muon momentum resolution at high momentum, a new procedure based on reconstructing the invariant mass of di-muons from boosted Zs is under development. Both procedures show an improvement in the momentum precision of Global Muons with respect to Tracker-only Muons. Recent developments in track-based alignment include a better treatment of the tails of residual distributions and accounting for correla...

  7. Splicing Express: a software suite for alternative splicing analysis using next-generation sequencing data

    Directory of Open Access Journals (Sweden)

    Jose E. Kroll

    2015-11-01

    Full Text Available Motivation. Alternative splicing events (ASEs are prevalent in the transcriptome of eukaryotic species and are known to influence many biological phenomena. The identification and quantification of these events are crucial for a better understanding of biological processes. Next-generation DNA sequencing technologies have allowed deep characterization of transcriptomes and made it possible to address these issues. ASEs analysis, however, represents a challenging task especially when many different samples need to be compared. Some popular tools for the analysis of ASEs are known to report thousands of events without annotations and/or graphical representations. A new tool for the identification and visualization of ASEs is here described, which can be used by biologists without a solid bioinformatics background.Results. A software suite named Splicing Express was created to perform ASEs analysis from transcriptome sequencing data derived from next-generation DNA sequencing platforms. Its major goal is to serve the needs of biomedical researchers who do not have bioinformatics skills. Splicing Express performs automatic annotation of transcriptome data (GTF files using gene coordinates available from the UCSC genome browser and allows the analysis of data from all available species. The identification of ASEs is done by a known algorithm previously implemented in another tool named Splooce. As a final result, Splicing Express creates a set of HTML files composed of graphics and tables designed to describe the expression profile of ASEs among all analyzed samples. By using RNA-Seq data from the Illumina Human Body Map and the Rat Body Map, we show that Splicing Express is able to perform all tasks in a straightforward way, identifying well-known specific events.Availability and Implementation.Splicing Express is written in Perl and is suitable to run only in UNIX-like systems. More details can be found at: http://www.bioinformatics-brazil.org/splicingexpress.

  8. Identification of common genetic variation that modulates alternative splicing.

    Directory of Open Access Journals (Sweden)

    Jeremy Hull

    2007-06-01

    Full Text Available Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs. In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron-exon boundary, although the distance between these SNPs and the intron-exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.

  9. Genomic and Transcriptomic Analysis Reveals Spliced Leader Trans-Splicing in Cryptomonads

    OpenAIRE

    Roy, Scott William

    2017-01-01

    Spliced leader trans-splicing (SLTS) is a poorly understood mechanism that is found in a diversity of eukaryotic lineages. In SLTS, a short RNA sequence is added near the 5′ ends of the transcripts of protein-coding genes by a modified spliceosomal reaction. Available data suggest that SLTS has evolved many times, and might be more likely to evolve in animals. That SLTS might be more likely to evolve in the context of the generally complex transcriptomes characteristic of animals suggests the...

  10. Sequence assembly

    DEFF Research Database (Denmark)

    Scheibye-Alsing, Karsten; Hoffmann, S.; Frankel, Annett Maria

    2009-01-01

    Despite the rapidly increasing number of sequenced and re-sequenced genomes, many issues regarding the computational assembly of large-scale sequencing data have remain unresolved. Computational assembly is crucial in large genome projects as well for the evolving high-throughput technologies...... and plays an important role in processing the information generated by these methods. Here, we provide a comprehensive overview of the current publicly available sequence assembly programs. We describe the basic principles of computational assembly along with the main concerns, such as repetitive sequences...... in genomic DNA, highly expressed genes and alternative transcripts in EST sequences. We summarize existing comparisons of different assemblers and provide a detailed descriptions and directions for download of assembly programs at: http://genome.ku.dk/resources/assembly/methods.html....

  11. A global analysis of C. elegans trans-splicing

    Science.gov (United States)

    Allen, Mary Ann; Hillier, LaDeana W.; Waterston, Robert H.; Blumenthal, Thomas

    2011-01-01

    Trans-splicing of one of two short leader RNAs, SL1 or SL2, occurs at the 5′ ends of pre-mRNAs of many C. elegans genes. We have exploited RNA-sequencing data from the modENCODE project to analyze the transcriptome of C. elegans for patterns of trans-splicing. Transcripts of ∼70% of genes are trans-spliced, similar to earlier estimates based on analysis of far fewer genes. The mRNAs of most trans-spliced genes are spliced to either SL1 or SL2, but most genes are not trans-spliced to both, indicating that SL1 and SL2 trans-splicing use different underlying mechanisms. SL2 trans-splicing occurs in order to separate the products of genes in operons genome wide. Shorter intercistronic distance is associated with greater use of SL2. Finally, increased use of SL1 trans-splicing to downstream operon genes can indicate the presence of an extra promoter in the intercistronic region, creating what has been termed a “hybrid” operon. Within hybrid operons the presence of the two promoters results in the use of the two SL classes: Transcription that originates at the promoter upstream of another gene creates a polycistronic pre-mRNA that receives SL2, whereas transcription that originates at the internal promoter creates transcripts that receive SL1. Overall, our data demonstrate that >17% of all C. elegans genes are in operons. PMID:21177958

  12. Regulation of alternative splicing in human obesity loci.

    Science.gov (United States)

    Kaminska, Dorota; Käkelä, Pirjo; Nikkola, Elina; Venesmaa, Sari; Ilves, Imre; Herzig, Karl-Heinz; Kolehmainen, Marjukka; Karhunen, Leila; Kuusisto, Johanna; Gylling, Helena; Pajukanta, Päivi; Laakso, Markku; Pihlajamäki, Jussi

    2016-10-01

    Multiple obesity susceptibility loci have been identified by genome-wide association studies, yet the mechanisms by which these loci influence obesity remain unclear. Alternative splicing could contribute to obesity by regulating the transcriptomic and proteomic diversity of genes in these loci. Based on a database search, 72 of the 136 genes at the 13 obesity loci encoded multiple protein isoforms. Thus, alternative splicing of these genes in adipose tissue samples was analyzed from the Metabolic Syndrome in Men population-based study and from two weight loss intervention studies (surgical and very low calorie diet). Alternative splicing was confirmed in 11 genes with PCR capillary electrophoresis in human subcutaneous adipose tissue. Interestingly, differential splicing of TRA2B, BAG6, and MSH5 was observed between lean individuals with normoglycemia and overweight individuals with type 2 diabetes. Of these genes, we detected fat depot-dependent splicing of TRA2B and BAG6 and weight loss-induced regulation of MSH5 splicing in the intervention studies. Finally, body mass index was a major determinant of TRA2B, BAG6, and MSH5 splicing in the combined data. This study provides evidence for alternative splicing in obesity loci, suggesting that alternative splicing at least in part mediates the obesity-associated risk in these loci. © 2016 The Obesity Society.

  13. Splice Site Mutations in the ATP7A Gene

    DEFF Research Database (Denmark)

    Skjørringe, Tina; Tümer, Zeynep; Møller, Lisbeth Birk

    2011-01-01

    Menkes disease (MD) is caused by mutations in the ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12...... mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation...

  14. Characterizing HIV-1 Splicing by Using Next-Generation Sequencing.

    Science.gov (United States)

    Emery, Ann; Zhou, Shuntai; Pollom, Elizabeth; Swanstrom, Ronald

    2017-03-15

    Full-length human immunodeficiency virus type 1 (HIV-1) RNA serves as the genome or as an mRNA, or this RNA undergoes splicing using four donors and 10 acceptors to create over 50 physiologically relevant transcripts in two size classes (1.8 kb and 4 kb). We developed an assay using Primer ID-tagged deep sequencing to quantify HIV-1 splicing. Using the lab strain NL4-3, we found that A5 (env/nef) is the most commonly used acceptor (about 50%) and A3 (tat) the least used (about 3%). Two small exons are made when a splice to acceptor A1 or A2 is followed by activation of donor D2 or D3, and the high-level use of D2 and D3 dramatically reduces the amount of vif and vpr transcripts. We observed distinct patterns of temperature sensitivity of splicing to acceptors A1 and A2. In addition, disruption of a conserved structure proximal to A1 caused a 10-fold reduction in all transcripts that utilized A1. Analysis of a panel of subtype B transmitted/founder viruses showed that splicing patterns are conserved, but with surprising variability of usage. A subtype C isolate was similar, while a simian immunodeficiency virus (SIV) isolate showed significant differences. We also observed transsplicing from a downstream donor on one transcript to an upstream acceptor on a different transcript, which we detected in 0.3% of 1.8-kb RNA reads. There were several examples of splicing suppression when the env intron was retained in the 4-kb size class. These results demonstrate the utility of this assay and identify new examples of HIV-1 splicing regulation. IMPORTANCE During HIV-1 replication, over 50 conserved spliced RNA variants are generated. The splicing assay described here uses new developments in deep-sequencing technology combined with Primer ID-tagged cDNA primers to efficiently quantify HIV-1 splicing at a depth that allows even low-frequency splice variants to be monitored. We have used this assay to examine several features of HIV-1 splicing and to identify new examples of

  15. Adenosine to Inosine editing frequency controlled by splicing efficiency.

    Science.gov (United States)

    Licht, Konstantin; Kapoor, Utkarsh; Mayrhofer, Elisa; Jantsch, Michael F

    2016-07-27

    Alternative splicing and adenosine to inosine (A to I) RNA-editing are major factors leading to co- and post-transcriptional modification of genetic information. Both, A to I editing and splicing occur in the nucleus. As editing sites are frequently defined by exon-intron basepairing, mRNA splicing efficiency should affect editing levels. Moreover, splicing rates affect nuclear retention and will therefore also influence the exposure of pre-mRNAs to the editing-competent nuclear environment. Here, we systematically test the influence of splice rates on RNA-editing using reporter genes but also endogenous substrates. We demonstrate for the first time that the extent of editing is controlled by splicing kinetics when editing is guided by intronic elements. In contrast, editing sites that are exclusively defined by exonic structures are almost unaffected by the splicing efficiency of nearby introns. In addition, we show that editing levels in pre- and mature mRNAs do not match. This phenomenon can in part be explained by the editing state of an RNA influencing its splicing rate but also by the binding of the editing enzyme ADAR that interferes with splicing. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Hybrid vehicle motor alignment

    Science.gov (United States)

    Levin, Michael Benjamin

    2001-07-03

    A rotor of an electric motor for a motor vehicle is aligned to an axis of rotation for a crankshaft of an internal combustion engine having an internal combustion engine and an electric motor. A locator is provided on the crankshaft, a piloting tool is located radially by the first locator to the crankshaft. A stator of the electric motor is aligned to a second locator provided on the piloting tool. The stator is secured to the engine block. The rotor is aligned to the crankshaft and secured thereto.

  17. Discriminative Shape Alignment

    DEFF Research Database (Denmark)

    Loog, M.; de Bruijne, M.

    2009-01-01

    The alignment of shape data to a common mean before its subsequent processing is an ubiquitous step within the area shape analysis. Current approaches to shape analysis or, as more specifically considered in this work, shape classification perform the alignment in a fully unsupervised way......, not taking into account that eventually the shapes are to be assigned to two or more different classes. This work introduces a discriminative variation to well-known Procrustes alignment and demonstrates its benefit over this classical method in shape classification tasks. The focus is on two...

  18. Belt Aligning Revisited

    Directory of Open Access Journals (Sweden)

    Yurchenko Vadim

    2017-01-01

    parts of the conveyor, the sides of the belt wear intensively. This results in reducing the life of the belt. The reasons for this phenomenon are well investigated, but the difficulty lies in the fact that they all act simultaneously. The belt misalignment prevention can be carried out in two ways: by minimizing the effect of causes and by aligning the belt. The construction of aligning devices and errors encountered in practice are considered in this paper. Self-aligning roller supports rotational in plan view are recommended as a means of combating the belt misalignment.

  19. Methods in ALFA Alignment

    CERN Document Server

    Melendez, Jordan

    2014-01-01

    This note presents two model-independent methods for use in the alignment of the ALFA forward detectors. Using a Monte Carlo simulated LHC run at \\beta = 90m and \\sqrt{s} = 7 TeV, the Kinematic Peak alignment method is utilized to reconstruct the Mandelstam momentum transfer variable t for single-diractive protons. The Hot Spot method uses fluctuations in the hitmap density to pinpoint particular regions in the detector that could signal a misalignment. Another method uses an error function fit to find the detector edge. With this information, the vertical alignment can be determined.

  20. Pre-mRNA Splicing in Plants: In Vivo Functions of RNA-Binding Proteins Implicated in the Splicing Process

    Directory of Open Access Journals (Sweden)

    Katja Meyer

    2015-07-01

    Full Text Available Alternative pre-messenger RNA splicing in higher plants emerges as an important layer of regulation upon exposure to exogenous and endogenous cues. Accordingly, mutants defective in RNA-binding proteins predicted to function in the splicing process show severe phenotypic alterations. Among those are developmental defects, impaired responses to pathogen threat or abiotic stress factors, and misregulation of the circadian timing system. A suite of splicing factors has been identified in the model plant Arabidopsis thaliana. Here we summarize recent insights on how defects in these splicing factors impair plant performance.

  1. Alternate trans splicing in Trypanosoma equiperdum: implications for splice site selection.

    Science.gov (United States)

    Layden, R E; Eisen, H

    1988-03-01

    We examined the structures of the 5' ends of mRNAs encoding variant surface glycoprotein 78 (VSG-78) and VSG-1(78) in Trypanosoma equiperdum. Several mRNA species were found for each gene, and all contained the 35-base miniexon (or spliced leader) sequence attached at different positions on their 5' ends. Thus, the generation of multiple messages for each VSG occurred by attachment of the miniexon at one of several 3' splice acceptor sites. The frequency with which individual splice sites were used varied from less than 1 to 95% of the RNA produced from a particular gene. We propose that the miniexon RNA and RNA from the VSG genes may interact via base pairing and that this in part specifies the use of particular acceptor sites. Sequences complementary to the miniexon primary transcript, termed the "med-comp site," were found in both genes and in several published sequences. Splice sites were most often used if they were the first site 3' of the med-comp site and contained a high pyrimidine content in the bases preceding the AG acceptor signal.

  2. Trans-splicing and operons in C. elegans.

    Science.gov (United States)

    Blumenthal, Thomas

    2012-11-20

    About 70% of C. elegans mRNAs are trans-spliced to one of two 22 nucleotide spliced leaders. SL1 is used to trim off the 5' ends of pre-mRNAs and replace them with the SL1 sequence. This processing event is very closely related to cis-splicing, or intron removal. The SL1 sequence is donated by a 100 nt small nuclear ribonucleoprotein particle (snRNP), the SL1 snRNP. This snRNP is structurally and functionally similar to the U snRNAs (U1, U2, U4, U5 and U6) that play key roles in intron removal and trans-splicing, except that the SL1 snRNP is consumed in the process. More than half of C. elegans pre-mRNAs are subject to SL1 trans-splicing, whereas ~30% are not trans-spliced. The remaining genes are trans-spliced by SL2, which is donated by a similar snRNP, the SL2 snRNP. SL2 recipients are all downstream genes in closely spaced gene clusters similar to bacterial operons. They are transcribed from a promoter at the 5' end of the cluster of between 2 and 8 genes. This transcription makes a polycistronic pre-mRNA that is co-transcriptionally processed by cleavage and polyadenylation at the 3' end of each gene, and this event is closely coupled to the SL2 trans-splicing event that occurs only ~100 nt further downstream. SL2 trans-splicing requires a sequence between the genes, the Ur element, that likely base pairs with the 5' splice site on the SL2 snRNP, in a manner analogous to the interaction between the 5' splice site in cis-splicing with the U1 snRNP. The key difference is that in trans-splicing, the snRNP contains the 5' splice site, whereas in cis-splicing the pre-mRNA does. Some operons, termed "hybrid operons", contain an additional promoter between two genes that can express the downstream gene or genes with a developmental profile that is different from that of the entire operon. The operons contain primarily genes required for rapid growth, including genes whose products are needed for mitochondrial function and the basic machinery of gene expression

  3. Small-molecule inhibition of HIV pre-mRNA splicing as a novel antiretroviral therapy to overcome drug resistance.

    Directory of Open Access Journals (Sweden)

    Nadia Bakkour

    2007-10-01

    Full Text Available The development of multidrug-resistant viruses compromises antiretroviral therapy efficacy and limits therapeutic options. Therefore, it is an ongoing task to identify new targets for antiretroviral therapy and to develop new drugs. Here, we show that an indole derivative (IDC16 that interferes with exonic splicing enhancer activity of the SR protein splicing factor SF2/ASF suppresses the production of key viral proteins, thereby compromising subsequent synthesis of full-length HIV-1 pre-mRNA and assembly of infectious particles. IDC16 inhibits replication of macrophage- and T cell-tropic laboratory strains, clinical isolates, and strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Importantly, drug treatment of primary blood cells did not alter splicing profiles of endogenous genes involved in cell cycle transition and apoptosis. Thus, human splicing factors represent novel and promising drug targets for the development of antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses.

  4. Spatially confined assembly of nanoparticles.

    Science.gov (United States)

    Jiang, Lin; Chen, Xiaodong; Lu, Nan; Chi, Lifeng

    2014-10-21

    The ability to assemble NPs into ordered structures that are expected to yield collective physical or chemical properties has afforded new and exciting opportunities in the field of nanotechnology. Among the various configurations of nanoparticle assemblies, two-dimensional (2D) NP patterns and one-dimensional (1D) NP arrays on surfaces are regarded as the ideal assembly configurations for many technological devices, for example, solar cells, magnetic memory, switching devices, and sensing devices, due to their unique transport phenomena and the cooperative properties of NPs in assemblies. To realize the potential applications of NP assemblies, especially in nanodevice-related applications, certain key issues must still be resolved, for example, ordering and alignment, manipulating and positioning in nanodevices, and multicomponent or hierarchical structures of NP assemblies for device integration. Additionally, the assembly of NPs with high precision and high levels of integration and uniformity for devices with scaled-down dimensions has become a key and challenging issue. Two-dimensional NP patterns and 1D NP arrays are obtained using traditional lithography techniques (top-down strategies) or interfacial assembly techniques (bottom-up strategies). However, a formidable challenge that persists is the controllable assembly of NPs in desired locations over large areas with high precision and high levels of integration. The difficulty of this assembly is due to the low efficiency of small features over large areas in lithography techniques or the inevitable structural defects that occur during the assembly process. The combination of self-assembly strategies with existing nanofabrication techniques could potentially provide effective and distinctive solutions for fabricating NPs with precise position control and high resolution. Furthermore, the synergistic combination of spatially mediated interactions between nanoparticles and prestructures on surfaces may play

  5. MUON DETECTORS: ALIGNMENT

    CERN Multimedia

    G.Gomez

    Since September, the muon alignment system shifted from a mode of hardware installation and commissioning to operation and data taking. All three optical subsystems (Barrel, Endcap and Link alignment) have recorded data before, during and after CRAFT, at different magnetic fields and during ramps of the magnet. This first data taking experience has several interesting goals: •    study detector deformations and movements under the influence of the huge magnetic forces; •    study the stability of detector structures and of the alignment system over long periods, •    study geometry reproducibility at equal fields (specially at 0T and 3.8T); •    reconstruct B=0T geometry and compare to nominal/survey geometries; •    reconstruct B=3.8T geometry and provide DT and CSC alignment records for CMSSW. However, the main goal is to recons...

  6. Functions for fission yeast splicing factors SpSlu7 and SpPrp18 in alternative splice-site choice and stress-specific regulated splicing.

    Directory of Open Access Journals (Sweden)

    Geetha Melangath

    Full Text Available Budding yeast spliceosomal factors ScSlu7 and ScPrp18 interact and mediate intron 3'ss choice during second step pre-mRNA splicing. The fission yeast genome with abundant multi-intronic transcripts, degenerate splice signals and SR proteins is an apt unicellular fungal model to deduce roles for core spliceosomal factors in alternative splice-site choice, intron retention and to study the cellular implications of regulated splicing. From our custom microarray data we deduce a stringent reproducible subset of S. pombe alternative events. We examined the role of factors SpSlu7 or SpPrp18 for these splice events and investigated the relationship to growth phase and stress. Wild-type log and stationary phase cells showed ats1+ exon 3 skipped and intron 3 retained transcripts. Interestingly the non-consensus 5'ss in ats1+ intron 3 caused SpSlu7 and SpPrp18 dependent intron retention. We validated the use of an alternative 5'ss in dtd1+ intron 1 and of an upstream alternative 3'ss in DUF3074 intron 1. The dtd1+ intron 1 non-canonical 5'ss yielded an alternative mRNA whose levels increased in stationary phase. Utilization of dtd1+ intron 1 sub-optimal 5' ss required functional SpPrp18 and SpSlu7 while compromise in SpSlu7 function alone hampered the selection of the DUF3074 intron 1 non canonical 3'ss. We analysed the relative abundance of these splice isoforms during mild thermal, oxidative and heavy metal stress and found stress-specific splice patterns for ats1+ and DUF3074 intron 1 some of which were SpSlu7 and SpPrp18 dependent. By studying ats1+ splice isoforms during compromised transcription elongation rates in wild-type, spslu7-2 and spprp18-5 mutant cells we found dynamic and intron context-specific effects in splice-site choice. Our work thus shows the combinatorial effects of splice site strength, core splicing factor functions and transcription elongation kinetics to dictate alternative splice patterns which in turn serve as an additional

  7. The deterministic optical alignment of the HERMES spectrograph

    Science.gov (United States)

    Gers, Luke; Staszak, Nicholas

    2014-07-01

    The High Efficiency and Resolution Multi Element Spectrograph (HERMES) is a four channel, VPH-grating spectrograph fed by two 400 fiber slit assemblies whose construction and commissioning has now been completed at the Anglo Australian Telescope (AAT). The size, weight, complexity, and scheduling constraints of the system necessitated that a fully integrated, deterministic, opto-mechanical alignment system be designed into the spectrograph before it was manufactured. This paper presents the principles about which the system was assembled and aligned, including the equipment and the metrology methods employed to complete the spectrograph integration.

  8. MUON DETECTORS: ALIGNMENT

    CERN Multimedia

    G. Gomez

    2012-01-01

      A new muon alignment has been produced for 2012 A+B data reconstruction. It uses the latest Tracker alignment and single-muon data samples to align both DTs and CSCs. Physics validation has been performed and shows a modest improvement in stand-alone muon momentum resolution in the barrel, where the alignment is essentially unchanged from the previous version. The reference-target track-based algorithm using only collision muons is employed for the first time to align the CSCs, and a substantial improvement in resolution is observed in the endcap and overlap regions for stand-alone muons. This new alignment is undergoing the approval process and is expected to be deployed as part of a new global tag in the beginning of December. The pT dependence of the φ-bias in curvature observed in Monte Carlo was traced to a relative vertical misalignment between the Tracker and barrel muon systems. Moving the barrel as a whole to match the Tracker cures this pT dependence, leaving only the &phi...

  9. Approaches to link RNA secondary structures with splicing regulation

    DEFF Research Database (Denmark)

    Plass, Mireya; Eyras, Eduardo

    2014-01-01

    In higher eukaryotes, alternative splicing is usually regulated by protein factors, which bind to the pre-mRNA and affect the recognition of splicing signals. There is recent evidence that the secondary structure of the pre-mRNA may also play an important role in this process, either by facilitat...

  10. Revealing the Determinants of Widespread Alternative Splicing Perturbation in Cancer

    Directory of Open Access Journals (Sweden)

    Yongsheng Li

    2017-10-01

    Full Text Available It is increasingly appreciated that alternative splicing plays a key role in generating functional specificity and diversity in cancer. However, the mechanisms by which cancer mutations perturb splicing remain unknown. Here, we developed a network-based strategy, DrAS-Net, to investigate more than 2.5 million variants across cancer types and link somatic mutations with cancer-specific splicing events. We identified more than 40,000 driver variant candidates and their 80,000 putative splicing targets deregulated in 33 cancer types and inferred their functional impact. Strikingly, tumors with splicing perturbations show reduced expression of immune system-related genes and increased expression of cell proliferation markers. Tumors harboring different mutations in the same gene often exhibit distinct splicing perturbations. Further stratification of 10,000 patients based on their mutation-splicing relationships identifies subtypes with distinct clinical features, including survival rates. Our work reveals how single-nucleotide changes can alter the repertoires of splicing isoforms, providing insights into oncogenic mechanisms for precision medicine.

  11. Alternative Splicing of STAT3 Is Affected by RNA Editing.

    Science.gov (United States)

    Goldberg, Lior; Abutbul-Amitai, Mor; Paret, Gideon; Nevo-Caspi, Yael

    2017-05-01

    A-to-I RNA editing, carried out by adenosine deaminase acting on RNA (ADAR) enzymes, is an epigenetic phenomenon of posttranscriptional modifications on pre-mRNA. RNA editing in intronic sequences may influence alternative splicing of flanking exons. We have previously shown that conditions that induce editing result in elevated expression of signal transducer and activator of transcription 3 (STAT3), preferentially the alternatively-spliced STAT3β isoform. Mechanisms regulating alternative splicing of STAT3 have not been elucidated. STAT3 undergoes A-to-I RNA editing in an intron residing in proximity to the alternatively spliced exon. We hypothesized that RNA editing plays a role in regulating alternative splicing toward STAT3β. In this study we extend our observation connecting RNA editing to the preferential induction of STAT3β expression. We study the involvement of ADAR1 in STAT3 editing and reveal the connection between editing and alternative splicing of STAT3. Deferoaxamine treatment caused the induction in STAT3 RNA editing and STAT3β expression. Silencing ADAR1 caused a decrease in STAT3 editing and expression with a preferential decrease in STAT3β. Cells transfected with a mutated minigene showed preferential splicing toward the STAT3β transcript. Editing in the STAT3 intron is performed by ADAR1 and affects STAT3 alternative splicing. These results suggest that RNA editing is one of the molecular mechanisms regulating the expression of STAT3β.

  12. 30 CFR 18.43 - Explosion-proof splice boxes.

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Explosion-proof splice boxes. 18.43 Section 18.43 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR TESTING, EVALUATION... Design Requirements § 18.43 Explosion-proof splice boxes. Internal connections shall be rigidly held and...

  13. Studying the composition of mRNPs in vitro using splicing-competent cell extracts.

    Science.gov (United States)

    Steckelberg, Anna-Lena; Gehring, Niels H

    2014-02-01

    The correct processing and faithful decoding of mRNAs during gene expression depends on the interaction with RNA-binding proteins (RBPs). The association of RBPs with pre-mRNAs starts during transcription by RNA polymerase II and undergoes constant remodeling during pre-mRNA processing and later steps of genes expression. Recently developed high throughput methods enabled to define RBP binding sites in vivo and to identify a large number of novel RBPs in eukaryotic cells. However, the detailed characterization of RBP-RNA interactions as well as the analysis of functional RNPs is greatly facilitated by well-defined in vitro systems. Here, we describe a versatile method to study the assembly and splicing-dependent remodeling of mRNPs in vitro. This method employs splicing-competent whole cell extracts (WCE) generated from transfected human embryonic kidney (HEK) 293 cells. FLAG-tagged proteins present in the WCE are incorporated into mRNPs in vitro and afterwards used to immunoprecipitate substrate RNAs. We outline the principles of purifying in vitro assembled mRNPs and provide detailed protocols for the preparation and use of whole cell extracts. Alternative purification strategies and RNA substrates are discussed. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Study and Development of a Laser Based Alignment System

    CERN Multimedia

    Stern, G

    2014-01-01

    CLIC (Compact Linear Collider) has tight requirements regarding pre-alignment of beam related components: 10 µm accuracy over a sliding window of 200 m along the 20 km of linac. To perform such an alignment, a new system is proposed combining laser beam as straight line reference and camera/shutter assemblies as sensors. The poster describes the alignment system and shows results regarding laser pointing stability with respect to time, shutter type, distance and environment. These results give a frame for future building and calibrating of sensors.

  15. Alignment of the ATLAS Inner Detector tracking system

    CERN Document Server

    Wang, J; The ATLAS collaboration

    2010-01-01

    The ATLAS Inner Detector (ID) consists of two silicon subsystems, the Pixel detector and the Semiconductor Tracker (SCT), complemented by the Transition Radiation Tracker (TRT) composed of drift tubes. After the assembly of the detector, the position of the individual modules is known with much worse accuracy than their intrinsic resolution. Therefore a track-based alignment procedure has to be applied. The baseline goal is to determine the position and orientation of the modules with such precision that the track parameters’ determination is not worsened by more than 20% with respect to that expected from the perfectly aligned detector. This is crucial for efficient track reconstruction and precise momentum measurement and vertex reconstruction. The alignment of the ID requires the determination of its almost 36,000 degrees of freedom(DoF) with high accuracy. Thus the demanded precision for the alignment of the silicon sensors is below 10 micrometer.The implementation of the track based alignment with the ...

  16. Curriculum Alignment Research Suggests that Alignment Can Improve Student Achievement

    Science.gov (United States)

    Squires, David

    2012-01-01

    Curriculum alignment research has developed showing the relationship among three alignment categories: the taught curriculum, the tested curriculum and the written curriculum. Each pair (for example, the taught and the written curriculum) shows a positive impact for aligning those results. Following this, alignment results from the Third…

  17. Some relations between two stages DNA splicing languages

    Science.gov (United States)

    Mudaber, Mohammad Hassan; Yusof, Yuhani; Mohamad, Mohd Sham

    2014-06-01

    A new symbolization of Yusof-Goode (Y-G) rule, which is associated with Y-G splicing system, was introduced by Yusof in 2012 under the framework of formal language theory. The purpose of this investigation is to present the biological process of DNA splicing in a translucent way. In this study, two stages splicing languages are introduced based on Y-G approach and some relations between stage one and stage two splicing languages are presented, given as theorems. Additionally, the existing relations between two stages splicing languages based on crossings and contexts of restriction enzymes factors with respect to two initial strings (having two cutting sites) and two rules are presented as subset.

  18. The Electrical Resistance of Rutherford-Type Superconducting Cable Splices

    CERN Document Server

    Heck, C; Fleiter, J; Bottura, L

    2015-01-01

    The electrical resistance of Large Hadron Collider main busbar cable lap splices produced by soft soldering has been measured with two independent methods as a function of intercable contact area and for splices made of cables with various defects. For defect-free lap splices, the resistance increases from 0.3 to 10 nΩ (at 4.3 K in self-field) when reducing the cable overlap length from 120 to 3 mm, as expected assuming that the resistance is inversely proportional to the intercable contact area. The resistance of bridge splices that connect side-by-side cables can be predicted from the lap splice resistances and the overlap areas involved.

  19. LHC DIPOLE MAGNET SPLICE RESISTANCE FROM SM18 DATA MINING

    CERN Document Server

    Reymond, H; Charrondiere, C; Lehmann Miotto, G; Rijllart, A; Scannicchio, D A

    2011-01-01

    The splice incident which happened during commissioning of the LHC on the 19th of September 2008 caused damage to several magnets and adjacent equipment. This raised not only the question of how it happened, but also about the state of all other splices. The inter magnet splices were immediately studied and new measurements recorded, but the internal magnet splices were still a concern. At the Chamonix meeting in January 2009, the CERN management decided to create a working group to analyse quench data of the magnet acceptance tests in an attempt to find indications for bad splices in the main dipoles. This resulted in a data-mining project that took about one year to complete. This presentation describes how the data was stored, extracted and analysed reusing existing LabVIEW™ based tools. We also present the encountered difficulties and the importance of combining measured data with operator notes in the logbook.

  20. Detecting Image Splicing Using Merged Features in Chroma Space

    Directory of Open Access Journals (Sweden)

    Bo Xu

    2014-01-01

    Full Text Available Image splicing is an image editing method to copy a part of an image and paste it onto another image, and it is commonly followed by postprocessing such as local/global blurring, compression, and resizing. To detect this kind of forgery, the image rich models, a feature set successfully used in the steganalysis is evaluated on the splicing image dataset at first, and the dominant submodel is selected as the first kind of feature. The selected feature and the DCT Markov features are used together to detect splicing forgery in the chroma channel, which is convinced effective in splicing detection. The experimental results indicate that the proposed method can detect splicing forgeries with lower error rate compared to the previous literature.

  1. RNA splicing factors as oncoproteins and tumor suppressors

    Science.gov (United States)

    Dvinge, Heidi; Kim, Eunhee; Abdel-Wahab, Omar; Bradley, Robert K.

    2016-01-01

    Preface The recent genomic characterization of cancers has revealed recurrent somatic point mutations and copy number changes affecting genes encoding RNA splicing factors. Initial studies of these ‘spliceosomal mutations’ suggest that the proteins bearing these mutations exhibit altered splice site and/or exon recognition preferences relative to their wild-type counterparts, resulting in cancer-specific mis-splicing. Such changes in the splicing machinery may create novel vulnerabilities in cancer cells that can be therapeutically exploited using compounds that can influence the splicing process. Further studies to dissect the biochemical, genomic, and biological effects of spliceosomal mutations are critical for the development of cancer therapies targeted to these mutations. PMID:27282250

  2. A TIMP-1 splice variant transcript

    DEFF Research Database (Denmark)

    Øbro, Nina Friesgård; Lademann, Ulrik Axel; Birkenkamp-Demtroder, Karin

    2008-01-01

    A splice variant of tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNA lacking exon 2 (TIMP-1-v2) has been identified in human cancer cells and in colorectal and breast cancer tumors. The purpose of this study was (1) to study the level of full length TIMP-1 and TIMP-1-v2 transcripts in color...... of TIMP-1 pre-mRNA to TIMP-1-v2 mRNA might be involved in regulating TIMP-1 expression.......A splice variant of tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNA lacking exon 2 (TIMP-1-v2) has been identified in human cancer cells and in colorectal and breast cancer tumors. The purpose of this study was (1) to study the level of full length TIMP-1 and TIMP-1-v2 transcripts...... in colorectal tumors; (2) to investigate if TIMP-1-v2 is translated to protein. Full length TIMP-1 and TIMP-1-v2 mRNA levels were compared between colorectal tumors and normal mucosa by Q-PCR. Both full length TIMP-1 and TIMP-1-v2 transcripts were upregulated in tumor tissue. However, the level of TIMP-1-v2...

  3. Stable assembly of HIV-1 export complexes occurs cotranscriptionally

    DEFF Research Database (Denmark)

    Nawroth, Isabel; Mueller, Florian; Basyuk, Eugenia

    2014-01-01

    The HIV-1 Rev protein mediates export of unspliced and singly spliced viral transcripts by binding to the Rev response element (RRE) and recruiting the cellular export factor CRM1. Here, we investigated the recruitment of Rev to the transcription sites of HIV-1 reporters that splice either post......- or cotranscriptionally. In both cases, we observed that Rev localized to the transcription sites of the reporters and recruited CRM1. Rev and CRM1 remained at the reporter transcription sites when cells were treated with the splicing inhibitor Spliceostatin A (SSA), showing that the proteins associate with RNA prior...... to or during early spliceosome assembly. Fluorescence recovery after photobleaching (FRAP) revealed that Rev and CRM1 have similar kinetics as the HIV-1 RNA, indicating that Rev, CRM1, and RRE-containing RNAs are released from the site of transcription in one single export complex. These results suggest...

  4. MaxAlign: maximizing usable data in an alignment

    DEFF Research Database (Denmark)

    Oliveira, Rodrigo Gouveia; Sackett, Peter Wad; Pedersen, Anders Gorm

    2007-01-01

    the alignment prior to such analyses. Specifically, it maximizes the number of nucleotide (or amino acid) symbols that are present in gap-free columns - the alignment area - by selecting the optimal subset of sequences to exclude from the alignment. MaxAlign can be used prior to phylogenetic and bioinformatical...... show that it is not advisable to exclude gapped columns from phylogenetic analyses unless MaxAlign is used first. Finally, we find that the sequences removed by MaxAlign from an alignment tend to be those that would otherwise be associated with low phylogenetic accuracy, and that the presence of gaps...

  5. Muscle assembly: a titanic achievement?

    Science.gov (United States)

    Gregorio, C C; Granzier, H; Sorimachi, H; Labeit, S

    1999-02-01

    The formation of perfectly aligned myofibrils in striated muscle represents a dramatic example of supramolecular assembly in eukaryotic cells. Recently, considerable progress has been made in deciphering the roles that titin, the third most abundant protein in muscle, has in this process. An increasing number of sarcomeric proteins (ligands) are being identified that bind to specific titin domains. Titin may serve as a molecular blueprint for sarcomere assembly and turnover by specifying the precise position of its ligands within each half-sarcomere in addition to functioning as a molecular spring that maintains the structural integrity of the contracting myofibrils.

  6. RNA Structural Alignments, Part I

    DEFF Research Database (Denmark)

    Havgaard, Jakob Hull; Gorodkin, Jan

    2014-01-01

    Simultaneous alignment and secondary structure prediction of RNA sequences is often referred to as "RNA structural alignment." A class of the methods for structural alignment is based on the principles proposed by Sankoff more than 25 years ago. The Sankoff algorithm simultaneously folds and aligns...

  7. MUON DETECTORS: ALIGNMENT

    CERN Multimedia

    G. Gomez

    2010-01-01

    For the last three months, the Muon Alignment group has focussed on providing a new, improved set of alignment constants for the end-of-year data reprocessing. These constants were delivered on time and approved by the CMS physics validation team on November 17. The new alignment incorporates several improvements over the previous one from March for nearly all sub-systems. Motivated by the loss of information from a hardware failure in May (an entire MAB was lost), the optical barrel alignment has moved from a modular, super-plane reconstruction, to a full, single loop calculation of the entire geometry for all DTs in stations 1, 2 and 3. This makes better use of the system redundancy, mitigating the effect of the information loss. Station 4 is factorised and added afterwards to make the system smaller (and therefore faster to run), and also because the MAB calibration at the MB4 zone is less precise. This new alignment procedure was tested at 0 T against photogrammetry resulting in precisions of the order...

  8. MUON DETECTORS: ALIGNMENT

    CERN Multimedia

    Gervasio Gomez

    2012-01-01

      The new alignment for the DT chambers has been successfully used in physics analysis starting with the 52X Global Tag. The remaining main areas of development over the next few months will be preparing a new track-based CSC alignment and producing realistic APEs (alignment position errors) and MC misalignment scenarios to match the latest muon alignment constants. Work on these items has been delayed from the intended timeline, mostly due to a large involvement of the muon alignment man-power in physics analyses over the first half of this year. As CMS keeps probing higher and higher energies, special attention must be paid to the reconstruction of very-high-energy muons. Recent muon POG reports from mid-June show a φ-dependence in curvature bias in Monte Carlo samples. This bias is observed already at the tracker level, where it is constant with muon pT, while it grows with pT as muon chamber information is added to the tracks. Similar studies show a much smaller effect in data, at le...

  9. MUON DETECTORS: ALIGNMENT

    CERN Multimedia

    M. Dallavalle

    2013-01-01

    A new Muon misalignment scenario for 2011 (7 TeV) Monte Carlo re-processing was re-leased. The scenario is based on running of standard track-based reference-target algorithm (exactly as in data) using single-muon simulated sample (with the transverse-momentum spectrum matching data). It used statistics similar to what was used for alignment with 2011 data, starting from an initially misaligned Muon geometry from uncertainties of hardware measurements and using the latest Tracker misalignment geometry. Validation of the scenario (with muons from Z decay and high-pT simulated muons) shows that it describes data well. The study of systematic uncertainties (dominant by now due to huge amount of data collected by CMS and used for muon alignment) is finalised. Realistic alignment position errors are being obtained from the estimated uncertainties and are expected to improve the muon reconstruction performance. Concerning the Hardware Alignment System, the upgrade of the Barrel Alignment is in progress. By now, d...

  10. Deciphering Transcriptome and Complex Alternative Splicing Transcripts in Mammary Gland Tissues from Cows Naturally Infected with Staphylococcus aureus Mastitis

    Science.gov (United States)

    Jiang, Qiang; Yang, Chun Hong; Zhang, Yan; Sun, Yan; Li, Rong Ling; Wang, Chang Fa; Zhong, Ji Feng; Huang, Jin Ming

    2016-01-01

    Alternative splicing (AS) contributes to the complexity of the mammalian proteome and plays an important role in diseases, including infectious diseases. The differential AS patterns of these transcript sequences between the healthy (HS3A) and mastitic (HS8A) cows naturally infected by Staphylococcus aureus were compared to understand the molecular mechanisms underlying mastitis resistance and susceptibility. In this study, using the Illumina paired-end RNA sequencing method, 1352 differentially expressed genes (DEGs) with higher than twofold changes were found in the HS3A and HS8A mammary gland tissues. Gene ontology and KEGG pathway analyses revealed that the cytokine–cytokine receptor interaction pathway is the most significantly enriched pathway. Approximately 16k annotated unigenes were respectively identified in two libraries, based on the bovine Bos taurus UMD3.1 sequence assembly and search. A total of 52.62% and 51.24% annotated unigenes were alternatively spliced in term of exon skipping, intron retention, alternative 5′ splicing and alternative 3ʹ splicing. Additionally, 1,317 AS unigenes were HS3A-specific, whereas 1,093 AS unigenes were HS8A-specific. Some immune-related genes, such as ITGB6, MYD88, ADA, ACKR1, and TNFRSF1B, and their potential relationships with mastitis were highlighted. From Chromosome 2, 4, 6, 7, 10, 13, 14, 17, and 20, 3.66% (HS3A) and 5.4% (HS8A) novel transcripts, which harbor known quantitative trait locus associated with clinical mastitis, were identified. Many DEGs in the healthy and mastitic mammary glands are involved in immune, defense, and inflammation responses. These DEGs, which exhibit diverse and specific splicing patterns and events, can endow dairy cattle with the potential complex genetic resistance against mastitis. PMID:27459697

  11. Orbital assembly and maintenance study

    Science.gov (United States)

    Gorman, D.; Grant, C.; Kyrias, G.; Lord, C.; Rombach, J.; Salis, M.; Skidmore, R.; Thomas, R.

    1975-01-01

    The requirements, conceptual design, tradeoffs, procedures, and techniques for orbital assembly of the support structure of the microwave power transmission system and the radio astronomy telescope are described. Thermal and stress analyses, packaging, alignment, and subsystems requirements are included along with manned vs. automated and transportation tradeoffs. Technical and operational concepts for the manned and automated maintenance of satellites were investigated and further developed results are presented.

  12. Splicing-related single nucleotide polymorphism of RAB, member of RAS oncogene family like 2B (RABL2B) jeopardises semen quality in Chinese Holstein bulls.

    Science.gov (United States)

    Wang, Xiuge; Cui, Xiaohui; Zhang, Yan; Hao, Haisheng; Ju, Zhihua; Liu, Deyu; Jiang, Qiang; Yang, Chunhong; Sun, Yan; Wang, Changfa; Huang, Jinming; Zhu, Huabin

    2017-11-01

    RAB, member of RAS oncogene family like 2B (RABL2B) is a member of a poorly characterised clade of the RAS GTPase superfamily, which plays an essential role in male fertility, sperm intraflagellar transport and tail assembly. In the present study, we identified a novel RABL2B splice variant in bovine testis and spermatozoa. This splice variant, designated RABL2B-TV, is characterised by exon 2 skipping. Moreover, a single nucleotide polymorphism (SNP), namely c.125G>A, was found within the exonic splicing enhancer (ESE) motif, indicating that the SNP caused the production of the RABL2B-TV aberrant splice variant. This was demonstrated by constructing a pSPL3 exon capturing vector with different genotypes and transfecting these vectors into murine Leydig tumour cell line (MLTC-1) cells. Expression of the RABL2B-TV transcript was lower in semen from high- versus low-performance bulls. Association analysis showed that sperm deformity rate was significantly lower in Chinese Holstein bulls with the GG or GA genotype than in bulls with the AA genotype (Pquality in bulls with different RABL2B genotypes is generated via an alternative splicing mechanism caused by a functional SNP within the ESE motif.

  13. Belt Aligning Revisited

    Science.gov (United States)

    Yurchenko, Vadim

    2017-11-01

    The misalignment causes the greatest damage to the conveyor belt. As a result of the interaction of the moving belt with the stationary parts of the conveyor, the sides of the belt wear intensively. This results in reducing the life of the belt. The reasons for this phenomenon are well investigated, but the difficulty lies in the fact that they all act simultaneously. The belt misalignment prevention can be carried out in two ways: by minimizing the effect of causes and by aligning the belt. The construction of aligning devices and errors encountered in practice are considered in this paper. Self-aligning roller supports rotational in plan view are recommended as a means of combating the belt misalignment.

  14. Comparison of three assembly strategies for a heterozygous seedless grapevine genome assembly.

    Science.gov (United States)

    Patel, Sagar; Lu, Zhixiu; Jin, Xiaozhu; Swaminathan, Padmapriya; Zeng, Erliang; Fennell, Anne Y

    2018-01-17

    De novo heterozygous assembly is an ongoing challenge requiring improved assembly approaches. In this study, three strategies were used to develop de novo Vitis vinifera 'Sultanina' genome assemblies for comparison with the inbred V. vinifera (PN40024 12X.v2) reference genome and a published Sultanina ALLPATHS-LG assembly (AP). The strategies were: 1) a default PLATANUS assembly (PLAT_d) for direct comparison with AP assembly, 2) an iterative merging strategy using METASSEMBLER to combine PLAT_d and AP assemblies (MERGE) and 3) PLATANUS parameter modifications plus GapCloser (PLAT*_GC). The three new assemblies were greater in size than the AP assembly. PLAT*_GC had the greatest number of scaffolds aligning with a minimum of 95% identity and ≥1000 bp alignment length to V. vinifera (PN40024 12X.v2) reference genome. SNP analysis also identified additional high quality SNPs. A greater number of sequence reads mapped back with zero-mismatch to the PLAT_d, MERGE, and PLAT*_GC (>94%) than was found in the AP assembly (87%) indicating a greater fidelity to the original sequence data in the new assemblies than in AP assembly. A de novo gene prediction conducted using seedless RNA-seq data predicted > 30,000 coding sequences for the three new de novo assemblies, with the greatest number (30,544) in PLAT*_GC and only 26,515 for the AP assembly. Transcription factor analysis indicated good family coverage, but some genes found in the VCOST.v3 annotation were not identified in any of the de novo assemblies, particularly some from  the MYB and ERF families. The PLAT_d and PLAT*_GC had a greater number of synteny blocks with the V. vinifera (PN40024 12X.v2) reference genome than AP or MERGE. PLAT*_GC provided the most contiguous assembly with only 1.2% scaffold N, in contrast to AP (10.7% N), PLAT_d (6.6% N) and Merge (6.4% N). A PLAT*_GC pseudo-chromosome assembly with chromosome alignment to the reference genome V. vinifera, (PN40024 12X.v2) provides new information

  15. Regulation of plant developmental processes by a novel splicing factor.

    Science.gov (United States)

    Ali, Gul Shad; Palusa, Saiprasad G; Golovkin, Maxim; Prasad, Jayendra; Manley, James L; Reddy, Anireddy S N

    2007-05-30

    Serine/arginine-rich (SR) proteins play important roles in constitutive and alternative splicing and other aspects of mRNA metabolism. We have previously isolated a unique plant SR protein (SR45) with atypical domain organization. However, the biological and molecular functions of this novel SR protein are not known. Here, we report biological and molecular functions of this protein. Using an in vitro splicing complementation assay, we showed that SR45 functions as an essential splicing factor. Furthermore, the alternative splicing pattern of transcripts of several other SR genes was altered in a mutant, sr45-1, suggesting that the observed phenotypic abnormalities in sr45-1 are likely due to altered levels of SR protein isoforms, which in turn modulate splicing of other pre-mRNAs. sr45-1 exhibited developmental abnormalities, including delayed flowering, narrow leaves and altered number of petals and stamens. The late flowering phenotype was observed under both long days and short days and was rescued by vernalization. FLC, a key flowering repressor, is up-regulated in sr45-1 demonstrating that SR45 influences the autonomous flowering pathway. Changes in the alternative splicing of SR genes and the phenotypic defects in the mutant were rescued by SR45 cDNA, further confirming that the observed defects in the mutant are due to the lack of SR45. These results indicate that SR45 is a novel plant-specific splicing factor that plays a crucial role in regulating developmental processes.

  16. Aberrant and alternative splicing in skeletal system disease.

    Science.gov (United States)

    Fan, Xin; Tang, Liling

    2013-10-01

    The main function of skeletal system is to support the body and help movement. A variety of factors can lead to skeletal system disease, including age, exercise, and of course genetic makeup and expression. Pre-mRNA splicing plays a crucial role in gene expression, by creating multiple protein variants with different biological functions. The recent studies show that several skeletal system diseases are related to pre-mRNA splicing. This review focuses on the relationship between pre-mRNA splicing and skeletal system disease. On the one hand, splice site mutation that leads to aberrant splicing often causes genetic skeletal system disease, like COL1A1, SEDL and LRP5. On the other hand, alternative splicing without genomic mutation may generate some marker protein isoforms, for example, FN, VEGF and CD44. Therefore, understanding the relationship between pre-mRNA splicing and skeletal system disease will aid in uncovering the mechanism of disease and contribute to the future development of gene therapy. © 2013 Elsevier B.V. All rights reserved.

  17. Alternative splicing regulation of APP exon 7 by RBFox proteins.

    Science.gov (United States)

    Alam, Shafiul; Suzuki, Hitoshi; Tsukahara, Toshifumi

    2014-12-01

    RBFox proteins are well-known alternative splicing regulators. We have shown previously that during neuronal differentiation of P19 cells induced by all-trans retinoic acid and cell aggregation, RBFox1 shows markedly increased temporal expression. To find its key splicing regulation, we examined the effect of RBFox1 on 33 previously reported and validated neuronal splicing events of P19 cells. We observed that alternative splicing of three genes, specifically, amyloid precursor protein (APP), disks large homolog 3 (DLG3), and G protein, alpha activating activity polypeptide O (GNAO1), was altered by transient RBFox1 expression in HEK293 and HeLa cells. Moreover, an RBFox1 mutant (RBFox1FA) that was unable to bind the target RNA sequence ((U)GCAUG) did not induce these splicing events. APP generates amyloid beta peptides that are involved in the pathology of Alzheimer's disease, and therefore we examined APP alternative splicing regulation by RBFox1 and other splicing regulators. Our results indicated that RBFox proteins promote the skipping of APP exon 7, but not the inclusion of exon 8. We made APP6789 minigenes and observed that two (U)GCAUG sequences, located upstream of exon 7 and in exon 7, functioned to induce skipping of exon 7 by RBFox proteins. Overall, RBFox proteins may shift APP from exon 7 containing isoforms, APP770 and APP751, toward the exon 7 lacking isoform, APP695, which is predominant in neural tissues. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Entropy driven self-assembly of nonamphiphilic colloidal membranes

    National Research Council Canada - National Science Library

    Edward Barry; Zvonimir Dogic; Noel A. Clark

    2010-01-01

    We demonstrate that homogeneous monodisperse rods in the presence of attractive interactions assemble into equilibrium 2D fluid-like membranes composed of a one-rod length thick monolayer of aligned rods...

  19. Functional analysis of alternative splicing of the FLOWERING LOCUS T orthologous gene in Chrysanthemum morifolium.

    Science.gov (United States)

    Mao, Yachao; Sun, Jing; Cao, Peipei; Zhang, Rong; Fu, Qike; Chen, Sumei; Chen, Fadi; Jiang, Jiafu

    2016-01-01

    As the junction of floral development pathways, the FLOWERING LOCUS T (FT) protein called 'florigen' plays an important role in the process of plant flowering through signal integration. We isolated four transcripts encoding different isoforms of a FT orthologous gene CmFTL1, from Chrysanthemum morifolium cultivar 'Jimba'. Sequence alignments suggested that the four transcripts are related to the intron 1. Expression analysis showed that four alternative splicing (AS) forms of CmFTL1 varied depending on the developmental stage of the flower. The functional complement experiment using an Arabidopsis mutant ft-10 revealed that the archetypal and AS forms of CmFTL1 had the function of complementing late flower phenotype in different levels. In addition, transgenic confirmation at transcript level showed CmFTL1 and CmFTL1ast coexist in the same tissue type at the same developmental stage, indicating a post-transcriptional modification of CmFTL1 in Arabidopsis. Moreover, ectopic expression of different AS forms in chrysanthemum resulted in the development of multiple altered phenotypes, varying degrees of early flowering. We found that an alternative splicing form (CmFTL1-astE134) without the exon 2 lacked the ability causing the earlier flower phenotype. The evidence in this study indicates that complex alternative processing of CmFTL1 transcripts in C. morifolium may be associated with flowering regulation and hold some potential for biotechnical engineering to create early-flowering phenotypes in ornamental cultivars.

  20. Alignment of the LHCb vertex locator

    Science.gov (United States)

    Gersabeck, M.

    2009-01-01

    LHCb will commence data taking as the first dedicated heavy flavour experiment at a hadron collider in 2008. A very high hit precision from its vertex detector (vertex locator, VELO) is essential to meet the tight requirements of vertex reconstruction in B-physics. The single hit precision of the VELO is better than 10 μm. However, the VELO is operated only 8 mm from the beam and must be retracted and reinserted each LHC fill. Hence, the detector places unique demands on its alignment algorithm. The partially assembled VELO system has already been tested in a beam test. The novel software alignment methods are presented together with their interplay with the metrology measurements. Results from Monte Carlo simulation studies are discussed and recent beam test results are shown that prove the method's precision at the micron level.

  1. The surveyors' quest for perfect alignment

    CERN Multimedia

    2003-01-01

    Photogrammetry of a CMS endcap and part of the hadronic calorimeter.The structure was covered with targets photographed by digital cameras. Perfect alignment.... Although CERN's surveyors do not claim to achieve it, they are constantly striving for it and deploy all necessary means to come as close as they can. In their highly specialised field of large-scale metrology, the solution lies in geodesy and photogrammetry, both of which are based on increasingly sophisticated instruments and systems. In civil engineering, these techniques are used for non-destructive inspection of bridges, dams and other structures, while industrial applications include dimensional verification and deformation measurement in large mechanical assemblies. The same techniques also come into play for the metrology of research tools such as large telescopes and of course, particle accelerators. Particle physics laboratories are especially demanding customers, and CERN has often asked for the impossible. As a result, the alignment metro...

  2. Oriented scanning is the leading mechanism underlying 5' splice site selection in mammals

    NARCIS (Netherlands)

    Borensztajn, Keren; Sobrier, Marie-Laure; Duquesnoy, Philippe; Fischer, Anne-Marie; Tapon-Bretaudière, Jacqueline; Amselem, Serge

    2006-01-01

    Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 5' splice sites are accurately identified remain controversial and incompletely resolved. The human F7

  3. Cost-effective sequencing of full-length cDNA clones powered by a de novo-reference hybrid assembly.

    Science.gov (United States)

    Kuroshu, Reginaldo M; Watanabe, Junichi; Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka; Kasahara, Masahiro

    2010-05-07

    Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence approximately 800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only approximately US$3 per clone, demonstrating a significant advantage over previous approaches.

  4. Cost-Effective Sequencing of Full-Length cDNA Clones Powered by a De Novo-Reference Hybrid Assembly

    Science.gov (United States)

    Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka

    2010-01-01

    Background Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. Methodology We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence ∼800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. Conclusions The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only ∼US$3 per clone, demonstrating a significant advantage over previous approaches. PMID:20479877

  5. Simple, generalizable route to highly aligned block copolymer thin films

    Science.gov (United States)

    Qiang, Zhe; Cavicchi, Kevin; Vogt, Bryan; University of Akron Team

    Macroscopic alignment of block copolymer domains in thin films is desired for many applications, such as cell responsive surfaces or optical polarizers. Alignment generally requires specialized tools that apply external fields, shear force gradient, or produce topological patterned substrates. This requirement limits the broad academic application of aligned BCPs. Here, we describe a simple modification of commonly utilized solvent vapor annealing (SVA) process for macroscopic alignment of BCPs. Adhering a flat, crosslinked elastomer pad to the BCP film leads to differential swelling between the elastomer pad and BCP to produce a shear force that aligns the ordered BCP domains. The role of elastomer properties, solvent quality, drying rate and degree of segregation of the block copolymer will be discussed to provide generalized rules for alignment with this technique. Cylindrical nanostructures formed in polystyrene-block-polydimethylsiloxane can be transformed into arrays of silica lines and increasing the thickness from a monolayer to bilayer can effectively halve the spacing of the lines. These results illustrate a generalized method for BCP alignment and a potential route for the generation of complex hierarchical assembled structures. A generalized method for block copolymer thin film alignment: solvent vapor annealing with shear.

  6. Splicing landscape of the eight collaborative cross founder strains.

    Science.gov (United States)

    Zheng, Christina L; Wilmot, Beth; Walter, Nicole Ar; Oberbeck, Denesa; Kawane, Sunita; Searles, Robert P; McWeeney, Shannon K; Hitzemann, Robert

    2015-02-05

    The Collaborative Cross (CC) is a large panel of genetically diverse recombinant inbred mouse strains specifically designed to provide a systems genetics resource for the study of complex traits. In part, the utility of the CC stems from the extensive genome-wide annotations of founder strain sequence and structural variation. Still missing, however, are transcriptome-specific annotations of the CC founder strains that could further enhance the utility of this resource. We provide a comprehensive survey of the splicing landscape of the 8 CC founder strains by leveraging the high level of alternative splicing within the brain. Using deep transcriptome sequencing, we found that a majority of the splicing landscape is conserved among the 8 strains, with ~65% of junctions being shared by at least 2 strains. We, however, found a large number of potential strain-specific splicing events as well, with an average of ~3000 and ~500 with ≥3 and ≥10 sequence read coverage, respectively, within each strain. To better understand strain-specific splicing within the CC founder strains, we defined criteria for and identified high-confidence strain-specific splicing events. These splicing events were defined as exon-exon junctions 1) found within only one strain, 2) with a read coverage ≥10, and 3) defined by a canonical splice site. With these criteria, a total of 1509 high-confidence strain-specific splicing events were identified, with the majority found within two of the wild-derived strains, CAST and PWK. Strikingly, the overwhelming majority, 94%, of these strain-specific splicing events are not yet annotated. Strain-specific splicing was also located within genomic regions recently reported to be over- and under-represented within CC populations. Phenotypic characterization of CC populations is increasing; thus these results will not only aid in further elucidating the transcriptomic architecture of the individual CC founder strains, but they will also help in guiding

  7. In-plane laser forming for high precision alignment

    NARCIS (Netherlands)

    Folkersma, Ger; Römer, Gerardus Richardus, Bernardus, Engelina; Brouwer, Dannis Michel; Huis in 't Veld, Bert

    2014-01-01

    Laser microforming is extensively used to align components with submicrometer accuracy, often after assembly. While laser-bending sheet metal is the most common laser-forming mechanism, the in-plane upsetting mechanism is preferred when a high actuator stiffness is required. A three-bridge planar

  8. Alternative splicing variations in mouse CAPS2: differential expression and functional properties of splicing variants

    Directory of Open Access Journals (Sweden)

    Furuichi Teiichi

    2007-04-01

    Full Text Available Abstract Background Ca2+-dependent activator protein 2 (CAPS2/CADPS2 is a secretory vesicle-associated protein involved in the release of neurotrophin. We recently reported that an aberrant, alternatively spliced CAPS2 mRNA that lacks exon 3 (CAPS2Δexon3 is detected in some patients with autism. Splicing variations in mouse CAPS2 and their expression and functions remain unclear. Results In this study, we defined 31 exons in the mouse CAPS2 gene and identified six alternative splicing variants, CAPS2a-f. CAPS2a is an isoform lacking exons 22 and 25, which encode part of the Munc13-1-homologous domain (MHD. CAPS2b lacks exon 25. CAPS2c lacks exons 11 and 22. CAPS2d, 2e, and 2f have C-terminal deletions from exon 14, exon 12, and exon 5, respectively. On the other hand, a mouse counterpart of CAPS2Δexon3 was not detected in the mouse tissues tested. CAPS2b was expressed exclusively in the brain, and the other isoforms were highly expressed in the brain, but also in some non-neural tissues. In the brain, all isoforms showed predominant expression patterns in the cerebellum. In the developing cerebellum, CAPS2b showed an up-regulated expression pattern, whereas the other isoforms exhibited transiently peaked expression patterns. CAPS2 proteins were mostly recovered in soluble fractions, but some were present in membrane fractions, except for CAPS2c and 2f, both of which lack the PH domain, suggesting that the PH domain is important for membrane association. In contrast to CAPS2a and 2b, CAPS2c showed slightly decreased BDNF-releasing activity, which is likely due to the C-terminal truncation of the PH domain in CAPS2c. Conclusion This study indicates that, in mouse, there are six splicing variants of CAPS2 (CAPS2a-f, and that these are subdivided into two groups: a long form containing the C-terminal MHD and a short form lacking the C-terminal MHD. These results demonstrate that the splicing variations correlate with their expression patterns and

  9. Identification, expression and functional characterization of M4L, a muscarinic acetylcholine M4 receptor splice variant.

    Directory of Open Access Journals (Sweden)

    Douglas A Schober

    Full Text Available Rodent genomic alignment sequences support a 2-exon model for muscarinic M4 receptor. Using this model a novel N-terminal extension was discovered in the human muscarinic acetylcholine M4 receptor. An open reading frame was discovered in the human, mouse and rat with a common ATG (methionine start codon that extended the N-terminus of the muscarinic acetylcholine M4 receptor subtype by 155 amino acids resulting in a longer variant. Transcriptional evidence for this splice variant was confirmed by RNA-Seq and RT-PCR experiments performed from human donor brain prefrontal cortices. We detected a human upstream exon indicating the translation of the mature longer M4 receptor transcript. The predicted size for the longer two-exon M4 receptor splice variant with the additional 155 amino acid N-terminal extension, designated M4L is 69.7 kDa compared to the 53 kDa canonical single exon M4 receptor (M4S. Western blot analysis from a mammalian overexpression system, and saturation radioligand binding with [3H]-NMS (N-methyl-scopolamine demonstrated the expression of this new splice variant. Comparative pharmacological characterization between the M4L and M4S receptors revealed that both the orthosteric and allosteric binding sites for both receptors were very similar despite the addition of an N-terminal extension.

  10. Automatic Word Alignment

    Science.gov (United States)

    2014-02-18

    Cited OTHER PUBLICATIONS Wu, Hua, etal., “Boosting Statistical Word Alignment Using Labeled and Unlabeled Data” Toshiba (China) research and...Labeled and Unlabeled Data” Toshiba {China) Research and Development Center (Jul. 1, 2006) pp. 913-920. * cited by examiner U .S. Patent F eb.18, 2014

  11. Advanced aligner orthodontics

    Directory of Open Access Journals (Sweden)

    Ojima Kenji

    2017-01-01

    Full Text Available Invisalign initially had limitations which have now been overcome.Advances in the quality of aligner materials, attachments and the introduction of a new force system, have expanded the range of treatment possibilities from severe crowding to more difficult extraction cases, open bite cases, and lower molar distalization cases.

  12. Aligning Responsible Business Practices

    DEFF Research Database (Denmark)

    Weller, Angeli E.

    2017-01-01

    This article offers an in-depth case study of a global high tech manufacturer that aligned its ethics and compliance, corporate social responsibility, and sustainability practices. Few large companies organize their responsible business practices this way, despite conceptual relevance and calls...... and managers interested in understanding how responsible business practices may be collectively organized....

  13. MUON DETECTORS: ALIGNMENT

    CERN Multimedia

    G. Gomez and Y. Pakhotin

    2012-01-01

      A new track-based alignment for the DT chambers is ready for deployment: an offline tag has already been produced which will become part of the 52X Global Tag. This alignment was validated within the muon alignment group both at low and high momentum using a W/Z skim sample. It shows an improved mass resolution for pairs of stand-alone muons, improved curvature resolution at high momentum, and improved DT segment extrapolation residuals. The validation workflow for high-momentum muons used to depend solely on the “split cosmics” method, looking at the curvature difference between muon tracks reconstructed in the upper or lower half of CMS. The validation has now been extended to include energetic muons decaying from heavily boosted Zs: the di-muon invariant mass for global and stand-alone muons is reconstructed, and the invariant mass resolution is compared for different alignments. The main areas of development over the next few months will be preparing a new track-based C...

  14. Aligning Mental Representations

    DEFF Research Database (Denmark)

    Kano Glückstad, Fumiko

    2013-01-01

    This work introduces a framework that implements asymmetric communication theory proposed by Sperber and Wilson [1]. The framework applies a generalization model known as the Bayesian model of generalization (BMG) [2] for aligning knowledge possessed by two communicating parties. The work focuses...

  15. Fiducialisation and initial alignment of CLIC component with micrometric accuracy

    CERN Document Server

    Mainaud Durand, Helene; Buzio, Marco; Caiazza, Domenico; Catalan Lasheras, Nuria; Cherif, Ahmed; Doytchinov, Iordan Petrov; Fuchs, Jean-Frederic; Gaddi, Andrea; Galindo Munoz, Natalia; Gayde, Jean-Christophe; Kamugasa, Solomon William; Modena, Michele; Novotny, Peter; Sanz, Claude; Severino, Giordana; Russenschuck, Stephan; Tshilumba, David; Vlachakis, Vasileios; Wendt, Manfred; Zorzetti, Silvia; CERN. Geneva. ATS Department

    2016-01-01

    We propose a new solution to fiducialise the three major components of the CLIC collider: quadrupoles, beam-position monitors (BPM), and accelerating structures (AS). This solution is based on the use of a copper-beryllium (CuBe) wire to locate the reference position, i.e. the symmetry axes of the components (their magnetic, respectively electromagnetic centre axis), and to determine their position in the common support assembly defining a local coordinate system, with respect to the fiducials. These alignment targets will be used later to align the support assembly in the tunnel. With such a method, several accelerator components of different types, supported by a dedicated adjustment system, can be simultaneously fiducialised and pre-aligned using the same wire, enabling a micrometric accuracy with help of a 3D coordinate measurement machine (CMM). Alternative solutions based on frequency scanning interferometry (FSI) and micro-triangulation are also under development, to perform such fiducialisation and in...

  16. Automated alignment of optical components for high-power diode lasers

    Science.gov (United States)

    Brecher, C.; Pyschny, N.; Haag, S.; Guerrero Lule, V.

    2012-03-01

    Despite major progress in developing brilliant laser sources a huge potential for cost reductions can be found in simpler setups and automated assembly processes, especially for large volume applications. In this presentation, a concept for flexible automation in optics assembly is presented which is based on standard micro assembly systems with relatively large workspace and modular micromanipulators to enhance the system with additional degrees of freedom and a very high motion resolution. The core component is a compact flexure-based micromanipulator especially designed for the alignment of micro optical components which will be described in detail. The manipulator has been applied in different scenarios to develop and investigate automated alignment processes. This paper focuses on the automated alignment of fast axis collimation (FAC) lenses which is a crucial step during the production of diode lasers. The handling and positioning system, the measuring arrangement for process feedback during active alignment as well as the alignment strategy will be described. The fine alignment of the FAC lens is performed with the micromanipulator under concurrent analysis of the far and the near field intensity distribution. An optimization of the image processing chains for the alignment of a FAC in front of a diode bar led to cycle times of less than 30 seconds. An outlook on other applications and future work regarding the development of automated assembly processes as well as new ideas for flexible assembly systems with desktop robots will close the talk.

  17. Bulkiness versus anisotropy: The optimal shape of polarizable Brownian nanoparticles for alignment in electric fields

    NARCIS (Netherlands)

    Kwaadgras, B.W.; Dijkstra, M.|info:eu-repo/dai/nl/123538807; van Roij, R.H.H.G.|info:eu-repo/dai/nl/152978984

    2012-01-01

    Self-assembly and alignment of anisotropic colloidal particles are important processes that can be influenced by external electric fields. However, dielectric nanoparticles are generally hard to align this way because of their small size and low polarizability. In this work, we employ the coupled

  18. Capillary self-alignment of mesoscopic foil components for sensor-systems-in-foil

    NARCIS (Netherlands)

    Arutinov, G.; Smits, E.C.P.; Mastrangeli, M.; Heck, G. van; Brand, J. van den; Schoo, H.F.M.; Dietzel, A.H.

    2012-01-01

    This paper reports on the effective use of capillary self-alignment for low-cost and time-efficient assembly of heterogeneous foil components into a smart electronic identification label. Particularly, we demonstrate the accurate (better than 50m) alignment of cm-sized functional foil dies. We

  19. Fast global sequence alignment technique

    KAUST Repository

    Bonny, Mohamed Talal

    2011-11-01

    Bioinformatics database is growing exponentially in size. Processing these large amount of data may take hours of time even if super computers are used. One of the most important processing tool in Bioinformatics is sequence alignment. We introduce fast alignment algorithm, called \\'Alignment By Scanning\\' (ABS), to provide an approximate alignment of two DNA sequences. We compare our algorithm with the wellknown sequence alignment algorithms, the \\'GAP\\' (which is heuristic) and the \\'Needleman-Wunsch\\' (which is optimal). The proposed algorithm achieves up to 51% enhancement in alignment score when it is compared with the GAP Algorithm. The evaluations are conducted using different lengths of DNA sequences. © 2011 IEEE.

  20. ABS: Sequence alignment by scanning

    KAUST Repository

    Bonny, Mohamed Talal

    2011-08-01

    Sequence alignment is an essential tool in almost any computational biology research. It processes large database sequences and considered to be high consumers of computation time. Heuristic algorithms are used to get approximate but fast results. We introduce fast alignment algorithm, called Alignment By Scanning (ABS), to provide an approximate alignment of two DNA sequences. We compare our algorithm with the well-known alignment algorithms, the FASTA (which is heuristic) and the \\'Needleman-Wunsch\\' (which is optimal). The proposed algorithm achieves up to 76% enhancement in alignment score when it is compared with the FASTA Algorithm. The evaluations are conducted using different lengths of DNA sequences. © 2011 IEEE.

  1. Alignment and Load Transfer in Carbon Nanotube and Dicyclopentadiene Composites

    Science.gov (United States)

    Severino, Joseph Vincent

    Individual carbon nanotubes (CNTs) are the strongest materials available but their macroscopic assemblies are weak. This work establishes a new thermosetting dicyclopentadiene (DCPD) and CNT composite that increases the strength of CNT assemblies. These high volume fraction and void free structures constitute advanced materials that could one day replace traditional composite systems. To further the understanding of physical interactions between polymer and CNTs, a novel "capstan" load transfer mechanism is also introduced. Self-supporting assemblies of interconnected carbon nanotubes were stretched, twisted and compressed to fashion composites by the infusion and polymerization of low viscosity DCPD based monomeric resins. The properties of the CNTs, polymer and composite were characterized with thermal gravimetric analysis (TGA), dynamic mechanical analysis (DMA) and Raman spectroscopy. The microstructure was analyzed by wide angle X-ray scattering (WAXS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Sheets were drawn at 15 m/min from a growth furnace to impart alignment then stretched to further modify alignment. The mechanical properties were determined in five orientations with respect to the growth direction. The strength was nearly three times higher along this growth direction than it was perpendicular, and modulus was nearly six times higher. Transverse stretching achieved 1.5 times the elongation but alignment was inferior due to CNT kinking that prevented alignment and consolidation. Composites yarns and sheets were investigated for the mechanical properties, microstructure and load transfer. The DCPD resin was found to wet the CNTs and lubricated deformation. This reduced loads during processing, and curing solidified the aligned and consolidated structure. The stretched and twisted composite yarns increased the failure stress 51%. In aligned composite sheet, the failure stress increased 200%. The increased stresses

  2. Regulation of Apoptosis by Alternative Pre-mRNA Splicing

    National Research Council Canada - National Science Library

    Schwerk, Christian; Schulze-Osthoff, Klaus

    2005-01-01

    .... A large number of apoptotic factors are regulated via alternative splicing, a process that allows for the production of discrete protein isoforms with often distinct functions from a common mRNA precursor...

  3. Dynamic integration of splicing within gene regulatory pathways

    National Research Council Canada - National Science Library

    Braunschweig, Ulrich; Gueroussov, Serge; Plocik, Alex M; Graveley, Brenton R; Blencowe, Benjamin J

    2013-01-01

    .... In addition to generating vast repertoires of RNAs and proteins, splicing has a profound impact on other gene regulatory layers, including mRNA transcription, turnover, transport, and translation...

  4. Alternative splicing of follicle-stimulating hormone receptor pre-mRNA: cloning and characterization of two alternatively spliced mRNA transcripts

    NARCIS (Netherlands)

    R. Kraaij (Robert); M. Verhoef-Post (Miriam); J.A. Grootegoed (Anton); A.P.N. Themmen (Axel)

    1998-01-01

    textabstractGlycoprotein hormone receptors contain a large extracellular domain that is encoded by multiple exons, facilitating the possibility of expressing alternatively spliced transcripts. We have cloned two new splice variants of the rat follicle-stimulating

  5. RNA-sequencing of a mouse-model of spinal muscular atrophy reveals tissue-wide changes in splicing of U12-dependent introns

    DEFF Research Database (Denmark)

    Doktor, Thomas Koed; Hua, Yimin; Andersen, Henriette Skovgaard

    2016-01-01

    Spinal Muscular Atrophy (SMA) is a neuromuscular disorder caused by insufficient levels of the Survival of Motor Neuron (SMN) protein. SMN is expressed ubiquitously and functions in RNA processing pathways that include trafficking of mRNA and assembly of snRNP complexes. Importantly, SMA severity...... is correlated with decreased snRNP assembly activity. In particular, the minor spliceosomal snRNPs are affected, and some U12-dependent introns have been reported to be aberrantly spliced in patient cells and animal models. SMA is characterized by loss of motor neurons, but the underlying mechanism is largely...... unknown. It is likely that aberrant splicing of genes expressed in motor neurons is involved in SMA pathogenesis, but increasing evidence indicates that pathologies also exist in other tissues. We present here a comprehensive RNA-seq study that covers multiple tissues in an SMA mouse model. We show...

  6. Androgen Receptor Splice Variants and Resistance to Taxane Chemotherapy

    Science.gov (United States)

    2017-10-01

    AWARD NUMBER: W81XWH-14-1-0480 TITLE: Androgen Receptor Splice Variants and Resistance to Taxane Chemotherapy PRINCIPAL INVESTIGATOR...Splice Variants and Resistance to Taxane Chemotherapy 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0480 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...During this reporting period, we have obtained approval for a no-cost extension of this award and change of research focus in the final year. This

  7. Roles of alternative splicing in modulating transcriptional regulation.

    Science.gov (United States)

    Li, Jin; Wang, Yang; Rao, Xi; Wang, Yue; Feng, Weixing; Liang, Hong; Liu, Yunlong

    2017-10-03

    The ability of a transcription factor to regulate its targets is modulated by a variety of genetic and epigenetic mechanisms. Alternative splicing can modulate gene function by adding or removing certain protein domains, and therefore affect the activity of protein. Reverse engineering of gene regulatory networks using gene expression profiles has proven valuable in dissecting the logical relationships among multiple proteins during the transcriptional regulation. However, it is unclear whether alternative splicing of certain proteins affects the activity of other transcription factors. In order to investigate the roles of alternative splicing during transcriptional regulation, we constructed a statistical model to infer whether the alternative splicing events of modulator proteins can affect the ability of key transcription factors in regulating the expression levels of their transcriptional targets. We tested our strategy in KIRC (Kidney Renal Clear Cell Carcinoma) using the RNA-seq data downloaded from TCGA (the Cancer Genomic Atlas). We identified 828of modulation relationships between the splicing levels of modulator proteins and activity levels of transcription factors. For instance, we found that the activity levels of GR (glucocorticoid receptor) protein, a key transcription factor in kidney, can be influenced by the splicing status of multiple proteins, including TP53, MDM2 (mouse double minute 2 homolog), RBM14 (RNA-binding protein 14) and SLK (STE20 like kinase). The influenced GR-targets are enriched by key cancer-related pathways, including p53 signaling pathway, TR/RXR activation, CAR/RXR activation, G1/S checkpoint regulation pathway, and G2/M DNA damage checkpoint regulation pathway. Our analysis suggests, for the first time, that exon inclusion levels of certain regulatory proteins can affect the activities of many transcription factors. Such analysis can potentially unravel a novel mechanism of how splicing variation influences the cellular

  8. Alternative splice variants in TIM barrel proteins from human genome correlate with the structural and evolutionary modularity of this versatile protein fold.

    Directory of Open Access Journals (Sweden)

    Adrián Ochoa-Leyva

    Full Text Available After the surprisingly low number of genes identified in the human genome, alternative splicing emerged as a major mechanism to generate protein diversity in higher eukaryotes. However, it is still not known if its prevalence along the genome evolution has contributed to the overall functional protein diversity or if it simply reflects splicing noise. The (βα8 barrel or TIM barrel is one of the most frequent, versatile, and ancient fold encountered among enzymes. Here, we analyze the structural modifications present in TIM barrel proteins from the human genome product of alternative splicing events. We found that 87% of all splicing events involved deletions; most of these events resulted in protein fragments that corresponded to the (βα2, (βα4, (βα5, (βα6, and (βα7 subdomains of TIM barrels. Because approximately 7% of all the splicing events involved internal β-strand substitutions, we decided, based on the genomic data, to design β-strand and α-helix substitutions in a well-studied TIM barrel enzyme. The biochemical characterization of one of the chimeric variants suggests that some of the splice variants in the human genome with β-strand substitutions may be evolving novel functions via either the oligomeric state or substrate specificity. We provide results of how the splice variants represent subdomains that correlate with the independently folding and evolving structural units previously reported. This work is the first to observe a link between the structural features of the barrel and a recurrent genetic mechanism. Our results suggest that it is reasonable to expect that a sizeable fraction of splice variants found in the human genome represent structurally viable functional proteins. Our data provide additional support for the hypothesis of the origin of the TIM barrel fold through the assembly of smaller subdomains. We suggest a model of how nature explores new proteins through alternative splicing as a mechanism to

  9. Magnetic Alignment of Block Copolymer Microdomains by Intrinsic Chain Anisotropy

    Science.gov (United States)

    Rokhlenko, Yekaterina; Gopinadhan, Manesh; Osuji, Chinedum O.; Zhang, Kai; O'Hern, Corey S.; Larson, Steven R.; Gopalan, Padma; Majewski, Paweł W.; Yager, Kevin G.

    2015-12-01

    We examine the role of intrinsic chain susceptibility anisotropy in magnetic field directed self-assembly of a block copolymer using in situ x-ray scattering. Alignment of a lamellar mesophase is observed on cooling across the disorder-order transition with the resulting orientational order inversely proportional to the cooling rate. We discuss the origin of the susceptibility anisotropy, Δ χ , that drives alignment and calculate its magnitude using coarse-grained molecular dynamics to sample conformations of surface-tethered chains, finding Δ χ ≈2 ×1 0-8. From field-dependent scattering data, we estimate that grains of ≈1.2 μ m are present during alignment. These results demonstrate that intrinsic anisotropy is sufficient to support strong field-induced mesophase alignment and suggest a versatile strategy for field control of orientational order in block copolymers.

  10. Chemical strategies for die/wafer submicron alignment and bonding.

    Energy Technology Data Exchange (ETDEWEB)

    Martin, James Ellis; Baca, Alicia I.; Chu, Dahwey; Rohwer, Lauren Elizabeth Shea

    2010-09-01

    This late-start LDRD explores chemical strategies that will enable sub-micron alignment accuracy of dies and wafers by exploiting the interfacial energies of chemical ligands. We have micropatterned commensurate features, such as 2-d arrays of micron-sized gold lines on the die to be bonded. Each gold line is functionalized with alkanethiol ligands before the die are brought into contact. The ligand interfacial energy is minimized when the lines on the die are brought into registration, due to favorable interactions between the complementary ligand tails. After registration is achieved, standard bonding techniques are used to create precision permanent bonds. We have computed the alignment forces and torque between two surfaces patterned with arrays of lines or square pads to illustrate how best to maximize the tendency to align. We also discuss complex, aperiodic patterns such as rectilinear pad assemblies, concentric circles, and spirals that point the way towards extremely precise alignment.

  11. Magnetic Alignment of Block Copolymer Microdomains by Intrinsic Chain Anisotropy.

    Science.gov (United States)

    Rokhlenko, Yekaterina; Gopinadhan, Manesh; Osuji, Chinedum O; Zhang, Kai; O'Hern, Corey S; Larson, Steven R; Gopalan, Padma; Majewski, Paweł W; Yager, Kevin G

    2015-12-18

    We examine the role of intrinsic chain susceptibility anisotropy in magnetic field directed self-assembly of a block copolymer using in situ x-ray scattering. Alignment of a lamellar mesophase is observed on cooling across the disorder-order transition with the resulting orientational order inversely proportional to the cooling rate. We discuss the origin of the susceptibility anisotropy, Δχ, that drives alignment and calculate its magnitude using coarse-grained molecular dynamics to sample conformations of surface-tethered chains, finding Δχ≈2×10^{-8}. From field-dependent scattering data, we estimate that grains of ≈1.2  μm are present during alignment. These results demonstrate that intrinsic anisotropy is sufficient to support strong field-induced mesophase alignment and suggest a versatile strategy for field control of orientational order in block copolymers.

  12. Orbit IMU alignment: Error analysis

    Science.gov (United States)

    Corson, R. W.

    1980-01-01

    A comprehensive accuracy analysis of orbit inertial measurement unit (IMU) alignments using the shuttle star trackers was completed and the results are presented. Monte Carlo techniques were used in a computer simulation of the IMU alignment hardware and software systems to: (1) determine the expected Space Transportation System 1 Flight (STS-1) manual mode IMU alignment accuracy; (2) investigate the accuracy of alignments in later shuttle flights when the automatic mode of star acquisition may be used; and (3) verify that an analytical model previously used for estimating the alignment error is a valid model. The analysis results do not differ significantly from expectations. The standard deviation in the IMU alignment error for STS-1 alignments was determined to the 68 arc seconds per axis. This corresponds to a 99.7% probability that the magnitude of the total alignment error is less than 258 arc seconds.

  13. RNA splicing is responsive to MBNL1 dose.

    Directory of Open Access Journals (Sweden)

    Sonali P Jog

    Full Text Available Myotonic dystrophy (DM1 is a highly variable, multi-system disorder resulting from the expansion of an untranslated CTG tract in DMPK. In DM1 expanded CUG repeat RNAs form hairpin secondary structures that bind and aberrantly sequester the RNA splice regulator, MBNL1. RNA splice defects resulting as a consequence of MBNL1 depletion have been shown to play a key role in the development of DM1 pathology. In patient populations, both the number and severity of DM1 symptoms increase broadly as a function of CTG tract length. However significant variability in the DM1 phenotype is observed in patients encoding similar CTG repeat numbers. Here we demonstrate that a gradual decrease in MBNL1 levels results both in the expansion of the repertoire of splice defects and an increase in the severity of the splice alterations. Thus, MBNL1 loss does not have an all or none outcome but rather shows a graded effect on the number and severity of the ensuing splice defects. Our results suggest that once a critical threshold is reached, relatively small dose variations of free MBNL1 levels, which may reflect modest changes in the size of the CUG tract or the extent of hairpin secondary structure formation, can significantly alter the number and severity of splice abnormalities and thus contribute to the phenotype variability observed in DM1 patients.

  14. Abnormalities in Alternative Splicing of Apoptotic Genes and Cardiovascular Diseases

    Directory of Open Access Journals (Sweden)

    Zodwa Dlamini

    2015-11-01

    Full Text Available Apoptosis is required for normal heart development in the embryo, but has also been shown to be an important factor in the occurrence of heart disease. Alternative splicing of apoptotic genes is currently emerging as a diagnostic and therapeutic target for heart disease. This review addresses the involvement of abnormalities in alternative splicing of apoptotic genes in cardiac disorders including cardiomyopathy, myocardial ischemia and heart failure. Many pro-apoptotic members of the Bcl-2 family have alternatively spliced isoforms that lack important active domains. These isoforms can play a negative regulatory role by binding to and inhibiting the pro-apoptotic forms. Alternative splicing is observed to be increased in various cardiovascular diseases with the level of alternate transcripts increasing elevated in diseased hearts compared to healthy subjects. In many cases these isoforms appear to be the underlying cause of the disease, while in others they may be induced in response to cardiovascular pathologies. Regardless of this, the detection of alternate splicing events in the heart can serve as useful diagnostic or prognostic tools, while those splicing events that seem to play a causative role in cardiovascular disease make attractive future drug targets.

  15. Splice site mutations in the ATP7A gene.

    Directory of Open Access Journals (Sweden)

    Tina Skjørringe

    Full Text Available Menkes disease (MD is caused by mutations in the ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12 mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation to in vivo results. Ninety-six percent of the mutations identified in 45 patients with classical MD were predicted to have a significant effect on splicing, which concurs with the absence of any detectable wild-type transcript in all 19 patients investigated in vivo. Sixty-seven percent of the mutations identified in 12 patients with milder phenotypes were predicted to have no significant effect on splicing, which concurs with the presence of wild-type transcript in 7 out of 9 patients investigated in vivo. Both the in silico predictions and the in vivo results support the hypothesis previously suggested by us and others, that the presence of some wild-type transcript is correlated to a milder phenotype.

  16. Width of gene expression profile drives alternative splicing.

    Directory of Open Access Journals (Sweden)

    Daniel Wegmann

    Full Text Available Alternative splicing generates an enormous amount of functional and proteomic diversity in metazoan organisms. This process is probably central to the macromolecular and cellular complexity of higher eukaryotes. While most studies have focused on the molecular mechanism triggering and controlling alternative splicing, as well as on its incidence in different species, its maintenance and evolution within populations has been little investigated. Here, we propose to address these questions by comparing the structural characteristics as well as the functional and transcriptional profiles of genes with monomorphic or polymorphic splicing, referred to as MS and PS genes, respectively. We find that MS and PS genes differ particularly in the number of tissues and cell types where they are expressed.We find a striking deficit of PS genes on the sex chromosomes, particularly on the Y chromosome where it is shown not to be due to the observed lower breadth of expression of genes on that chromosome. The development of a simple model of evolution of cis-regulated alternative splicing leads to predictions in agreement with these observations. It further predicts the conditions for the emergence and the maintenance of cis-regulated alternative splicing, which are both favored by the tissue specific expression of splicing variants. We finally propose that the width of the gene expression profile is an essential factor for the acquisition of new transcript isoforms that could later be maintained by a new form of balancing selection.

  17. Digital holographic microtomography of fusion spliced optical fibers

    Science.gov (United States)

    Deng, Yating; Xiao, Wen; Ma, Xichao; Pan, Feng

    2017-03-01

    In this paper, we report three-dimensional(3D) measurement results of structural parameters of fusion spliced optical fibers using digital holographic microtomography. A holographic setup in microscopy configuration with the sample-fixed and setup-rotating scheme is established. A series of holograms is recorded from various incident angles. Then the filtered backprojection algorithm is applied to reconstruct the 3D refractive index (RI) distributions of the fusion spliced optical fibers inserted in the index-matching liquid. Experimental results exhibit the internal and external shapes of three kinds of fusion splices between different fibers, including a single-mode fiber(SMF) and a multimode fiber, an SMF and a panda polarization maintaining fiber (Panda PMF), and an SMF and a bow-tie polarization maintaining fiber (Bow-Tie PMF). With 3D maps of RI, it is intuitive to observe internal structural details of fused fibers and evaluate the splicing quality. This paper describes a powerful method for non-invasive microscopic measurement of fiber splicing. Furthermore, it provides the possibility of detecting fiber splicing loss by 3D structures.

  18. A comprehensive evaluation of alignment algorithms in the context of RNA-seq.

    Directory of Open Access Journals (Sweden)

    Robert Lindner

    Full Text Available Transcriptome sequencing (RNA-Seq overcomes limitations of previously used RNA quantification methods and provides one experimental framework for both high-throughput characterization and quantification of transcripts at the nucleotide level. The first step and a major challenge in the analysis of such experiments is the mapping of sequencing reads to a transcriptomic origin including the identification of splicing events. In recent years, a large number of such mapping algorithms have been developed, all of which have in common that they require algorithms for aligning a vast number of reads to genomic or transcriptomic sequences. Although the FM-index based aligner Bowtie has become a de facto standard within mapping pipelines, a much larger number of possible alignment algorithms have been developed also including other variants of FM-index based aligners. Accordingly, developers and users of RNA-seq mapping pipelines have the choice among a large number of available alignment algorithms. To provide guidance in the choice of alignment algorithms for these purposes, we evaluated the performance of 14 widely used alignment programs from three different algorithmic classes: algorithms using either hashing of the reference transcriptome, hashing of reads, or a compressed FM-index representation of the genome. Here, special emphasis was placed on both precision and recall and the performance for different read lengths and numbers of mismatches and indels in a read. Our results clearly showed the significant reduction in memory footprint and runtime provided by FM-index based aligners at a precision and recall comparable to the best hash table based aligners. Furthermore, the recently developed Bowtie 2 alignment algorithm shows a remarkable tolerance to both sequencing errors and indels, thus, essentially making hash-based aligners obsolete.

  19. Catalyzing alignment processes

    DEFF Research Database (Denmark)

    Lauridsen, Erik Hagelskjær; Jørgensen, Ulrik

    2004-01-01

    This paper describes how environmental management systems (EMS) spur the circulation of processes that support the constitution of environmental issues as specific environ¬mental objects and objectives. EMS catalyzes alignmentprocesses that produce coherence among the different elements involved...... time and in combination with other social processes establish more aligned and standardized environmental performance between countries. However, examples of the introduction of environmental management suggests that EMS’ only plays a minor role in developing the actual environmental objectives...

  20. Alignment of concerns

    DEFF Research Database (Denmark)

    Andersen, Tariq Osman; Bansler, Jørgen P.; Kensing, Finn

    E-health promises to enable and support active patient participation in chronic care. However, these fairly recent innovations are complicated matters and emphasize significant challenges, such as patients’ and clinicians’ different ways of conceptualizing disease and illness. Informed by insight...... from medical phenomenology and our own empirical work in telemonitoring and medical care of heart patients, we propose a design rationale for e-health systems conceptualized as the ‘alignment of concerns’....

  1. Exonic splicing enhancer-dependent selection of the bovine papillomavirus type 1 nucleotide 3225 3' splice site can be rescued in a cell lacking splicing factor ASF/SF2 through activation of the phosphatidylinositol 3-kinase/Akt pathway.

    Science.gov (United States)

    Liu, Xuefeng; Mayeda, Akila; Tao, Mingfang; Zheng, Zhi-Ming

    2003-02-01

    Bovine papillomavirus type 1 (BPV-1) late pre-mRNAs are spliced in keratinocytes in a differentiation-specific manner: the late leader 5' splice site alternatively splices to a proximal 3' splice site (at nucleotide 3225) to express L2 or to a distal 3' splice site (at nucleotide 3605) to express L1. Two exonic splicing enhancers, each containing two ASF/SF2 (alternative splicing factor/splicing factor 2) binding sites, are located between the two 3' splice sites and have been identified as regulating alternative 3' splice site usage. The present report demonstrates for the first time that ASF/SF2 is required under physiological conditions for the expression of BPV-1 late RNAs and for selection of the proximal 3' splice site for BPV-1 RNA splicing in DT40-ASF cells, a genetically engineered chicken B-cell line that expresses only human ASF/SF2 controlled by a tetracycline-repressible promoter. Depletion of ASF/SF2 from the cells by tetracycline greatly decreased viral RNA expression and RNA splicing at the proximal 3' splice site while increasing use of the distal 3' splice site in the remaining viral RNAs. Activation of cells lacking ASF/SF2 through anti-immunoglobulin M-B-cell receptor cross-linking rescued viral RNA expression and splicing at the proximal 3' splice site and enhanced Akt phosphorylation and expression of the phosphorylated serine/arginine-rich (SR) proteins SRp30s (especially SC35) and SRp40. Treatment with wortmannin, a specific phosphatidylinositol 3-kinase/Akt kinase inhibitor, completely blocked the activation-induced activities. ASF/SF2 thus plays an important role in viral RNA expression and splicing at the proximal 3' splice site, but activation-rescued viral RNA expression and splicing in ASF/SF2-depleted cells is mediated through the phosphatidylinositol 3-kinase/Akt pathway and is associated with the enhanced expression of other SR proteins.

  2. Genomic and Transcriptomic Analysis Reveals Spliced Leader Trans-Splicing in Cryptomonads.

    Science.gov (United States)

    Roy, Scott William

    2017-03-01

    Spliced leader trans-splicing (SLTS) is a poorly understood mechanism that is found in a diversity of eukaryotic lineages. In SLTS, a short RNA sequence is added near the 5' ends of the transcripts of protein-coding genes by a modified spliceosomal reaction. Available data suggest that SLTS has evolved many times, and might be more likely to evolve in animals. That SLTS might be more likely to evolve in the context of the generally complex transcriptomes characteristic of animals suggests the possibility that SLTS functions in gene regulation or transcriptome diversification, however no general novel function for SLTS is known. Here, I report SLTS in a lineage of cellularly complex unicellular eukaryotes. Cryptomonads are a group of eukaryotic algae that acquired photosynthetic capacity by secondary endosymbiosis of a red alga, and that retain a reduced copy of the nucleus of the engulfed alga. I estimate that at least one-fifth of genes in the model cryptomonad Guillardia theta and its relative Hanusia phi undergo SLTS. I show that hundreds of genes in G. theta generate alternative transcripts by SLTS at alternative sites, however I find little evidence for alternative protein production by alternative SLTS splicing. Interestingly, I find no evidence for substantial operon structure in the G. theta genome, in contrast to previous findings in other lineages with SLTS. These results extend SLTS to another major group of eukaryotes, and heighten the mystery of the evolution of SLTS and its association with cellular and transcriptomic complexity. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  3. Seeking the perfect alignment

    CERN Multimedia

    2002-01-01

    The first full-scale tests of the ATLAS Muon Spectrometer are about to begin in Prévessin. The set-up includes several layers of Monitored Drift Tubes Chambers (MDTs) and will allow tests of the performance of the detectors and of their highly accurate alignment system.   Monitored Drift Chambers in Building 887 in Prévessin, where they are just about to be tested. Muon chambers are keeping the ATLAS Muon Spectrometer team quite busy this summer. Now that most people go on holiday, the beam and alignment tests for these chambers are just starting. These chambers will measure with high accuracy the momentum of high-energy muons, and this implies very demanding requirements for their alignment. The MDT chambers consist of drift tubes, which are gas-filled metal tubes, 3 cm in diameter, with wires running down their axes. With high voltage between the wire and the tube wall, the ionisation due to traversing muons is detected as electrical pulses. With careful timing of the pulses, the position of the muon t...

  4. Human splicing factor ASF/SF2 encodes for a repressor domain required for its inhibitory activity on pre-mRNA splicing.

    Science.gov (United States)

    Dauksaite, Vita; Akusjärvi, Göran

    2002-04-12

    The essential splicing factor ASF/SF2 activates or represses splicing depending on where on the pre-mRNA it binds. We have shown previously that ASF/SF2 inhibits adenovirus IIIa pre-mRNA splicing by binding to an intronic repressor element. Here we used MS2-ASF/SF2 fusion proteins to show that the second RNA binding domain (RBD2) is both necessary and sufficient for the splicing repressor function of ASF/SF2. Furthermore, we show that the completely conserved SWQDLKD motif in ASF/SF2-RBD2 is essential for splicing repression. Importantly, this heptapeptide motif is unlikely to be directly involved in RNA binding given its position within the predicted structure of RBD2. The activity of the ASF/SF2-RBD2 domain in splicing was position-dependent. Thus, tethering RBD2 to the IIIa intron resulted in splicing repression, whereas RBD2 binding at the second exon had no effect on IIIa splicing. The splicing repressor activity of RBD2 was not unique to the IIIa pre-mRNA, as binding of RBD2 at an intronic position in the rabbit beta-globin pre-mRNA also resulted in splicing inhibition. Taken together, our results suggest that ASF/SF2 encode distinct domains responsible for its function as a splicing enhancer or splicing repressor protein.

  5. AR Alternative Splicing and Prostate Cancer Progression

    Science.gov (United States)

    2013-07-01

    patients with castration-resistant prostate cancer. Cancer Res 2009;69:2912–8. 15. Culig Z, Bartsch G. Androgen axis in prostate cancer. J Cell Biochem...such as MLPA are useful for identifying deletions or duplications that involve probe-binding sites, this study has illustrated that unbiased...the AR locus is illustrated at the top. Paired-end sequence reads were mapped to the hg19 build of the human genome using Burrows --Wheeler Alignment

  6. Alignment as a Teacher Variable

    Science.gov (United States)

    Porter, Andrew C.; Smithson, John; Blank, Rolf; Zeidner, Timothy

    2007-01-01

    With the exception of the procedures developed by Porter and colleagues (Porter, 2002), other methods of defining and measuring alignment are essentially limited to alignment between tests and standards. Porter's procedures have been generalized to investigating the alignment between content standards, tests, textbooks, and even classroom…

  7. Semiautomated improvement of RNA alignments

    DEFF Research Database (Denmark)

    Andersen, Ebbe Sloth; Lind-Thomsen, Allan; Knudsen, Bjarne

    2007-01-01

    We have developed a semiautomated RNA sequence editor (SARSE) that integrates tools for analyzing RNA alignments. The editor highlights different properties of the alignment by color, and its integrated analysis tools prevent the introduction of errors when doing alignment editing. SARSE readily...

  8. Probing Nucleic Acid Interactions and Pre-mRNA Splicing by Förster Resonance Energy Transfer (FRET Microscopy

    Directory of Open Access Journals (Sweden)

    David Staněk

    2012-11-01

    Full Text Available Förster resonance energy transfer (FRET microscopy is a powerful technique routinely used to monitor interactions between biomolecules. Here, we focus on the techniques that are used for investigating the structure and interactions of nucleic acids (NAs. We present a brief overview of the most commonly used FRET microscopy techniques, their advantages and drawbacks. We list experimental approaches recently used for either in vitro or in vivo studies. Next, we summarize how FRET contributed to the understanding of pre-mRNA splicing and spliceosome assembly.

  9. Assembly Modulated by Particle Position and Shape: A New Concept in Self-Assembly.

    Science.gov (United States)

    Tavacoli, Joe W; Heuvingh, Julien; Du Roure, Olivia

    2017-11-10

    In this communication we outline how the bespoke arrangements and design of micron-sized superparamagnetic shapes provide levers to modulate their assembly under homogeneous magnetic fields. We label this new approach, 'assembly modulated by particle position and shape' (APPS). Specifically, using rectangular lattices of superparamagnetic micron-sized cuboids, we construct distinct microstructures by adjusting lattice pitch and angle of array with respect to a magnetic field. Broadly, we find two modes of assembly: (1) immediate 2D jamming of the cuboids as they rotate to align with the applied field (rotation-induced jamming) and (2) aggregation via translation after their full alignment (dipole-dipole assembly). The boundary between these two assembly pathways is independent on field strength being solely a function of the cuboid's dimensions, lattice pitch, and array angle with respect to field-a relationship which we capture, along with other features of the assembly process, in a 'phase diagram'. In doing so, we set out initial design rules to build custom made assemblies. Moreover, these assemblies can be made flexible thanks to the hinged contacts of their particle building blocks. This flexibility, combined with the superparamagnetic nature of the architectures, renders our assembly method particularly appropriate for the construction of complex actuators at a scale hitherto not possible.

  10. Expression of alternatively spliced sodium channel alpha-subunit genes. Unique splicing patterns are observed in dorsal root ganglia.

    Science.gov (United States)

    Raymond, Christopher K; Castle, John; Garrett-Engele, Philip; Armour, Christopher D; Kan, Zhengyan; Tsinoremas, Nicholas; Johnson, Jason M

    2004-10-29

    Molecular medicine requires the precise definition of drug targets, and tools are now in place to provide genome-wide information on the expression and alternative splicing patterns of any known gene. DNA microarrays were used to monitor transcript levels of the nine well-characterized alpha-subunit sodium channel genes across a broad range of tissues from cynomolgus monkey, a non-human primate model. Alternative splicing of human transcripts for a subset of the genes that are expressed in dorsal root ganglia, SCN8A (Na(v)1.6), SCN9A (Na(v)1.7), and SCN11A (Na(v)1.9) was characterized in detail. Genomic sequence analysis among gene family paralogs and between cross-species orthologs suggested specific alternative splicing events within transcripts of these genes, all of which were experimentally confirmed in human tissues. Quantitative PCR revealed that certain alternative splice events are uniquely expressed in dorsal root ganglia. In addition to characterization of human transcripts, alternatively spliced sodium channel transcripts were monitored in a rat model for neuropathic pain. Consistent down-regulation of all transcripts was observed, as well as significant changes in the splicing patterns of SCN8A and SCN9A.

  11. Survey and alignment of VEPP-4M

    Energy Technology Data Exchange (ETDEWEB)

    Levashov, Y.I.; Bokov, M.A.; Pupkov, Y.A. [Budker Inst. of Nuclear Physics, Novosibirsk (Russian Federation)

    1996-01-01

    VEPP-4 is a 6 GeV electron-positron collider, and it has been operated since 1980. The magnetic system of the VEPP-4 was modified a few years ago to increase the luminosity and to extend the possibility for the experiments on nuclear physics and synchrotron radiation (SR). The new magnet lattice is fully symmetrical with respect to the interaction point (IP). Two regular cells in the center of both half-rings were replaced by the special insertions accomodating 14 SR beamlines. A new superconducting detector is being assembled now. The layout of the VEPP-4M is shown. The alignment system of the VEPP-4M has been changed on the basis of experience and the new apprehension of alignment goals. The main parameters of the VEPP-4M are shown. The accuracy required for positioning VEPP-4M components is given by the detailed tolerances. The alignment concept is explained. Each component of the magnet system has at least two fiducial marks, and the network monument has exactly the same reference socket as that used for the fiducial marks. The network consists of 94 permanent monuments fixed on the tunnel wall by brackets. Planimetric network and altimetric network, instrumentation and the results are reported. (K.I.)

  12. SmD1 Modulates the miRNA Pathway Independently of Its Pre-mRNA Splicing Function.

    Directory of Open Access Journals (Sweden)

    Xiao-Peng Xiong

    2015-08-01

    Full Text Available microRNAs (miRNAs are a class of endogenous regulatory RNAs that play a key role in myriad biological processes. Upon transcription, primary miRNA transcripts are sequentially processed by Drosha and Dicer ribonucleases into ~22-24 nt miRNAs. Subsequently, miRNAs are incorporated into the RNA-induced silencing complexes (RISCs that contain Argonaute (AGO family proteins and guide RISC to target RNAs via complementary base pairing, leading to post-transcriptional gene silencing by a combination of translation inhibition and mRNA destabilization. Select pre-mRNA splicing factors have been implicated in small RNA-mediated gene silencing pathways in fission yeast, worms, flies and mammals, but the underlying molecular mechanisms are not well understood. Here, we show that SmD1, a core component of the Drosophila small nuclear ribonucleoprotein particle (snRNP implicated in splicing, is required for miRNA biogenesis and function. SmD1 interacts with both the microprocessor component Pasha and pri-miRNAs, and is indispensable for optimal miRNA biogenesis. Depletion of SmD1 impairs the assembly and function of the miRISC without significantly affecting the expression of major canonical miRNA pathway components. Moreover, SmD1 physically and functionally associates with components of the miRISC, including AGO1 and GW182. Notably, miRNA defects resulting from SmD1 silencing can be uncoupled from defects in pre-mRNA splicing, and the miRNA and splicing machineries are physically and functionally distinct entities. Finally, photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP analysis identifies numerous SmD1-binding events across the transcriptome and reveals direct SmD1-miRNA interactions. Our study suggests that SmD1 plays a direct role in miRNA-mediated gene silencing independently of its pre-mRNA splicing activity and indicates that the dual roles of splicing factors in post-transcriptional gene regulation may be

  13. The CMS Muon System Alignment

    CERN Document Server

    Martinez Ruiz-Del-Arbol, P

    2009-01-01

    The alignment of the muon system of CMS is performed using different techniques: photogrammetry measurements, optical alignment and alignment with tracks. For track-based alignment, several methods are employed, ranging from a hit and impact point (HIP) algorithm and a procedure exploiting chamber overlaps to a global fit method based on the Millepede approach. For start-up alignment as long as available integrated luminosity is still significantly limiting the size of the muon sample from collisions, cosmic muon and beam halo signatures play a very strong role. During the last commissioning runs in 2008 the first aligned geometries have been produced and validated with data. The CMS offline computing infrastructure has been used in order to perform improved reconstructions. We present the computational aspects related to the calculation of alignment constants at the CERN Analysis Facility (CAF), the production and population of databases and the validation and performance in the official reconstruction. Also...

  14. Regulation of plant developmental processes by a novel splicing factor.

    Directory of Open Access Journals (Sweden)

    Gul Shad Ali

    Full Text Available Serine/arginine-rich (SR proteins play important roles in constitutive and alternative splicing and other aspects of mRNA metabolism. We have previously isolated a unique plant SR protein (SR45 with atypical domain organization. However, the biological and molecular functions of this novel SR protein are not known. Here, we report biological and molecular functions of this protein. Using an in vitro splicing complementation assay, we showed that SR45 functions as an essential splicing factor. Furthermore, the alternative splicing pattern of transcripts of several other SR genes was altered in a mutant, sr45-1, suggesting that the observed phenotypic abnormalities in sr45-1 are likely due to altered levels of SR protein isoforms, which in turn modulate splicing of other pre-mRNAs. sr45-1 exhibited developmental abnormalities, including delayed flowering, narrow leaves and altered number of petals and stamens. The late flowering phenotype was observed under both long days and short days and was rescued by vernalization. FLC, a key flowering repressor, is up-regulated in sr45-1 demonstrating that SR45 influences the autonomous flowering pathway. Changes in the alternative splicing of SR genes and the phenotypic defects in the mutant were rescued by SR45 cDNA, further confirming that the observed defects in the mutant are due to the lack of SR45. These results indicate that SR45 is a novel plant-specific splicing factor that plays a crucial role in regulating developmental processes.

  15. Alternative splicing events are a late feature of pathology in a mouse model of spinal muscular atrophy.

    Directory of Open Access Journals (Sweden)

    Dirk Bäumer

    2009-12-01

    Full Text Available Spinal muscular atrophy is a severe motor neuron disease caused by inactivating mutations in the SMN1 gene leading to reduced levels of full-length functional SMN protein. SMN is a critical mediator of spliceosomal protein assembly, and complete loss or drastic reduction in protein leads to loss of cell viability. However, the reason for selective motor neuron degeneration when SMN is reduced to levels which are tolerated by all other cell types is not currently understood. Widespread splicing abnormalities have recently been reported at end-stage in a mouse model of SMA, leading to the proposition that disruption of efficient splicing is the primary mechanism of motor neuron death. However, it remains unclear whether splicing abnormalities are present during early stages of the disease, which would be a requirement for a direct role in disease pathogenesis. We performed exon-array analysis of RNA from SMN deficient mouse spinal cord at 3 time points, pre-symptomatic (P1, early symptomatic (P7, and late-symptomatic (P13. Compared to littermate control mice, SMA mice showed a time-dependent increase in the number of exons showing differential expression, with minimal differences between genotypes at P1 and P7, but substantial variation in late-symptomatic (P13 mice. Gene ontology analysis revealed differences in pathways associated with neuronal development as well as cellular injury. Validation of selected targets by RT-PCR confirmed the array findings and was in keeping with a shift between physiologically occurring mRNA isoforms. We conclude that the majority of splicing changes occur late in SMA and may represent a secondary effect of cell injury, though we cannot rule out significant early changes in a small number of transcripts crucial to motor neuron survival.

  16. PEDOT as an alignment layer and electrode in AFLC devices

    Science.gov (United States)

    Medialdea, D. P.; Bennis, N.; Cerrolaza, B.; Quintana, X.; Otón, J. M.; Arredondo, B.; Romero, B.; Geday, M. A.

    2009-03-01

    PEDOT [Poly (3, 4-ethylenedioxythiophene) poly (styrenesulfonate)] is a material typically used organic light emission displays (OLEDs) manufactured as a conductor, or rather, as a hole injector. In this paper, three different issues have been studied: the use of rubbed PEDOT as alignment layer, the resistivity of the applied PEDOT layers, i.e., its potential as a replacement of ITO electrodes, and the induction of asymmetry in the electro-optical response of liquid crystal cells by assembling the display with dissimilar coated aligning surfaces — PEDOT on one side and buffed nylon on the counter electrode

  17. SOAP2: an improved ultrafast tool for short read alignment

    DEFF Research Database (Denmark)

    Li, Ruiqiang; Yu, Chang; Li, Yingrui

    2009-01-01

    for indexing the reference sequence in the main memory. We tested it on the whole human genome and found that this new algorithm reduced memory usage from 14.7 to 5.4 GB and improved alignment speed by 20-30 times. SOAP2 is compatible with both single- and paired-end reads. Additionally, this tool now supports...... multiple text and compressed file formats. A consensus builder has also been developed for consensus assembly and SNP detection from alignment of short reads on a reference genome. AVAILABILITY: http://soap.genomics.org.cn....

  18. Convergent origins and rapid evolution of spliced leader trans-splicing in metazoa: insights from the ctenophora and hydrozoa.

    Science.gov (United States)

    Derelle, Romain; Momose, Tsuyoshi; Manuel, Michael; Da Silva, Corinne; Wincker, Patrick; Houliston, Evelyn

    2010-04-01

    Replacement of mRNA 5' UTR sequences by short sequences trans-spliced from specialized, noncoding, spliced leader (SL) RNAs is an enigmatic phenomenon, occurring in a set of distantly related animal groups including urochordates, nematodes, flatworms, and hydra, as well as in Euglenozoa and dinoflagellates. Whether SL trans-splicing has a common evolutionary origin and biological function among different organisms remains unclear. We have undertaken a systematic identification of SL exons in cDNA sequence data sets from non-bilaterian metazoan species and their closest unicellular relatives. SL exons were identified in ctenophores and in hydrozoan cnidarians, but not in other cnidarians, placozoans, or sponges, or in animal unicellular relatives. Mapping of SL absence/presence obtained from this and previous studies onto current phylogenetic trees favors an evolutionary scenario involving multiple origins for SLs during eumetazoan evolution rather than loss from a common ancestor. In both ctenophore and hydrozoan species, multiple SL sequences were identified, showing high sequence diversity. Detailed analysis of a large data set generated for the hydrozoan Clytia hemisphaerica revealed trans-splicing of given mRNAs by multiple alternative SLs. No evidence was found for a common identity of trans-spliced mRNAs between different hydrozoans. One feature found specifically to characterize SL-spliced mRNAs in hydrozoans, however, was a marked adenosine enrichment immediately 3' of the SL acceptor splice site. Our findings of high sequence divergence and apparently indiscriminate use of SLs in hydrozoans, along with recent findings in other taxa, indicate that SL genes have evolved rapidly in parallel in diverse animal groups, with constraint on SL exon sequence evolution being apparently rare.

  19. General Assembly

    CERN Multimedia

    Staff Association

    2016-01-01

    5th April, 2016 – Ordinary General Assembly of the Staff Association! In the first semester of each year, the Staff Association (SA) invites its members to attend and participate in the Ordinary General Assembly (OGA). This year the OGA will be held on Tuesday, April 5th 2016 from 11:00 to 12:00 in BE Auditorium, Meyrin (6-2-024). During the Ordinary General Assembly, the activity and financial reports of the SA are presented and submitted for approval to the members. This is the occasion to get a global view on the activities of the SA, its financial management, and an opportunity to express one’s opinion, including taking part in the votes. Other points are listed on the agenda, as proposed by the Staff Council. Who can vote? Only “ordinary” members (MPE) of the SA can vote. Associated members (MPA) of the SA and/or affiliated pensioners have a right to vote on those topics that are of direct interest to them. Who can give his/her opinion? The Ordinary General Asse...

  20. Two new splice variants in porcine PPARGC1A

    Directory of Open Access Journals (Sweden)

    Peelman Luc J

    2008-12-01

    Full Text Available Abstract Background Peroxisome proliferator-activated receptor γ coactivator 1α (PPARGC1A is a coactivator with a vital and central role in fat and energy metabolism. It is considered to be a candidate gene for meat quality in pigs and is involved in the development of obesity and diabetes in humans. How its many functions are regulated, is however still largely unclear. Therefore a transcription profile of PPARGC1A in 32 tissues and 4 embryonic developmental stages in the pig was constructed by screening its cDNA for possible splice variants with exon-spanning primers. Findings This led to the discovery of 2 new splice variants in the pig, which were subsequently also detected in human tissues. In these variants, exon 8 was either completely or partly (the last 66 bp were conserved spliced out, potentially coding for a much shorter protein of respectively 337 and 359 amino acids (aa, of which the first 291 aa would be the same compared to the complete protein (796 aa. Conclusion Considering the functional domains of the PPARGC1A protein, it is very likely these splice variants considerably affect the function of the protein and alternative splicing could be one of the mechanisms by which the diverse functions of PPARGC1A are regulated.

  1. Alternative Splicing Regulates Targeting of Malate Dehydrogenase in Yarrowia lipolytica

    Science.gov (United States)

    Kabran, Philomène; Rossignol, Tristan; Gaillardin, Claude; Nicaud, Jean-Marc; Neuvéglise, Cécile

    2012-01-01

    Alternative pre-mRNA splicing is a major mechanism contributing to the proteome complexity of most eukaryotes, especially mammals. In less complex organisms, such as yeasts, the numbers of genes that contain introns are low and cases of alternative splicing (AS) with functional implications are rare. We report the first case of AS with functional consequences in the yeast Yarrowia lipolytica. The splicing pattern was found to govern the cellular localization of malate dehydrogenase, an enzyme of the central carbon metabolism. This ubiquitous enzyme is involved in the tricarboxylic acid cycle in mitochondria and in the glyoxylate cycle, which takes place in peroxisomes and the cytosol. In Saccharomyces cerevisiae, three genes encode three compartment-specific enzymes. In contrast, only two genes exist in Y. lipolytica. One gene (YlMDH1, YALI0D16753g) encodes a predicted mitochondrial protein, whereas the second gene (YlMDH2, YALI0E14190g) generates the cytosolic and peroxisomal forms through the alternative use of two 3′-splice sites in the second intron. Both splicing variants were detected in cDNA libraries obtained from cells grown under different conditions. Mutants expressing the individual YlMdh2p isoforms tagged with fluorescent proteins confirmed that they localized to either the cytosolic or the peroxisomal compartment. PMID:22368181

  2. Comparison of splice sites in mammals and chicken.

    Science.gov (United States)

    Abril, Josep F; Castelo, Robert; Guigó, Roderic

    2005-01-01

    We have carried out an initial analysis of the dynamics of the recent evolution of the splice-sites sequences on a large collection of human, rodent (mouse and rat), and chicken introns. Our results indicate that the sequences of splice sites are largely homogeneous within tetrapoda. We have also found that orthologous splice signals between human and rodents and within rodents are more conserved than unrelated splice sites, but the additional conservation can be explained mostly by background intron conservation. In contrast, additional conservation over background is detectable in orthologous mammalian and chicken splice sites. Our results also indicate that the U2 and U12 intron classes seem to have evolved independently since the split of mammals and birds; we have not been able to find a convincing case of interconversion between these two classes in our collections of orthologous introns. Similarly, we have not found a single case of switching between AT-AC and GT-AG subtypes within U12 introns, suggesting that this event has been a rare occurrence in recent evolutionary times. Switching between GT-AG and the noncanonical GC-AG U2 subtypes, on the contrary, does not appear to be unusual; in particular, T to C mutations appear to be relatively well tolerated in GT-AG introns with very strong donor sites.

  3. Control of human papillomavirus gene expression by alternative splicing.

    Science.gov (United States)

    Graham, Sheila V; Faizo, Arwa Ali A

    2017-03-02

    Human papillomaviruses possess circular double stranded DNA genomes of around 8kb in size from which multiple mRNAs are synthesized during an infectious life cycle. Although at least three viral promoters are used to initiate transcription, viral mRNAs are largely the product of processing of pre-mRNAs by alternative splicing and polyadenylation. The HPV life cycle and viral gene expression are tightly linked to differentiation of the epithelium the virus infects: there is an orchestrated production of viral mRNAs and proteins. In this review we describe viral mRNA expression and the roles of the SR and hnRNP proteins that respectively positively and negatively regulate splicing. We discuss HPV regulation of splicing factors and detail the evidence that the papillomavirus E2 protein has splicing-related activities. We highlight the possibility that HPV-mediated control of splicing in differentiating epithelial cells may be necessary to accomplish the viral replication cycle. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  4. A Challenging Pie to Splice: Drugging the Spliceosome.

    Science.gov (United States)

    León, Brian; Kashyap, Manoj K; Chan, Warren C; Krug, Kelsey A; Castro, Januario E; La Clair, James J; Burkart, Michael D

    2017-09-25

    Since its discovery in 1977, the study of alternative RNA splicing has revealed a plethora of mechanisms that had never before been documented in nature. Understanding these transitions and their outcome at the level of the cell and organism has become one of the great frontiers of modern chemical biology. Until 2007, this field remained in the hands of RNA biologists. However, the recent identification of natural product and synthetic modulators of RNA splicing has opened new access to this field, allowing for the first time a chemical-based interrogation of RNA splicing processes. Simultaneously, we have begun to understand the vital importance of splicing in disease, which offers a new platform for molecular discovery and therapy. As with many natural systems, gaining clear mechanistic detail at the molecular level is key towards understanding the operation of any biological machine. This minireview presents recent lessons learned in this emerging field of RNA splicing chemistry and chemical biology. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Pareto optimal pairwise sequence alignment.

    Science.gov (United States)

    DeRonne, Kevin W; Karypis, George

    2013-01-01

    Sequence alignment using evolutionary profiles is a commonly employed tool when investigating a protein. Many profile-profile scoring functions have been developed for use in such alignments, but there has not yet been a comprehensive study of Pareto optimal pairwise alignments for combining multiple such functions. We show that the problem of generating Pareto optimal pairwise alignments has an optimal substructure property, and develop an efficient algorithm for generating Pareto optimal frontiers of pairwise alignments. All possible sets of two, three, and four profile scoring functions are used from a pool of 11 functions and applied to 588 pairs of proteins in the ce_ref data set. The performance of the best objective combinations on ce_ref is also evaluated on an independent set of 913 protein pairs extracted from the BAliBASE RV11 data set. Our dynamic-programming-based heuristic approach produces approximated Pareto optimal frontiers of pairwise alignments that contain comparable alignments to those on the exact frontier, but on average in less than 1/58th the time in the case of four objectives. Our results show that the Pareto frontiers contain alignments whose quality is better than the alignments obtained by single objectives. However, the task of identifying a single high-quality alignment among those in the Pareto frontier remains challenging.

  6. All about alignment

    CERN Multimedia

    2006-01-01

    The ALICE absorbers, iron wall and superstructure have been installed with great precision. The ALICE front absorber, positioned in the centre of the detector, has been installed and aligned. Weighing more than 400 tonnes, the ALICE absorbers and the surrounding support structures have been installed and aligned with a precision of 1-2 mm, hardly an easy task but a very important one. The ALICE absorbers are made of three parts: the front absorber, a 35-tonne cone-shaped structure, and two small-angle absorbers, long straight cylinder sections weighing 18 and 40 tonnes. The three pieces lined up have a total length of about 17 m. In addition to these, ALICE technicians have installed a 300-tonne iron filter wall made of blocks that fit together like large Lego pieces and a surrounding metal support structure to hold the tracking and trigger chambers. The absorbers house the vacuum chamber and are also the reference surface for the positioning of the tracking and trigger chambers. For this reason, the ab...

  7. Two Influential Primate Classifications Logically Aligned.

    Science.gov (United States)

    Franz, Nico M; Pier, Naomi M; Reeder, Deeann M; Chen, Mingmin; Yu, Shizhuo; Kianmajd, Parisa; Bowers, Shawn; Ludäscher, Bertram

    2016-07-01

    Classifications and phylogenies of perceived natural entities change in the light of new evidence. Taxonomic changes, translated into Code-compliant names, frequently lead to name:meaning dissociations across succeeding treatments. Classification standards such as the Mammal Species of the World (MSW) may experience significant levels of taxonomic change from one edition to the next, with potential costs to long-term, large-scale information integration. This circumstance challenges the biodiversity and phylogenetic data communities to express taxonomic congruence and incongruence in ways that both humans and machines can process, that is, to logically represent taxonomic alignments across multiple classifications. We demonstrate that such alignments are feasible for two classifications of primates corresponding to the second and third MSW editions. Our approach has three main components: (i) use of taxonomic concept labels, that is name sec. author (where sec. means according to), to assemble each concept hierarchy separately via parent/child relationships; (ii) articulation of select concepts across the two hierarchies with user-provided Region Connection Calculus (RCC-5) relationships; and (iii) the use of an Answer Set Programming toolkit to infer and visualize logically consistent alignments of these input constraints. Our use case entails the Primates sec. Groves (1993; MSW2-317 taxonomic concepts; 233 at the species level) and Primates sec. Groves (2005; MSW3-483 taxonomic concepts; 376 at the species level). Using 402 RCC-5 input articulations, the reasoning process yields a single, consistent alignment and 153,111 Maximally Informative Relations that constitute a comprehensive meaning resolution map for every concept pair in the Primates sec. MSW2/MSW3. The complete alignment, and various partitions thereof, facilitate quantitative analyses of name:meaning dissociation, revealing that nearly one in three taxonomic names are not reliable across treatments

  8. Cadmium restores in vitro splicing activity inhibited by zinc-depletion

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Myeong Jin; Ayaki, Hitoshi; Kitamura, Keiko; Nishio, Hisahide [Kobe University Graduate School of Medicine, Department of Public Health, Kobe (Japan); Goji, Junko [Hyogo College of Medicine, Department of Public Health, Hyogo (Japan)

    2006-10-15

    Zinc (Zn)-depletion inhibits the second step of RNA splicing, namely exon-ligation. To investigate the effects of cadmium (Cd) and other metal ions on RNA splicing inhibited by Zn-depletion, we measured in vitro splicing activities in the presence of these metals. Zn-depletion in the splicing reaction mixture was achieved by addition of a Zn-chelator, 1,10-phenanthroline. Cd(II) at 1, 5 and 10 {mu}M restored the splicing activity to 2, 24 and 72% of that in the control reaction mixture, while higher concentrations of Cd(II) decreased the splicing activity, and more than 50 {mu}M Cd(II) showed a complete absence of spliced products. Hg(II) also restored the splicing activity, albeit to a lesser extent, since 5 and 10 {mu}M Hg(II) restored the splicing activity to 3 and 4% of the control value. The other metal ions examined in this study, Co(II), Cu(II), Mg(II) and Mn(II), did not show any restoration of the splicing activity. We concluded that Cd(II) could restore the in vitro splicing activity inhibited by Zn-depletion, although higher concentrations of Cd(II) prevented progress of the RNA splicing reaction. These results suggest that Cd(II) has a bifunctional property regarding RNA splicing, and is stimulatory at low concentrations and inhibitory at high concentrations. (orig.)

  9. The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms

    KAUST Repository

    Park, Hyo-Young

    2017-04-21

    The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms. These proteins also move rapidly and continuously in the nuclei, and their movements are affected by ATP depletion. The U2AF65 proteins are splicing factors that interact with SF1 and U2AF35 proteins to promote U2snRNP for the recognition of the pre-mRNA 3\\' splice site during early spliceosome assembly. We have determined the subcellular localization and movement of these proteins\\' Arabidopsis homologs. It was found that Arabidopsis U2AF65 homologs, AtU2AF65a, and AtU2AF65b proteins interact with AtU2AF35a and AtU2AF35b, which are Arabidopsis U2AF35 homologs. We have examined the mobility of these proteins including AtSF1 using fluorescence recovery after photobleaching and fluorescence loss in photobleaching analyses. These proteins displayed dynamic movements in nuclei and their movements were affected by ATP depletion. We have also demonstrated that these proteins shuttle between nuclei and cytoplasms, suggesting that they may also function in cytoplasm. These results indicate that such splicing factors show very similar characteristics to their human counterparts, suggesting evolutionary conservation.

  10. HnRNP L and hnRNP LL antagonistically modulate PTB-mediated splicing suppression of CHRNA1 pre-mRNA.

    Science.gov (United States)

    Rahman, Mohammad Alinoor; Masuda, Akio; Ohe, Kenji; Ito, Mikako; Hutchinson, David O; Mayeda, Akila; Engel, Andrew G; Ohno, Kinji

    2013-10-14

    CHRNA1 gene, encoding the muscle nicotinic acetylcholine receptor alpha subunit, harbors an inframe exon P3A. Inclusion of exon P3A disables assembly of the acetylcholine receptor subunits. A single nucleotide mutation in exon P3A identified in congenital myasthenic syndrome causes exclusive inclusion of exon P3A. The mutation gains a de novo binding affinity for a splicing enhancing RNA-binding protein, hnRNP LL, and displaces binding of a splicing suppressing RNA-binding protein, hnRNP L. The hnRNP L binds to another splicing repressor PTB through the proline-rich region and promotes PTB binding to the polypyrimidine tract upstream of exon P3A, whereas hnRNP LL lacking the proline-rich region cannot bind to PTB. Interaction of hnRNP L with PTB inhibits association of U2AF(65) and U1 snRNP with the upstream and downstream of P3A, respectively, which causes a defect in exon P3A definition. HnRNP L and hnRNP LL thus antagonistically modulate PTB-mediated splicing suppression of exon P3A.

  11. Onorbit IMU alignment error budget

    Science.gov (United States)

    Corson, R. W.

    1980-01-01

    The Star Tracker, Crew Optical Alignment Sight (COAS), and Inertial Measurement Unit (IMU) from a complex navigation system with a multitude of error sources were combined. A complete list of the system errors is presented. The errors were combined in a rational way to yield an estimate of the IMU alignment accuracy for STS-1. The expected standard deviation in the IMU alignment error for STS-1 type alignments was determined to be 72 arc seconds per axis for star tracker alignments and 188 arc seconds per axis for COAS alignments. These estimates are based on current knowledge of the star tracker, COAS, IMU, and navigation base error specifications, and were partially verified by preliminary Monte Carlo analysis.

  12. Widespread evolutionary conservation of alternatively spliced exons in caenorhabditis

    DEFF Research Database (Denmark)

    Irimia, Manuel; Rukov, Jakob L; Penny, David

    2007-01-01

    Alternative splicing (AS) contributes to increased transcriptome and proteome diversity in various eukaryotic lineages. Previous studies showed low levels of conservation of alternatively spliced (cassette) exons within mammals and within dipterans. We report a strikingly different pattern...... in Caenorhabditis nematodes-more than 92% of cassette exons from Caenorhabditis elegans are conserved in Caenorhabditis briggsae and/or Caenorhabditis remanei. High levels of conservation extend to minor-form exons (present in a minority of transcripts) and are particularly pronounced for exons showing complex...... patterns of splicing. The functionality of the vast majority of cassette exons is underscored by various other features. We suggest that differences in conservation between lineages reflect differences in levels of functionality and further suggest that these differences are due to differences in intron...

  13. Minor class splicing shapes the zebrafish transcriptome during development

    DEFF Research Database (Denmark)

    Markmiller, Sebastian; Cloonan, Nicole; Lardelli, Rea M

    2014-01-01

    as the U11/U12 di-snRNP 65-kDa protein, a unique component of the U12-type spliceosome. The biochemical impact of the mutation in clbn is the formation of aberrant U11- and U12-containing small nuclear ribonucleoproteins that impair the efficiency of U12-type splicing. Using RNA sequencing and microarrays....... Analysis of its transcriptome reveals efficient mRNA processing as a critical process for the growth and proliferation of cells during vertebrate development.......Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities...

  14. Identifying splicing regulatory elements with de Bruijn graphs.

    Science.gov (United States)

    Badr, Eman; Heath, Lenwood S

    2014-12-01

    Splicing regulatory elements (SREs) are short, degenerate sequences on pre-mRNA molecules that enhance or inhibit the splicing process via the binding of splicing factors, proteins that regulate the functioning of the spliceosome. Existing methods for identifying SREs in a genome are either experimental or computational. Here, we propose a formalism based on de Bruijn graphs that combines genomic structure, word count enrichment analysis, and experimental evidence to identify SREs found in exons. In our approach, SREs are not restricted to a fixed length (i.e., k-mers, for a fixed k). As a result, we identify 2001 putative exonic enhancers and 3080 putative exonic silencers for human genes, with lengths varying from 6 to 15 nucleotides. Many of the predicted SREs overlap with experimentally verified binding sites. Our model provides a novel method to predict variable length putative regulatory elements computationally for further experimental investigation.

  15. Stabilized cyclopropane analogs of the splicing inhibitor FD-895.

    Science.gov (United States)

    Villa, Reymundo; Kashyap, Manoj Kumar; Kumar, Deepak; Kipps, Thomas J; Castro, Januario E; La Clair, James J; Burkart, Michael D

    2013-09-12

    Targeting the spliceosome with small molecule inhibitors provides a new avenue to target cancer by intercepting alternate splicing pathways. Although our understanding of alternate mRNA splicing remains poorly understood, it provides an escape pathway for many cancers resistant to current therapeutics. These findings have encouraged recent academic and industrial efforts to develop natural product spliceosome inhibitors, including FD-895 (1a), pladienolide B (1b), and pladienolide D (1c), into next-generation anticancer drugs. The present study describes the application of semisynthesis and total synthesis to reveal key structure-activity relationships for the spliceosome inhibition by 1a. This information is applied to deliver new analogs with improved stability and potent activity at inhibiting splicing in patient derived cell lines.

  16. Alternative splice variants of the human PD-1 gene

    DEFF Research Database (Denmark)

    Nielsen, Christian; Ohm-Laursen, Line; Barington, Torben

    2005-01-01

    PD-1 is an immunoregulatory receptor expressed on the surface of activated T cells, B cells, and monocytes. We describe four alternatively spliced PD-1 mRNA transcripts (PD-1Deltaex2, PD-1Deltaex3, PD-1Deltaex2,3, and PD-1Deltaex2,3,4) in addition to the full length isoform. PD-1Deltaex2 and PD-1......Deltaex3 are generated by alternative splicing where exon 2 (extracellular IgV-like domain) and exon 3 (transmembrane domain) respectively are spliced out. PD-1Deltaex3 is therefore likely to encode a soluble form of PD-1. PD-1Deltaex2,3 lacks exon 2 and 3. These three variants have unaffected open...

  17. Global Genetic Robustness of the Alternative Splicing Machinery in Caenorhabditis elegans

    NARCIS (Netherlands)

    Li, Yang; Breitling, Rainer; Snoek, L. Basten; van der Velde, K. Joeri; Swertz, Morris A.; Riksen, Joost; Jansen, Ritsert C.; Kammenga, Jan E.; Borevitz, J.

    Alternative splicing is considered a major mechanism for creating multicellular diversity from a limited repertoire of genes. Here, we performed the first study of genetic variation controlling alternative splicing patterns by comprehensively identifying quantitative trait loci affecting the

  18. Nascent-seq indicates widespread cotranscriptional pre-mRNA splicing in Drosophila

    National Research Council Canada - National Science Library

    Khodor, Yevgenia L; Rodriguez, Joseph; Abruzzi, Katharine C; Tang, Chih-Hang Anthony; Marr, 2nd, Michael T; Rosbash, Michael

    2011-01-01

    ..., with ∼3% being almost completely retained in nascent pre-mRNA. Although individual introns showed slight but statistically significant differences in splicing efficiency, similar global levels of splicing were seen from both sources...

  19. Cloning, expression and alternative splicing of the novel isoform of hTCP11 gene

    DEFF Research Database (Denmark)

    Ma, Yong-xin; Zhang, Si-zhong; Wu, Qia-qing

    2003-01-01

    To identify a novel isoform of hTCP11 gene and investigate its expression and alternative splicing.......To identify a novel isoform of hTCP11 gene and investigate its expression and alternative splicing....

  20. Magnetic self-assembly of small parts

    Science.gov (United States)

    Shetye, Sheetal B.

    Modern society's propensity for miniaturized end-user products is compelling electronic manufacturers to assemble and package different micro-scale, multi-technology components in more efficient and cost-effective manners. As the size of the components gets smaller, issues such as part sticking and alignment precision create challenges that slow the throughput of conventional robotic pick-n-place systems. As an alternative, various self-assembly approaches have been proposed to manipulate micro to millimeter scale components in a parallel fashion without human or robotic intervention. In this dissertation, magnetic self-assembly (MSA) is demonstrated as a highly efficient, completely parallel process for assembly of millimeter scale components. MSA is achieved by integrating permanent micromagnets onto component bonding surfaces using wafer-level microfabrication processes. Embedded bonded powder methods are used for fabrication of the magnets. The magnets are then magnetized using pulse magnetization methods, and the wafers are then singulated to form individual components. When the components are randomly mixed together, self-assembly occurs when the intermagnetic forces overcome the mixing forces. Analytical and finite element methods (FEM) are used to study the force interactions between the micromagnets. The multifunctional aspects of MSA are presented through demonstration of part-to-part and part-to-substrate assembly of 1 mm x 1mm x 0.5 mm silicon components. Part-to-part assembly is demonstrated by batch assembly of free-floating parts in a liquid environment with the assembly yield of different magnetic patterns varying from 88% to 90% in 20 s. Part-to-substrate assembly is demonstrated by assembling an ordered array onto a fixed substrate in a dry environment with the assembly yield varying from 86% to 99%. In both cases, diverse magnetic shapes/patterns are used to control the alignment and angular orientation of the components. A mathematical model is

  1. The alignment strategy of HADES

    Energy Technology Data Exchange (ETDEWEB)

    Pechenova, O., E-mail: O.Pechenova@gsi.de [Institut für Kernphysik, Johann Wolfgang Goethe-Universität, 60438 Frankfurt (Germany); Pechenov, V. [GSI Helmholtzzentrum für Schwerionenforschung, 64291 Darmstadt (Germany); Galatyuk, T. [GSI Helmholtzzentrum für Schwerionenforschung, 64291 Darmstadt (Germany); Technische Universität Darmstadt, D-64289 Darmstadt (Germany); Hennino, T. [Institut de Physique Nucléaire (UMR 8608), CNRS/IN2P3 – Université Paris Sud, F-91406 Orsay Cedex (France); Holzmann, R. [GSI Helmholtzzentrum für Schwerionenforschung, 64291 Darmstadt (Germany); Kornakov, G. [Departamento de Física de Partículas, Univ. de Santiago de Compostela, 15706 Santiago de Compostela (Spain); Technische Universität Darmstadt, D-64289 Darmstadt (Germany); Markert, J.; Müntz, C. [Institut für Kernphysik, Johann Wolfgang Goethe-Universität, 60438 Frankfurt (Germany); Salabura, P. [Smoluchowski Institute of Physics, Jagiellonian University of Kraków, 30-059 Kraków (Poland); Schmah, A. [Lawrence Berkeley National Laboratory, Berkeley (United States); Schwab, E. [GSI Helmholtzzentrum für Schwerionenforschung, 64291 Darmstadt (Germany); Stroth, J. [Institut für Kernphysik, Johann Wolfgang Goethe-Universität, 60438 Frankfurt (Germany); GSI Helmholtzzentrum für Schwerionenforschung, 64291 Darmstadt (Germany)

    2015-06-11

    The global as well as intrinsic alignment of any spectrometer impacts directly on its performance and the quality of the achievable physics results. An overview of the current alignment procedure of the DiElectron Spectrometer HADES is presented with an emphasis on its main features and its accuracy. The sequence of all steps and procedures is given, including details on photogrammetric and track-based alignment.

  2. Lunar Alignments - Identification and Analysis

    Science.gov (United States)

    González-García, A. César

    Lunar alignments are difficult to establish given the apparent lack of written accounts clearly pointing toward lunar alignments for individual temples. While some individual cases are reviewed and highlighted, the weight of the proof must fall on statistical sampling. Some definitions for the lunar alignments are provided in order to clarify the targets, and thus, some new tools are provided to try to test the lunar hypothesis in several cases, especially in megalithic astronomy.

  3. Alignment of helical membrane protein sequences using AlignMe.

    Directory of Open Access Journals (Sweden)

    Marcus Stamm

    Full Text Available Few sequence alignment methods have been designed specifically for integral membrane proteins, even though these important proteins have distinct evolutionary and structural properties that might affect their alignments. Existing approaches typically consider membrane-related information either by using membrane-specific substitution matrices or by assigning distinct penalties for gap creation in transmembrane and non-transmembrane regions. Here, we ask whether favoring matching of predicted transmembrane segments within a standard dynamic programming algorithm can improve the accuracy of pairwise membrane protein sequence alignments. We tested various strategies using a specifically designed program called AlignMe. An updated set of homologous membrane protein structures, called HOMEP2, was used as a reference for optimizing the gap penalties. The best of the membrane-protein optimized approaches were then tested on an independent reference set of membrane protein sequence alignments from the BAliBASE collection. When secondary structure (S matching was combined with evolutionary information (using a position-specific substitution matrix (P, in an approach we called AlignMePS, the resultant pairwise alignments were typically among the most accurate over a broad range of sequence similarities when compared to available methods. Matching transmembrane predictions (T, in addition to evolutionary information, and secondary-structure predictions, in an approach called AlignMePST, generally reduces the accuracy of the alignments of closely-related proteins in the BAliBASE set relative to AlignMePS, but may be useful in cases of extremely distantly related proteins for which sequence information is less informative. The open source AlignMe code is available at https://sourceforge.net/projects/alignme/, and at http://www.forrestlab.org, along with an online server and the HOMEP2 data set.

  4. GraphAlignment: Bayesian pairwise alignment of biological networks

    Directory of Open Access Journals (Sweden)

    Kolář Michal

    2012-11-01

    Full Text Available Abstract Background With increased experimental availability and accuracy of bio-molecular networks, tools for their comparative and evolutionary analysis are needed. A key component for such studies is the alignment of networks. Results We introduce the Bioconductor package GraphAlignment for pairwise alignment of bio-molecular networks. The alignment incorporates information both from network vertices and network edges and is based on an explicit evolutionary model, allowing inference of all scoring parameters directly from empirical data. We compare the performance of our algorithm to an alternative algorithm, Græmlin 2.0. On simulated data, GraphAlignment outperforms Græmlin 2.0 in several benchmarks except for computational complexity. When there is little or no noise in the data, GraphAlignment is slower than Græmlin 2.0. It is faster than Græmlin 2.0 when processing noisy data containing spurious vertex associations. Its typical case complexity grows approximately as O(N2.6. On empirical bacterial protein-protein interaction networks (PIN and gene co-expression networks, GraphAlignment outperforms Græmlin 2.0 with respect to coverage and specificity, albeit by a small margin. On large eukaryotic PIN, Græmlin 2.0 outperforms GraphAlignment. Conclusions The GraphAlignment algorithm is robust to spurious vertex associations, correctly resolves paralogs, and shows very good performance in identification of homologous vertices defined by high vertex and/or interaction similarity. The simplicity and generality of GraphAlignment edge scoring makes the algorithm an appropriate choice for global alignment of networks.

  5. Genome-wide survey of allele-specific splicing in humans

    Directory of Open Access Journals (Sweden)

    Scheffler Konrad

    2008-06-01

    Full Text Available Abstract Background Accurate mRNA splicing depends on multiple regulatory signals encoded in the transcribed RNA sequence. Many examples of mutations within human splice regulatory regions that alter splicing qualitatively or quantitatively have been reported and allelic differences in mRNA splicing are likely to be a common and important source of phenotypic diversity at the molecular level, in addition to their contribution to genetic disease susceptibility. However, because the effect of a mutation on the efficiency of mRNA splicing is often difficult to predict, many mutations that cause disease through an effect on splicing are likely to remain undiscovered. Results We have combined a genome-wide scan for sequence polymorphisms likely to affect mRNA splicing with analysis of publicly available Expressed Sequence Tag (EST and exon array data. The genome-wide scan uses published tools and identified 30,977 SNPs located within donor and acceptor splice sites, branch points and exonic splicing enhancer elements. For 1,185 candidate splicing polymorphisms the difference in splicing between alternative alleles was corroborated by publicly available exon array data from 166 lymphoblastoid cell lines. We developed a novel probabilistic method to infer allele-specific splicing from EST data. The method uses SNPs and alternative mRNA isoforms mapped to EST sequences and models both regulated alternative splicing as well as allele-specific splicing. We have also estimated heritability of splicing and report that a greater proportion of genes show evidence of splicing heritability than show heritability of overall gene expression level. Our results provide an extensive resource that can be used to assess the possible effect on splicing of human polymorphisms in putative splice-regulatory sites. Conclusion We report a set of genes showing evidence of allele-specific splicing from an integrated analysis of genomic polymorphisms, EST data and exon array

  6. Alternative splicing of the AGAMOUS orthologous gene in double flower of Magnolia stellata (Magnoliaceae).

    Science.gov (United States)

    Zhang, Bo; Liu, Zhi-Xiong; Ma, Jiang; Song, Yi; Chen, Fa-Ju

    2015-12-01

    Magnolia stellata is a woody ornamental shrub with more petaloid tepals than related plants from family Magnoliaceae. Recent studies revealed that expression changes in an AGAMOUS (AG) orthologous gene could resulted in double flowers with increased numbers of petals. We isolated three transcripts encoding different isoforms of a single AG orthologous gene, MastAG, mastag_2 and mastag_3, from M. stellata. Sequence alignments and Southern blot analyses suggested that MastAG was a single-copy gene in M. stellata genomes, and that mastag_2 and mastag_3 were abnormally spliced isoforms of MastAG. An 144bp exon skipping in MastAG results in the truncated mastag_2 protein lacking the completely I domain and 18 aa of the K1 subdomain, whereas an 165bp exon skipping of MastAG produces a truncated mastag_3 protein lacking 6 aa of the K3 subdomain and the completely C terminal region. Expression analyses showed that three alternative splicing (AS) isoforms expressed only in developing stamens and carpels. Functional analyses revealed that MastAG could mimic the endogenous AG to specify carpel identity, but failed to regulate stamen development in an Arabidopsis ag-1 mutant. Moreover, the key domain or subdomain deletions represented by mastag_2 and mastag_3 resulted in loss of C-function. However, ectopic expression of mastag_2 in Arabidopsis produced flowers with sepals converted into carpeloid organs, but without petals and stamens, whereas ectopic expression of mastag_3 in Arabidopsis could mimic the flower phenotype of the ag mutant and produced double flowers with homeotic transformation of stamens into petals and carpels into another ag flower. Our results also suggest that mastag_3 holds some potential for biotechnical engineering to create multi-petal phenotypes in commercial ornamental cultivars. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  7. Assembling consumption

    DEFF Research Database (Denmark)

    Assembling Consumption marks a definitive step in the institutionalisation of qualitative business research. By gathering leading scholars and educators who study markets, marketing and consumption through the lenses of philosophy, sociology and anthropology, this book clarifies and applies...... the investigative tools offered by assemblage theory, actor-network theory and non-representational theory. Clear theoretical explanation and methodological innovation, alongside empirical applications of these emerging frameworks will offer readers new and refreshing perspectives on consumer culture and market...... societies. This is an essential reading for both seasoned scholars and advanced students of markets, economies and social forms of consumption....

  8. The exon junction complex differentially marks spliced junctions.

    Science.gov (United States)

    Saulière, Jérôme; Haque, Nazmul; Harms, Scot; Barbosa, Isabelle; Blanchette, Marco; Le Hir, Hervé

    2010-10-01

    The exon junction complex (EJC), which is deposited onto mRNAs as a consequence of splicing, is involved in multiple post-transcriptional events in metazoa. Here, using Drosophila melanogaster cells, we show that only some introns trigger EJC-dependent nonsense-mediated mRNA decay and that EJC association with particular spliced junctions depends on RNA cis-acting sequences. This study provides the first evidence to our knowledge that EJC deposition is not constitutive but instead is a regulated process.

  9. Chapter 1. Regulation of HIV-1 alternative RNA splicing and its role in virus replication.

    Science.gov (United States)

    Stoltzfus, C Martin

    2009-01-01

    Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNA species, both completely and incompletely spliced, are produced by alternative splicing of the primary viral RNA transcript. In addition, about half of the viral RNA remains unspliced and is transported to the cytoplasm where it is used both as mRNA and as genomic RNA. In general, the identities of the completely and incompletely spliced HIV-1 mRNA species are determined by the proximity of the open reading frames to the 5'-end of the mRNAs. The relative abundance of the mRNAs encoding the HIV-1 gene products is determined by the frequency of splicing at the different alternative 3'-splice sites. This chapter will highlight studies showing how HIV-1 uses exon definition to control the level of splicing at each of its 3'-splice sites through a combination of positively acting exonic splicing enhancer (ESE) elements, negatively acting exonic and intronic splicing silencer elements (ESS and ISS elements, respectively), and the 5'-splice sites of the regulated exons. Each of these splicing elements represent binding sites for cellular factors whose levels in the infected cell can determine the dominance of the positive or negative elements on HIV-1 alternative splicing. Both mutations of HIV-1 splicing elements and overexpression or inhibition of cellular splicing factors that bind to these elements have been used to show that disruption of regulated splicing inhibits HIV-1 replication. These studies have provided strong rationale for the investigation and development of antiviral drugs that specifically inhibit HIV-1 RNA splicing.

  10. Can the HIV-1 splicing machinery be targeted for drug discovery?

    Directory of Open Access Journals (Sweden)

    Dlamini Z

    2017-03-01

    Full Text Available Zodwa Dlamini, Rodney Hull Research, Innovation & Engagements Portfolio, Mangosuthu University of Technology, Durban, South Africa Abstract: HIV-1 is able to express multiple protein types and isoforms from a single 9 kb mRNA transcript. These proteins are also expressed at particular stages of viral development, and this is achieved through the control of alternative splicing and the export of these transcripts from the nucleus. The nuclear export is controlled by the HIV protein Rev being required to transport incompletely spliced and partially spliced mRNA from the nucleus where they are normally retained. This implies a close relationship between the control of alternate splicing and the nuclear export of mRNA in the control of HIV-1 viral proliferation. This review discusses both the processes. The specificity and regulation of splicing in HIV-1 is controlled by the use of specific splice sites as well as exonic splicing enhancer and exonic splicing silencer sequences. The use of these silencer and enhancer sequences is dependent on the serine arginine family of proteins as well as the heterogeneous nuclear ribonucleoprotein family of proteins that bind to these sequences and increase or decrease splicing. Since alternative splicing is such a critical factor in viral development, it presents itself as a promising drug target. This review aims to discuss the inhibition of splicing, which would stall viral development, as an anti-HIV therapeutic strategy. In this review, the most recent knowledge of splicing in human immunodeficiency viral development and the latest therapeutic strategies targeting human immunodeficiency viral splicing are discussed. Keywords: alternative splicing, exonic splicing enhancer, exonic specific silencer, splicing based therapies, SR proteins, hnRNP, Rev, Tat, Vpr

  11. An erythroid differentiation-specific splicing switch in protein 4.1R mediated by the interaction of SF2/ASF with an exonic splicing enhancer.

    Science.gov (United States)

    Yang, Guang; Huang, Shu-Ching; Wu, Jane Y; Benz, Edward J

    2005-03-01

    Protein 4.1R is a vital component of the red blood cell membrane cytoskeleton. Promotion of cytoskeletal junctional complex stability requires an erythroid differentiation stage-specific splicing switch promoting inclusion of exon 16 within the spectrin/actin binding domain. We showed earlier that an intricate combination of positive and negative RNA elements controls exon 16 splicing. In this report, we further identified 3 putative exonic splicing enhancers within exon 16 and investigated the function of the sequence CAGACAT in the regulation of exon 16 splicing. Mutation of these sequences leads to increased exclusion of exon 16 in both in vivo and in vitro splicing assays, indicating that CAGACAT is a functional exonic splicing enhancer. UV cross-linking further detects an approximately 33-kDa protein that specifically binds to the CAGACAT-containing transcript. An anti-SF2/ASF antibody specifically immunoprecipitates the approximately 33-kDa protein. Furthermore, SF2/ASF stimulates exon 16 inclusion in both in vitro complementation assays and minigene-transfected mouse erythroleukemia cells (MELCs). Finally, SF2/ASF expression is up-regulated and correlates with exon 16 inclusion in differentiated MELCs. These results suggest that increased splicing factor 2/alternative splicing factor (SF2/ASF) expression in differentiated mouse erythroleukemia mediates a differentiation stage-specific exon 16 splicing switch through its interaction with the exonic splicing enhancer.

  12. Analysis of a botryllid enriched-full-length cDNA library: insight into the evolution of spliced leader trans-splicing in tunicates.

    Science.gov (United States)

    Gasparini, Fabio; Shimeld, Sebastian M

    2011-03-01

    In some animals, mRNA may be modified after transcription by the addition of a 5' spliced leader sequence. This is known as spliced leader (SL) trans-splicing, and is of uncertain function and evolutionary origin. Here, we report the identification of SL trans-splicing in the colonial ascidian Botryllus schlosseri. Combining our own expressed sequence tag (EST) data with additional data from GenBank, we identify the dominant spliced leader sequence and show it to be similar to that of other ascidians and to that of Oikopleura dioica, a basally diverging tunicate. Gene Ontology analysis of B. schlosseri ESTs with and without a 5' spliced leader shows that genes encoding ribosomal proteins tend not to be trans-spliced, a character shared with the ascidian Ciona intestinalis. We also examine individual cases of genes that produce mRNAs that are SL trans-spliced in B. schlosseri but not in C. intestinalis. We conclude that SL trans-splicing evolved early in the tunicate lineage and shows stability over considerable evolutionary time. However, SL trans-splicing may be gained or lost in individual genes.

  13. Evaluation of alignment algorithms for discovery and identification of pathogens using RNA-Seq.

    Directory of Open Access Journals (Sweden)

    Ivan Borozan

    Full Text Available Next-generation sequencing technologies provide an unparallelled opportunity for the characterization and discovery of known and novel viruses. Because viruses are known to have the highest mutation rates when compared to eukaryotic and bacterial organisms, we assess the extent to which eleven well-known alignment algorithms (BLAST, BLAT, BWA, BWA-SW, BWA-MEM, BFAST, Bowtie2, Novoalign, GSNAP, SHRiMP2 and STAR can be used for characterizing mutated and non-mutated viral sequences--including those that exhibit RNA splicing--in transcriptome samples. To evaluate aligners objectively we developed a realistic RNA-Seq simulation and evaluation framework (RiSER and propose a new combined score to rank aligners for viral characterization in terms of their precision, sensitivity and alignment accuracy. We used RiSER to simulate both human and viral read sequences and suggest the best set of aligners for viral sequence characterization in human transcriptome samples. Our results show that significant and substantial differences exist between aligners and that a digital-subtraction-based viral identification framework can and should use different aligners for different parts of the process. We determine the extent to which mutated viral sequences can be effectively characterized and show that more sensitive aligners such as BLAST, BFAST, SHRiMP2, BWA-SW and GSNAP can accurately characterize substantially divergent viral sequences with up to 15% overall sequence mutation rate. We believe that the results presented here will be useful to researchers choosing aligners for viral sequence characterization using next-generation sequencing data.

  14. Prediction of molecular alignment of nucleic acids in aligned media

    NARCIS (Netherlands)

    Wu, B.; Petersen, M.; Girard, F.C.; Tessari, M.; Wijmenga, S.S.

    2006-01-01

    We demonstrate - using the data base of all deposited DNA and RNA structures aligned in Pf1-medium and RDC refined - that for nucleic acids in a Pf1-medium the electrostatic alignment tensor can be predicted reliably and accurately via a simple and fast calculation based on the gyration tensor

  15. General Assembly

    CERN Multimedia

    Staff Association

    2015-01-01

    Mardi 5 mai à 11 h 00 Salle 13-2-005 Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : 1- Adoption de l’ordre du jour. 2- Approbation du procès-verbal de l’Assemblée générale ordinaire du 22 mai 2014. 3- Présentation et approbation du rapport d’activités 2014. 4- Présentation et approbation du rapport financier 2014. 5- Présentation et approbation du rapport des vérificateurs aux comptes pour 2014. 6- Programme 2015. 7- Présentation et approbation du projet de budget 2015 et taux de cotisation pour 2015. 8- Pas de modifications aux Statuts de l'Association du personnel proposée. 9- Élections des membres de la Commission é...

  16. General assembly

    CERN Multimedia

    Staff Association

    2015-01-01

    Mardi 5 mai à 11 h 00 Salle 13-2-005 Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : Adoption de l’ordre du jour. Approbation du procès-verbal de l’Assemblée générale ordinaire du 22 mai 2014. Présentation et approbation du rapport d’activités 2014. Présentation et approbation du rapport financier 2014. Présentation et approbation du rapport des vérificateurs aux comptes pour 2014. Programme 2015. Présentation et approbation du projet de budget 2015 et taux de cotisation pour 2015. Pas de modifications aux Statuts de l'Association du personnel proposée. Élections des membres de la Commission électorale. &am...

  17. General Assembly

    CERN Multimedia

    Staff Association

    2016-01-01

    Mardi 5 avril à 11 h 00 BE Auditorium Meyrin (6-2-024) Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : Adoption de l’ordre du jour. Approbation du procès-verbal de l’Assemblée générale ordinaire du 5 mai 2015. Présentation et approbation du rapport d’activités 2015. Présentation et approbation du rapport financier 2015. Présentation et approbation du rapport des vérificateurs aux comptes pour 2015. Programme de travail 2016. Présentation et approbation du projet de budget 2016 Approbation du taux de cotisation pour 2017. Modifications aux Statuts de l'Association du personnel proposée. Élections des membres de la Commissio...

  18. General Assembly

    CERN Multimedia

    Staff Association

    2017-01-01

    Conformément aux statuts de l’Association du personnel, une Assemblée générale ordinaire est organisée une fois par année (article IV.2.1). Projet d’ordre du jour : Adoption de l’ordre du jour. Approbation du procès-verbal de l’Assemblée générale ordinaire du 5 avril 2016. Présentation et approbation du rapport d’activités 2016. Présentation et approbation du rapport financier 2016. Présentation et approbation du rapport des vérificateurs aux comptes pour 2016. Programme de travail 2017. Présentation et approbation du projet de budget 2017 Approbation du taux de cotisation pour 2018. Modifications aux Statuts de l'Association du personnel proposées. Élections des membres de la Commission électorale. Élections des vérifica...

  19. ISVASE: identification of sequence variant associated with splicing event using RNA-seq data.

    Science.gov (United States)

    Aljohi, Hasan Awad; Liu, Wanfei; Lin, Qiang; Yu, Jun; Hu, Songnian

    2017-06-28

    Exon recognition and splicing precisely and efficiently by spliceosome is the key to generate mature mRNAs. About one third or a half of disease-related mutations affect RNA splicing. Software PVAAS has been developed to identify variants associated with aberrant splicing by directly using RNA-seq data. However, it bases on the assumption that annotated splicing site is normal splicing, which is not true in fact. We develop the ISVASE, a tool for specifically identifying sequence variants associated with splicing events (SVASE) by using RNA-seq data. Comparing with PVAAS, our tool has several advantages, such as multi-pass stringent rule-dependent filters and statistical filters, only using split-reads, independent sequence variant identification in each part of splicing (junction), sequence variant detection for both of known and novel splicing event, additional exon-exon junction shift event detection if known splicing events provided, splicing signal evaluation, known DNA mutation and/or RNA editing data supported, higher precision and consistency, and short running time. Using a realistic RNA-seq dataset, we performed a case study to illustrate the functionality and effectiveness of our method. Moreover, the output of SVASEs can be used for downstream analysis such as splicing regulatory element study and sequence variant functional analysis. ISVASE is useful for researchers interested in sequence variants (DNA mutation and/or RNA editing) associated with splicing events. The package is freely available at https://sourceforge.net/projects/isvase/ .

  20. Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes

    Science.gov (United States)

    Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

    2013-01-01

    DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893

  1. Interplay between DMD point mutations and splicing signals in Dystrophinopathy phenotypes.

    Directory of Open Access Journals (Sweden)

    Jonàs Juan-Mateu

    Full Text Available DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements.

  2. High-throughput sequence alignment using Graphics Processing Units

    Directory of Open Access Journals (Sweden)

    Trapnell Cole

    2007-12-01

    Full Text Available Abstract Background The recent availability of new, less expensive high-throughput DNA sequencing technologies has yielded a dramatic increase in the volume of sequence data that must be analyzed. These data are being generated for several purposes, including genotyping, genome resequencing, metagenomics, and de novo genome assembly projects. Sequence alignment programs such as MUMmer have proven essential for analysis of these data, but researchers will need ever faster, high-throughput alignment tools running on inexpensive hardware to keep up with new sequence technologies. Results This paper describes MUMmerGPU, an open-source high-throughput parallel pairwise local sequence alignment program that runs on commodity Graphics Processing Units (GPUs in common workstations. MUMmerGPU uses the new Compute Unified Device Architecture (CUDA from nVidia to align multiple query sequences against a single reference sequence stored as a suffix tree. By processing the queries in parallel on the highly parallel graphics card, MUMmerGPU achieves more than a 10-fold speedup over a serial CPU version of the sequence alignment kernel, and outperforms the exact alignment component of MUMmer on a high end CPU by 3.5-fold in total application time when aligning reads from recent sequencing projects using Solexa/Illumina, 454, and Sanger sequencing technologies. Conclusion MUMmerGPU is a low cost, ultra-fast sequence alignment program designed to handle the increasing volume of data produced by new, high-throughput sequencing technologies. MUMmerGPU demonstrates that even memory-intensive applications can run significantly faster on the relatively low-cost GPU than on the CPU.

  3. High-throughput sequence alignment using Graphics Processing Units.

    Science.gov (United States)

    Schatz, Michael C; Trapnell, Cole; Delcher, Arthur L; Varshney, Amitabh

    2007-12-10

    The recent availability of new, less expensive high-throughput DNA sequencing technologies has yielded a dramatic increase in the volume of sequence data that must be analyzed. These data are being generated for several purposes, including genotyping, genome resequencing, metagenomics, and de novo genome assembly projects. Sequence alignment programs such as MUMmer have proven essential for analysis of these data, but researchers will need ever faster, high-throughput alignment tools running on inexpensive hardware to keep up with new sequence technologies. This paper describes MUMmerGPU, an open-source high-throughput parallel pairwise local sequence alignment program that runs on commodity Graphics Processing Units (GPUs) in common workstations. MUMmerGPU uses the new Compute Unified Device Architecture (CUDA) from nVidia to align multiple query sequences against a single reference sequence stored as a suffix tree. By processing the queries in parallel on the highly parallel graphics card, MUMmerGPU achieves more than a 10-fold speedup over a serial CPU version of the sequence alignment kernel, and outperforms the exact alignment component of MUMmer on a high end CPU by 3.5-fold in total application time when aligning reads from recent sequencing projects using Solexa/Illumina, 454, and Sanger sequencing technologies. MUMmerGPU is a low cost, ultra-fast sequence alignment program designed to handle the increasing volume of data produced by new, high-throughput sequencing technologies. MUMmerGPU demonstrates that even memory-intensive applications can run significantly faster on the relatively low-cost GPU than on the CPU.

  4. Intradomain Textures in Block Copolymers: Multizone Alignment and Biaxiality

    Science.gov (United States)

    Prasad, Ishan; Seo, Youngmi; Hall, Lisa M.; Grason, Gregory M.

    2017-06-01

    Block copolymer (BCP) melt assembly has been studied for decades, focusing largely on self-organized spatial patterns of periodically ordered segment density. Here, we demonstrate that underlying the well-known composition profiles (i.e., ordered lamella, cylinders, spheres, and networks) are generic and heterogeneous patterns of segment orientation that couple strongly to morphology, even in the absence of specific factors that promote intra or interchain segment alignment. We employ both self-consistent field theory and coarse-grained simulation methods to measure polar and nematic order parameters of segments in a freely jointed chain model of diblock melts. We show that BCP morphologies have a multizone texture, with segments predominantly aligned normal and parallel to interdomain interfaces in the respective brush and interfacial regions of the microdomain. Further, morphologies with anisotropically curved interfaces (i.e., cylinders and networks) exhibit biaxial order that is aligned to the principal curvature axes of the interface.

  5. From Cryptic Toward Canonical Pre-mRNA Splicing in Pompe Disease: a Pipeline for the Development of Antisense Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Atze J Bergsma

    2016-01-01

    Full Text Available While 9% of human pathogenic variants have an established effect on pre-mRNA splicing, it is suspected that an additional 20% of otherwise classified variants also affect splicing. Aberrant splicing includes disruption of splice sites or regulatory elements, or creation or strengthening of cryptic splice sites. For the majority of variants, it is poorly understood to what extent and how these may affect splicing. We have identified cryptic splicing in an unbiased manner. Three types of cryptic splicing were analyzed in the context of pathogenic variants in the acid α-glucosidase gene causing Pompe disease. These involved newly formed deep intronic or exonic cryptic splice sites, and a natural cryptic splice that was utilized due to weakening of a canonical splice site. Antisense oligonucleotides that targeted the identified cryptic splice sites repressed cryptic splicing at the expense of canonical splicing in all three cases, as shown by reverse-transcriptase-quantitative polymerase chain reaction analysis and by enhancement of acid α-glucosidase enzymatic activity. This argues for a competition model for available splice sites, including intact or weakened canonical sites and natural or newly formed cryptic sites. The pipeline described here can detect cryptic splicing and correct canonical splicing using antisense oligonucleotides to restore the gene defect.

  6. The Semantics of Ontology Alignment

    Science.gov (United States)

    2004-08-01

    AUG 2004 2. REPORT TYPE 3. DATES COVERED 00-00-2004 to 00-00-2004 4. TITLE AND SUBTITLE The Semantics of Ontology Alignment 5a. CONTRACT NUMBER...ABSTRACT unclassified c. THIS PAGE unclassified Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 1 The Semantics of Ontology Alignment

  7. Lexical alignment in triadic communication

    Directory of Open Access Journals (Sweden)

    Anouschka eFoltz

    2015-02-01

    Full Text Available Lexical alignment refers to the adoption of one’s interlocutor’s lexical items. Accounts of the mechanisms underlying such lexical alignment differ (among other aspects in the role assigned to addressee-centered behavior. In this study, we used a triadic communicative situation to test which factors may modulate the extent to which participants’ lexical alignment reflects addressee-centered behavior. Pairs of naïve participants played a picture matching game and received information about the order in which pictures were to be matched from a voice over headphones. On critical trials, participants did or did not hear a name for the picture to be matched next over headphones. Importantly, when the voice over headphones provided a name, it did not match the name that the interlocutor had previously used to describe the object. Participants overwhelmingly used the word that the voice over headphones provided. This result points to non-addressee-centered behavior and is discussed in terms of disrupting alignment with the interlocutor as well as in terms of establishing alignment with the voice over headphones. In addition, the type of picture (line drawing vs. tangram shape independently modulated lexical alignment, such that participants showed more lexical alignment to their interlocutor for (more ambiguous tangram shapes compared to line drawings. Overall, the results point to a rather large role for non-addressee-centered behavior during lexical alignment.

  8. Rotational alignment in soft nuclei

    Energy Technology Data Exchange (ETDEWEB)

    Nojarov, R. (Bylgarska Akademiya na Naukite, Sofia. Inst. po Yadrena Fizika i Yadrena Energetika)

    1983-12-08

    It is shown that in transitional odd-A nuclei, where the rotation-aligned coupling scheme usually takes place, the low collective angular momentum states of the decoupled band are not completely aligned due to core softness. This is illustrated on the example of La-nuclei.

  9. Collective alignment of nanorods in thin Newtonian films

    Science.gov (United States)

    Gu, Yu; Burtovyy, Ruslan; Townsend, James; Owens, Jeffery; Luzinov, Igor; Kornev, Konstantin

    2013-11-01

    We provide a complete analytical description of the alignment kinetics of magnetic nanorods in magnetic field. Nickel nanorods were formed by template electrochemical deposition in alumina membranes from a dispersion in a water-glycerol mixture. To ensure uniformity of the dispersion, the surface of the nickel nanorods was covered with polyvinylpyrrolidone (PVP). A 40-70 nm coating prevented aggregation of nanoroda. These modifications allowed us to control alignment of the nanorods in a magnetic field and test the proposed theory. An orientational distribution function of nanorods was introduced. We demonstrated that the 0.04% volume fraction of nanorods in the glycerol-water mixture behaves as a system of non-interacting particles. However, the kinetics of alignment of a nanorod assembly does not follow the predictions of the single-nanorod theory. The distribution function theory explains the kinetics of alignment of a nanorod assembly and shows the significance of the initial distribution of nanorods in the film. It can be used to develop an experimental protocol for controlled ordering of magnetic nanorods in thin films. This work was supported by the Air Force Office of Scientific Research, Grant numbers FA9550-12-1-0459 and FA8650-09-D-507 5900.

  10. Rules and tools to predict the splicing effects of exonic and intronic mutations.

    Science.gov (United States)

    Ohno, Kinji; Takeda, Jun-Ichi; Masuda, Akio

    2017-09-26

    Development of next generation sequencing technologies has enabled detection of extensive arrays of germline and somatic single nucleotide variations (SNVs) in human diseases. SNVs affecting intronic GT-AG dinucleotides invariably compromise pre-mRNA splicing. Most exonic SNVs introduce missense/nonsense codons, but some affect auxiliary splicing cis-elements or generate cryptic GT-AG dinucleotides. Similarly, most intronic SNVs are silent, but some affect canonical and auxiliary splicing cis-elements or generate cryptic GT-AG dinucleotides. However, prediction of the splicing effects of SNVs is challenging. The splicing effects of SNVs generating cryptic AG or disrupting canonical AG can be inferred from the AG-scanning model. Similarly, the splicing effects of SNVs affecting the first nucleotide G of an exon can be inferred from AG-dependence of the 3' splice site (ss). A variety of tools have been developed for predicting the splicing effects of SNVs affecting the 5' ss, as well as exonic and intronic splicing enhancers/silencers. In contrast, only two tools, the Human Splicing Finder and the SVM-BP finder, are available for predicting the position of the branch point sequence. Similarly, IntSplice and Splicing based Analysis of Variants (SPANR) are the only tools to predict the splicing effects of intronic SNVs. The rules and tools introduced in this review are mostly based on observations of a limited number of genes, and no rule or tool can ensure 100% accuracy. Experimental validation is always required before any clinically relevant conclusions are drawn. Development of efficient tools to predict aberrant splicing, however, will facilitate our understanding of splicing pathomechanisms in human diseases. For further resources related to this article, please visit the WIREs website. © 2017 Wiley Periodicals, Inc.

  11. Long-range RNA pairings contribute to mutually exclusive splicing

    Science.gov (United States)

    Yue, Yuan; Yang, Yun; Dai, Lanzhi; Cao, Guozheng; Chen, Ran; Hong, Weiling; Liu, Baoping; Shi, Yang; Meng, Yijun; Shi, Feng; Xiao, Mu; Jin, Yongfeng

    2016-01-01

    Mutually exclusive splicing is an important means of increasing the protein repertoire, by which the Down's syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 different isoforms in Drosophila melanogaster. However, the regulatory mechanisms remain obscure due to the complexity of the Dscam exon cluster. Here, we reveal a molecular model for the regulation of the mutually exclusive splicing of the serpent pre-mRNA based on competition between upstream and downstream RNA pairings. Such dual RNA pairings confer fine tuning of the inclusion of alternative exons. Moreover, we demonstrate that the splicing outcome of alternative exons is mediated in relative pairing strength-correlated mode. Combined comparative genomics analysis and experimental evidence revealed similar bidirectional structural architectures in exon clusters 4 and 9 of the Dscam gene. Our findings provide a novel mechanistic framework for the regulation of mutually exclusive splicing and may offer potentially applicable insights into long-range RNA–RNA interactions in gene regulatory networks. PMID:26554032

  12. Splicing and local reinforcement of concrete filled FRP tubes.

    Science.gov (United States)

    2014-01-01

    This report includes fulfillment of Task 1 of a multi-task contract to further enhance concrete filled FRP tubes, or : the Bridge in a Backpack. Task 1 investigates and develops a feasible solution for splicing the concrete filled FRP : tubes. This w...

  13. Splicing regulator SLU7 is essential for maintaining liver homeostasis.

    Science.gov (United States)

    Elizalde, María; Urtasun, Raquel; Azkona, María; Latasa, María U; Goñi, Saioa; García-Irigoyen, Oihane; Uriarte, Iker; Segura, Victor; Collantes, María; Di Scala, Mariana; Lujambio, Amaia; Prieto, Jesús; Ávila, Matías A; Berasain, Carmen

    2014-07-01

    A precise equilibrium between cellular differentiation and proliferation is fundamental for tissue homeostasis. Maintaining this balance is particularly important for the liver, a highly differentiated organ with systemic metabolic functions that is endowed with unparalleled regenerative potential. Carcinogenesis in the liver develops as the result of hepatocellular de-differentiation and uncontrolled proliferation. Here, we identified SLU7, which encodes a pre-mRNA splicing regulator that is inhibited in hepatocarcinoma, as a pivotal gene for hepatocellular homeostasis. SLU7 knockdown in human liver cells and mouse liver resulted in profound changes in pre-mRNA splicing and gene expression, leading to impaired glucose and lipid metabolism, refractoriness to key metabolic hormones, and reversion to a fetal-like gene expression pattern. Additionally, loss of SLU7 also increased hepatocellular proliferation and induced a switch to a tumor-like glycolytic phenotype. Slu7 governed the splicing and/or expression of multiple genes essential for hepatocellular differentiation, including serine/arginine-rich splicing factor 3 (Srsf3) and hepatocyte nuclear factor 4α (Hnf4α), and was critical for cAMP-regulated gene transcription. Together, out data indicate that SLU7 is central regulator of hepatocyte identity and quiescence.

  14. Splicing aberrations caused by constitutional RB1 gene mutations in ...

    Indian Academy of Sciences (India)

    Analysis of RB1 mRNA from blood leukocytes of patients with retinoblastoma identified the effects of mutations involving consensus splice site, exonic substitution and whole-exon deletions identified in genomic DNA of these patients. In addition, this study identified mutations in cases in which no mutations were detectable ...

  15. Long-range RNA pairings contribute to mutually exclusive splicing.

    Science.gov (United States)

    Yue, Yuan; Yang, Yun; Dai, Lanzhi; Cao, Guozheng; Chen, Ran; Hong, Weiling; Liu, Baoping; Shi, Yang; Meng, Yijun; Shi, Feng; Xiao, Mu; Jin, Yongfeng

    2016-01-01

    Mutually exclusive splicing is an important means of increasing the protein repertoire, by which the Down's syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 different isoforms in Drosophila melanogaster. However, the regulatory mechanisms remain obscure due to the complexity of the Dscam exon cluster. Here, we reveal a molecular model for the regulation of the mutually exclusive splicing of the serpent pre-mRNA based on competition between upstream and downstream RNA pairings. Such dual RNA pairings confer fine tuning of the inclusion of alternative exons. Moreover, we demonstrate that the splicing outcome of alternative exons is mediated in relative pairing strength-correlated mode. Combined comparative genomics analysis and experimental evidence revealed similar bidirectional structural architectures in exon clusters 4 and 9 of the Dscam gene. Our findings provide a novel mechanistic framework for the regulation of mutually exclusive splicing and may offer potentially applicable insights into long-range RNA-RNA interactions in gene regulatory networks. © 2015 Yue et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  16. Deep learning of the tissue-regulated splicing code.

    Science.gov (United States)

    Leung, Michael K K; Xiong, Hui Yuan; Lee, Leo J; Frey, Brendan J

    2014-06-15

    Alternative splicing (AS) is a regulated process that directs the generation of different transcripts from single genes. A computational model that can accurately predict splicing patterns based on genomic features and cellular context is highly desirable, both in understanding this widespread phenomenon, and in exploring the effects of genetic variations on AS. Using a deep neural network, we developed a model inferred from mouse RNA-Seq data that can predict splicing patterns in individual tissues and differences in splicing patterns across tissues. Our architecture uses hidden variables that jointly represent features in genomic sequences and tissue types when making predictions. A graphics processing unit was used to greatly reduce the training time of our models with millions of parameters. We show that the deep architecture surpasses the performance of the previous Bayesian method for predicting AS patterns. With the proper optimization procedure and selection of hyperparameters, we demonstrate that deep architectures can be beneficial, even with a moderately sparse dataset. An analysis of what the model has learned in terms of the genomic features is presented. © The Author 2014. Published by Oxford University Press.

  17. Magnetic alignment and the Poisson alignment reference system

    Science.gov (United States)

    Griffith, L. V.; Schenz, R. F.; Sommargren, G. E.

    1990-08-01

    Three distinct metrological operations are necessary to align a free-electron laser (FEL): the magnetic axis must be located, a straight line reference (SLR) must be generated, and the magnetic axis must be related to the SLR. This article begins with a review of the motivation for developing an alignment system that will assure better than 100-μm accuracy in the alignment of the magnetic axis throughout an FEL. The 100-μm accuracy is an error circle about an ideal axis for 300 m or more. The article describes techniques for identifying the magnetic axes of solenoids, quadrupoles, and wiggler poles. Propagation of a laser beam is described to the extent of revealing sources of nonlinearity in the beam. Development of a straight-line reference based on the Poisson line, a diffraction effect, is described in detail. Spheres in a large-diameter laser beam create Poisson lines and thus provide a necessary mechanism for gauging between the magnetic axis and the SLR. Procedures for installing FEL components and calibrating alignment fiducials to the magnetic axes of the components are also described. The Poisson alignment reference system should be accurate to 25 μm over 300 m, which is believed to be a factor-of-4 improvement over earlier techniques. An error budget shows that only 25% of the total budgeted tolerance is used for the alignment reference system, so the remaining tolerances should fall within the allowable range for FEL alignment.

  18. Alignment-Annotator web server: rendering and annotating sequence alignments.

    Science.gov (United States)

    Gille, Christoph; Fähling, Michael; Weyand, Birgit; Wieland, Thomas; Gille, Andreas

    2014-07-01

    Alignment-Annotator is a novel web service designed to generate interactive views of annotated nucleotide and amino acid sequence alignments (i) de novo and (ii) embedded in other software. All computations are performed at server side. Interactivity is implemented in HTML5, a language native to web browsers. The alignment is initially displayed using default settings and can be modified with the graphical user interfaces. For example, individual sequences can be reordered or deleted using drag and drop, amino acid color code schemes can be applied and annotations can be added. Annotations can be made manually or imported (BioDAS servers, the UniProt, the Catalytic Site Atlas and the PDB). Some edits take immediate effect while others require server interaction and may take a few seconds to execute. The final alignment document can be downloaded as a zip-archive containing the HTML files. Because of the use of HTML the resulting interactive alignment can be viewed on any platform including Windows, Mac OS X, Linux, Android and iOS in any standard web browser. Importantly, no plugins nor Java are required and therefore Alignment-Anotator represents the first interactive browser-based alignment visualization. http://www.bioinformatics.org/strap/aa/ and http://strap.charite.de/aa/. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Testing the tidal alignment model of galaxy intrinsic alignment

    Energy Technology Data Exchange (ETDEWEB)

    Blazek, Jonathan; Seljak, Uroš [Department of Physics and Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720 (United States); McQuinn, Matthew, E-mail: blazek@berkeley.edu, E-mail: mmcquinn@berkeley.edu, E-mail: useljak@berkeley.edu [Department of Astronomy, University of California, Berkeley, CA 94720 (United States)

    2011-05-01

    Weak gravitational lensing has become a powerful probe of large-scale structure and cosmological parameters. Precision weak lensing measurements require an understanding of the intrinsic alignment of galaxy ellipticities, which can in turn inform models of galaxy formation. It is hypothesized that elliptical galaxies align with the background tidal field and that this alignment mechanism dominates the correlation between ellipticities on cosmological scales (in the absence of lensing). We use recent large-scale structure measurements from the Sloan Digital Sky Survey to test this picture with several statistics: (1) the correlation between ellipticity and galaxy overdensity, w{sub g+}; (2) the intrinsic alignment auto-correlation functions; (3) the correlation functions of curl-free, E, and divergence-free, B, modes, the latter of which is zero in the linear tidal alignment theory; (4) the alignment correlation function, w{sub g}(r{sub p},θ), a recently developed statistic that generalizes the galaxy correlation function to account for the angle between the galaxy separation vector and the principle axis of ellipticity. We show that recent measurements are largely consistent with the tidal alignment model and discuss dependence on galaxy luminosity. In addition, we show that at linear order the tidal alignment model predicts that the angular dependence of w{sub g}(r{sub p},θ) is simply w{sub g+}(r{sub p})cos (2θ) and that this dependence is consistent with recent measurements. We also study how stochastic nonlinear contributions to galaxy ellipticity impact these statistics. We find that a significant fraction of the observed LRG ellipticity can be explained by alignment with the tidal field on scales ∼> 10 \\hMpc. These considerations are relevant to galaxy formation and evolution.

  20. Pediatric sagittal alignment.

    Science.gov (United States)

    Mac-Thiong, Jean-Marc; Labelle, Hubert; Roussouly, Pierre

    2011-09-01

    There is a wide variation in the regional parameters used to describe the spine and sacro-pelvis in children and adolescents. There is a slight tendency for thoracic kyphosis and lumbar lordosis to increase with age. Pelvic incidence and pelvic tilt also tend to increase during growth, while sacral slope remains relatively stable. Strong knowledge of the close relationships between adjacent anatomical regions of the spine and sacro-pelvis is the key when evaluating and interpreting sagittal spino-pelvic alignment. The scheme of correlations between adjacent regional parameters needs to be preserved in order to maintain a balanced posture. The net resultant from these relationships between adjacent anatomical regions is best represented by parameters of sagittal global balance. C7 plumbline tends to move backwards from childhood to adulthood, where it stabilizes or slightly moves forward secondary to degenerative changes. C7 plumbline in front of both hip axis and center of the upper sacral endplate occurs in 29% of subjects aged 3-10 years, 12% of subjects aged between 10 and 18 years, and 14% of subjects aged 18 years or older. Therefore, although most normal subjects stand with a C7 plumbline behind the hip axis, a C7 plumbline in front of both hip axis and sacrum can be seen in normal individuals. However, progressive forward displacement of C7 plumbline should raise a suspicion for the risk of developing spinal pathology.

  1. Investigation of tissue-specific human orthologous alternative splice events in pig

    DEFF Research Database (Denmark)

    Hillig, Ann-Britt Nygaard; Jørgensen, Claus Bøttcher; Salicio, Susanna Cirera

    2010-01-01

    investigated alternative splice events detected in humans, in orthologous pig genes. A total of 17 genes with predicted exon skipping events were selected for further studies. The splice events for the selected genes were experimentally verified using real-time quantitative PCR analysis (qPCR) with splice......Alternative splicing of pre-mRNA can contribute to differences between tissues or cells either by regulating gene expression or creating proteins with various functions encoded by one gene. The number of investigated alternative splice events in pig has so far been limited. In this study we have...

  2. The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer

    DEFF Research Database (Denmark)

    Hartung, Anne-Mette; Swensen, Jeff; Uriz, Inaki E

    2016-01-01

    the function of a critical Exonic Splicing Enhancer (ESE) and creation of an Exonic Splicing Silencer (ESS). We show that this vulnerability of HRAS exon 2 is caused by a weak 3' splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE....... Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also...

  3. Electromechanical behaviour of REBCO tape lap splices under transverse compressive loading

    CERN Document Server

    Grether, A; Ballarino, A.; Bottura, L.

    2016-01-01

    We have studied the influence of transverse compressive stress on the resistance and critical current (Ic) of soldered REBCO tape lap splices. Internal contact resistances dominate the overall REBCO lap splice resistances. Application of transverse compressive stress up to 250 MPa during the resistance measurements does not alter the resistance and Ic of the soldered REBCO splices that were studied. The resistance of unsoldered REBCO tape lap splices depends strongly on the contact pressure. At a transverse compressive stress of 100 MPa to which Roebel cables are typically exposed in high field magnets, the crossover splice contact resistance is comparable to the internal tape resistances.

  4. Alignment between Protostellar Outflows and Filamentary Structure

    Energy Technology Data Exchange (ETDEWEB)

    Stephens, Ian W.; Dunham, Michael M.; Myers, Philip C.; Pokhrel, Riwaj; Sadavoy, Sarah I.; Lee, Katherine I.; Goodman, Alyssa A. [Harvard-Smithsonian Center for Astrophysics, 60 Garden Street, Cambridge, MA (United States); Vorobyov, Eduard I. [Institute of Fluid Mechanics and Heat Transfer, TU Wien, Vienna, A-1060 (Austria); Tobin, John J. [Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, 440 W. Brooks Street, Norman, OK 73019 (United States); Pineda, Jaime E. [Max-Planck-Institut für extraterrestrische Physik, Giessenbachstrasse 1, 85748 Garching (Germany); Offner, Stella S. R. [Department of Astronomy, University of Massachusetts, Amherst, MA 01003 (United States); Kristensen, Lars E. [Centre for Star and Planet Formation, Niels Bohr Institute and Natural History Museum of Denmark, University of Copenhagen, Øster Voldgade 5-7, DK-1350 Copenhagen K (Denmark); Jørgensen, Jes K. [Niels Bohr Institute and Center for Star and Planet Formation, Copenhagen University, DK-1350 Copenhagen K. (Denmark); Bourke, Tyler L. [SKA Organization, Jodrell Bank Observatory, Lower Withington, Macclesfield, Cheshire SK11 9DL (United Kingdom); Arce, Héctor G. [Department of Astronomy, Yale University, New Haven, CT 06520 (United States); Plunkett, Adele L., E-mail: ian.stephens@cfa.harvard.edu [European Southern Observatory, Av. Alonso de Cordova 3107, Vitacura, Santiago de Chile (Chile)

    2017-09-01

    We present new Submillimeter Array (SMA) observations of CO(2–1) outflows toward young, embedded protostars in the Perseus molecular cloud as part of the Mass Assembly of Stellar Systems and their Evolution with the SMA (MASSES) survey. For 57 Perseus protostars, we characterize the orientation of the outflow angles and compare them with the orientation of the local filaments as derived from Herschel observations. We find that the relative angles between outflows and filaments are inconsistent with purely parallel or purely perpendicular distributions. Instead, the observed distribution of outflow-filament angles are more consistent with either randomly aligned angles or a mix of projected parallel and perpendicular angles. A mix of parallel and perpendicular angles requires perpendicular alignment to be more common by a factor of ∼3. Our results show that the observed distributions probably hold regardless of the protostar’s multiplicity, age, or the host core’s opacity. These observations indicate that the angular momentum axis of a protostar may be independent of the large-scale structure. We discuss the significance of independent protostellar rotation axes in the general picture of filament-based star formation.

  5. The splicing fate of plant SPO11 genes

    Directory of Open Access Journals (Sweden)

    Thorben eSprink

    2014-05-01

    Full Text Available Towards the global understanding of plant meiosis, it seems to be essential to decipher why all as yet sequenced plants need or at least encode for two different meiotic SPO11 genes. This is in contrast to mammals and fungi, where only one SPO11 is present. Both SPO11 in plants are essential for the initiation of double strand breaks (DSBs during the meiotic prophase. In nearly all eukaryotic organisms DSB induction by SPO11 leads to meiotic DSB repair, thereby ensuring the formation of a necessary number of crossovers (CO as physical connections between the allelic chromosomes. We aim to investigate the specific functions and evolution of both SPO11 genes in land plants. Therefore, we identified and cloned the respective orthologous genes from Brassica rapa, Carica papaya, Oryza sativa and Physcomitrella patens. In parallel we determined the full length cDNA sequences of SPO11-1 and -2 from all of these plants by RT-PCR. During these experiments we observed that the analyzed plants exhibit a pattern of aberrant splicing products of both SPO11 mRNAs. Such an aberrant splicing has previously been described for Arabidopsis and therefore seems to be conserved throughout evolution. Most of the splicing forms of SPO11-1 and -2 seem to be non functional as they either showed intron retention or shortened exons accompanied by a frameshift leading to premature termination codons (PTCs in most cases. Nevertheless, we could detect one putative functional alternatively spliced mRNA for SPO11-1 and -2 each, indicating that splicing of SPO11 does not depend only on the gene sequence but also on the plant species and that it might play a regulatory role.

  6. Spliced leader-based metatranscriptomic analyses lead to recognition of hidden genomic features in dinoflagellates.

    Science.gov (United States)

    Lin, Senjie; Zhang, Huan; Zhuang, Yunyun; Tran, Bao; Gill, John

    2010-11-16

    Environmental transcriptomics (metatranscriptomics) for a specific lineage of eukaryotic microbes (e.g., Dinoflagellata) would be instrumental for unraveling the genetic mechanisms by which these microbes respond to the natural environment, but it has not been exploited because of technical difficulties. Using the recently discovered dinoflagellate mRNA-specific spliced leader as a selective primer, we constructed cDNA libraries (e-cDNAs) from one marine and two freshwater plankton assemblages. Small-scale sequencing of the e-cDNAs revealed functionally diverse transcriptomes proven to be of dinoflagellate origin. A set of dinoflagellate common genes and transcripts of dominant dinoflagellate species were identified. Further analyses of the dataset prompted us to delve into the existing, largely unannotated dinoflagellate EST datasets (DinoEST). Consequently, all four nucleosome core histones, two histone modification proteins, and a nucleosome assembly protein were detected, clearly indicating the presence of nucleosome-like machinery long thought not to exist in dinoflagellates. The isolation of rhodopsin from taxonomically and ecotypically diverse dinoflagellates and its structural similarity and phylogenetic affinity to xanthorhodopsin suggest a common genetic potential in dinoflagellates to use solar energy nonphotosynthetically. Furthermore, we found 55 cytoplasmic ribosomal proteins (RPs) from the e-cDNAs and 24 more from DinoEST, showing that the dinoflagellate phylum possesses all 79 eukaryotic RPs. Our results suggest that a sophisticated eukaryotic molecular machine operates in dinoflagellates that likely encodes many more unsuspected physiological capabilities and, meanwhile, demonstrate that unique spliced leaders are useful for profiling lineage-specific microbial transcriptomes in situ.

  7. cAMP-dependent reorganization of the Cajal bodies and splicing machinery in cultured Schwann cells.

    Science.gov (United States)

    Fernandez, Rosario; Pena, Emma; Navascues, Joaquin; Casafont, Iñigo; Lafarga, Miguel; Berciano, Maria T

    2002-12-01

    It is well established that forskolin-induced elevation of cAMP results in activation of DNA synthesis in Schwann cell cultures. This promitotic response is partially mediated by the Cdk2, which is required for the transition from the G1 to the S phase of the cell cycle. In the present study, we analyze the effects of cAMP elevation in cultured Schwann cells on the transcriptional activity and on the organization of two nuclear compartments involved in pre-mRNA processing: Cajal bodies (CBs) and splicing factor compartments. Our immunofluorescence and quantitative studies show that forskolin treatment induces a 5.6-fold increase in the proportion of S phase Schwann cells, detected by a short pulse (20 min) of BrdU incorporation. This increase in DNA synthesis correlates with an activation of global transcription, as is indicated by the higher nuclear incorporation of BrU in nascent RNA. Forskolin treatment significantly increases the percentage of Schwann cells containing typical CBs, which concentrate spliceosomal snRNPs and the survival motor neuron (SMN) protein. This increase in the number of CBs closely correlates with the activation of transcription. Moreover, the occurrence of CBs is significantly higher in BrdU (+) cells than in BrdU (-) cells, indicating that entry in the S phase promotes the formation of CBs. During the S phase, Schwann cell nuclei display higher Cdk2 nuclear staining and concentrate this kinase in CBs. Forskolin also induces a redistribution of the pre-mRNA splicing factors in Schwann cells. Primary cultures of Schwann cells provide an excellent physiological model to demonstrate that the assembly of CBs is a transcription- and replication-dependent cellular event. Moreover, the S phase accumulation of Cdk2 observed in Schwann cells supports a functional link between CBs and DNA replication, which is mediated by the possible participation of CBs in the regulatory control of histone gene expression. Copyright 2002 Wiley-Liss, Inc.

  8. Cotranscriptional recruitment of yeast TRAMP complex to intronic sequences promotes optimal pre-mRNA splicing

    Science.gov (United States)

    Kong, Ka-Yiu Edwin; Tang, Hei-Man Vincent; Pan, Kewu; Huang, Zhe; Lee, Tsz-Hang Jimmy; Hinnebusch, Alan G.; Wong, Chi-Ming

    2014-01-01

    Most unwanted RNA transcripts in the nucleus of eukaryotic cells, such as splicing-defective pre-mRNAs and spliced-out introns, are rapidly degraded by the nuclear exosome. In budding yeast, a number of these unwanted RNA transcripts, including spliced-out introns, are first recognized by the nuclear exosome cofactor Trf4/5p-Air1/2p-Mtr4p polyadenylation (TRAMP) complex before subsequent nuclear-exosome-mediated degradation. However, it remains unclear when spliced-out introns are recognized by TRAMP, and whether TRAMP may have any potential roles in pre-mRNA splicing. Here, we demonstrated that TRAMP is cotranscriptionally recruited to nascent RNA transcripts, with particular enrichment at intronic sequences. Deletion of TRAMP components led to further accumulation of unspliced pre-mRNAs even in a yeast strain defective in nuclear exosome activity, suggesting a novel stimulatory role of TRAMP in splicing. We also uncovered new genetic and physical interactions between TRAMP and several splicing factors, and further showed that TRAMP is required for optimal recruitment of the splicing factor Msl5p. Our study provided the first evidence that TRAMP facilitates pre-mRNA splicing, and we interpreted this as a fail-safe mechanism to ensure the cotranscriptional recruitment of TRAMP before or during splicing to prepare for the subsequent targeting of spliced-out introns to rapid degradation by the nuclear exosome. PMID:24097436

  9. Effect of Chord Splice Joints on Force Distribution and Deformations in Trusses with Punched Metal Plate Fasteners

    DEFF Research Database (Denmark)

    Ellegaard, Peter

    2007-01-01

    - their real behaviour is semi-rigid. The influence of splice joints on the distribution of member forces and rotations in the splice joints is investigated in this paper. A finite element program, TrussLab, where the splice joints are given semi-rigid properties is used to analyse the effect of splice joints...

  10. From Cryptic Toward Canonical Pre-mRNA Splicing in Pompe Disease: A Pipeline for the Development of Antisense Oligonucleotides

    NARCIS (Netherlands)

    A.J. Bergsma (Atze); S.L.M. in 't Groen (Stijn); F.W. Verheijen (Frans); A.T. van der Ploeg (Ans); W.W.M.P. Pijnappel (Pim)

    2016-01-01

    textabstractWhile 9% of human pathogenic variants have an established effect on pre-mRNA splicing, it is suspected that an additional 20% of otherwise classified variants also affect splicing. Aberrant splicing includes disruption of splice sites or regulatory elements, or creation or strengthening

  11. A new view of transcriptome complexity and regulation through the lens of local splicing variations.

    Science.gov (United States)

    Vaquero-Garcia, Jorge; Barrera, Alejandro; Gazzara, Matthew R; González-Vallinas, Juan; Lahens, Nicholas F; Hogenesch, John B; Lynch, Kristen W; Barash, Yoseph

    2016-02-01

    Alternative splicing (AS) can critically affect gene function and disease, yet mapping splicing variations remains a challenge. Here, we propose a new approach to define and quantify mRNA splicing in units of local splicing variations (LSVs). LSVs capture previously defined types of alternative splicing as well as more complex transcript variations. Building the first genome wide map of LSVs from twelve mouse tissues, we find complex LSVs constitute over 30% of tissue dependent transcript variations and affect specific protein families. We show the prevalence of complex LSVs is conserved in humans and identify hundreds of LSVs that are specific to brain subregions or altered in Alzheimer's patients. Amongst those are novel isoforms in the Camk2 family and a novel poison exon in Ptbp1, a key splice factor in neurogenesis. We anticipate the approach presented here will advance the ability to relate tissue-specific splice variation to genetic variation, phenotype, and disease.

  12. Effect of fiber blending ratios of cotton/polyester yarns on retained splice diameter

    Science.gov (United States)

    Çelik, H. İ.; Kaynak, H. K.

    2017-10-01

    The most important performance parameters of splicing are obtaining adequate strength and appearance at the splice point for all processing requirements. The diameter of spliced portion effects not only appearance of the splice joints but also physical characteristics such as packing density, strength, specific volume of the yarn. In this study, the effect of cotton/polyester fiber blend ratios on spliced portion diameter at different slicing air pressures was investigated. For this aim, three yarn samples 100% cotton, 80-20% CO-PES and 50- 50% CO-PES were produced with 40/1 Ne. Each yarn samples was spliced at three different pressures; 4 bar, 5 bar and 6 bar. The diameters of spliced portion and retained yarns were measured by using ImageJ program and the results were analyzed statistically.

  13. Splicing factor 1 modulates dietary restriction and TORC1 pathway longevity in C. elegans

    DEFF Research Database (Denmark)

    Heintz, Caroline; Doktor, Thomas K; Lanjuin, Anne

    2017-01-01

    RNA (pre-mRNA) splicing is a fundamental link between gene expression and the proteome, and deregulation of the splicing machinery is linked to several age-related chronic illnesses. However, the role of splicing homeostasis in healthy ageing remains unclear. Here we demonstrate that pre-mRNA splicing...... homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans. Using transcriptomics and in-depth splicing analysis in young and old animals fed ad libitum or subjected to dietary restriction, we find defects in global pre-mRNA splicing with age that are reduced by dietary restriction...... via splicing factor 1 (SFA-1; the C. elegans homologue of SF1, also known as branchpoint binding protein, BBP). We show that SFA-1 is specifically required for lifespan extension by dietary restriction and by modulation of the TORC1 pathway components AMPK, RAGA-1 and RSKS-1/S6 kinase. We also...

  14. Aligning for Innovation - Alignment Strategy to Drive Innovation

    Science.gov (United States)

    Johnson, Hurel; Teltschik, David; Bussey, Horace, Jr.; Moy, James

    2010-01-01

    With the sudden need for innovation that will help the country achieve its long-term space exploration objectives, the question of whether NASA is aligned effectively to drive the innovation that it so desperately needs to take space exploration to the next level should be entertained. Authors such as Robert Kaplan and David North have noted that companies that use a formal system for implementing strategy consistently outperform their peers. They have outlined a six-stage management systems model for implementing strategy, which includes the aligning of the organization towards its objectives. This involves the alignment of the organization from the top down. This presentation will explore the impacts of existing U.S. industrial policy on technological innovation; assess the current NASA organizational alignment and its impacts on driving technological innovation; and finally suggest an alternative approach that may drive the innovation needed to take the world to the next level of space exploration, with NASA truly leading the way.

  15. CAFE: aCcelerated Alignment-FrEe sequence analysis.

    Science.gov (United States)

    Lu, Yang Young; Tang, Kujin; Ren, Jie; Fuhrman, Jed A; Waterman, Michael S; Sun, Fengzhu

    2017-07-03

    Alignment-free genome and metagenome comparisons are increasingly important with the development of next generation sequencing (NGS) technologies. Recently developed state-of-the-art k-mer based alignment-free dissimilarity measures including CVTree, $d_2^*$ and $d_2^S$ are more computationally expensive than measures based solely on the k-mer frequencies. Here, we report a standalone software, aCcelerated Alignment-FrEe sequence analysis (CAFE), for efficient calculation of 28 alignment-free dissimilarity measures. CAFE allows for both assembled genome sequences and unassembled NGS shotgun reads as input, and wraps the output in a standard PHYLIP format. In downstream analyses, CAFE can also be used to visualize the pairwise dissimilarity measures, including dendrograms, heatmap, principal coordinate analysis and network display. CAFE serves as a general k-mer based alignment-free analysis platform for studying the relationships among genomes and metagenomes, and is freely available at https://github.com/younglululu/CAFE. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Magnetic axis alignment and the Poisson alignment reference system

    Science.gov (United States)

    Griffith, Lee V.; Schenz, Richard F.; Sommargren, Gary E.

    1989-01-01

    Three distinct metrological operations are necessary to align a free-electron laser (FEL): the magnetic axis must be located, a straight line reference (SLR) must be generated, and the magnetic axis must be related to the SLR. This paper begins with a review of the motivation for developing an alignment system that will assure better than 100 micrometer accuracy in the alignment of the magnetic axis throughout an FEL. The paper describes techniques for identifying the magnetic axis of solenoids, quadrupoles, and wiggler poles. Propagation of a laser beam is described to the extent of revealing sources of nonlinearity in the beam. Development and use of the Poisson line, a diffraction effect, is described in detail. Spheres in a large-diameter laser beam create Poisson lines and thus provide a necessary mechanism for gauging between the magnetic axis and the SLR. Procedures for installing FEL components and calibrating alignment fiducials to the magnetic axes of the components are also described. An error budget shows that the Poisson alignment reference system will make it possible to meet the alignment tolerances for an FEL.

  17. Clustering exact matches of pairwise sequence alignments by weighted linear regression

    Directory of Open Access Journals (Sweden)

    Liao Li

    2008-02-01

    Full Text Available Abstract Background At intermediate stages of genome assembly projects, when a number of contigs have been generated and their validity needs to be verified, it is desirable to align these contigs to a reference genome when it is available. The interest is not to analyze a detailed alignment between a contig and the reference genome at the base level, but rather to have a rough estimate of where the contig aligns to the reference genome, specifically, by identifying the starting and ending positions of such a region. This information is very useful in ordering the contigs, facilitating post-assembly analysis such as gap closure and resolving repeats. There exist programs, such as BLAST and MUMmer, that can quickly align and identify high similarity segments between two sequences, which, when seen in a dot plot, tend to agglomerate along a diagonal but can also be disrupted by gaps or shifted away from the main diagonal due to mismatches between the contig and the reference. It is a tedious and practically impossible task to visually inspect the dot plot to identify the regions covered by a large number of contigs from sequence assembly projects. A forced global alignment between a contig and the reference is not only time consuming but often meaningless. Results We have developed an algorithm that uses the coordinates of all the exact matches or high similarity local alignments, clusters them with respect to the main diagonal in the dot plot using a weighted linear regression technique, and identifies the starting and ending coordinates of the region of interest. Conclusion This algorithm complements existing pairwise sequence alignment packages by replacing the time-consuming seed extension phase with a weighted linear regression for the alignment seeds. It was experimentally shown that the gain in execution time can be outstanding without compromising the accuracy. This method should be of great utility to sequence assembly and genome

  18. A 38 nt region and its flanking sequences within gag of Friend murine leukemia virus are crucial for splicing at the correct 5' and 3' splice sites.

    Science.gov (United States)

    Machinaga, Akihito; Takase-Yoden, Sayaka

    2014-01-01

    The genome of the Friend murine leukemia virus (Fr-MLV) contains a 5' splice site (5'ss) located at 205 nt and a 3'ss located at 5489 nt. In our previous studies, it was shown that if the HindIII-BglII (879-1904 bp) fragment within gag is deleted from the proA8m1 vector, which carries the entire Fr-MLV sequence, then cryptic splicing of env-mRNA occurs. Here, attempts were made to identify the genomic segment(s) in this region that is/are essential to correct splicing. First, vectors with a serially truncated HindIII-BglII fragment were constructed. The vector, in which a 38 bp fragment (1612-1649 bp) is deleted or reversed in proA8m1, only produced splice variants. It was found that a 38 nt region within gag contains important elements that positively regulate splicing at the correct splice sites. Further analyses of a series of vectors carrying the 38 bp fragment and its flanking sequences showed that a region (1183-1611 nt) upstream of the 38 nt fragment also contains sequences that positively or negatively influence splicing at the correct splice sites. The SphI-NdeI (5140-5400 bp) fragment just upstream of the 3'ss was deleted from vectors that carried the 38 bp fragment and its flanking sequences, which yielded correctly spliced mRNA; interestingly, these deleted vectors showed cryptic splicing. These findings suggest that the 5140-5400 nt region located just upstream of the 3'ss is required for the splicing function of the 38 nt fragment and its flanking sequences. © 2013 The Societies and Wiley Publishing Asia Pty Ltd.

  19. AtRTD2: A Reference Transcript Dataset for accurate quantification of alternative splicing and expression changes in Arabidopsis thaliana RNA-seq data

    KAUST Repository

    Zhang, Runxuan

    2016-05-06

    Background Alternative splicing is the major post-transcriptional mechanism by which gene expression is regulated and affects a wide range of processes and responses in most eukaryotic organisms. RNA-sequencing (RNA-seq) can generate genome-wide quantification of individual transcript isoforms to identify changes in expression and alternative splicing. RNA-seq is an essential modern tool but its ability to accurately quantify transcript isoforms depends on the diversity, completeness and quality of the transcript information. Results We have developed a new Reference Transcript Dataset for Arabidopsis (AtRTD2) for RNA-seq analysis containing over 82k non-redundant transcripts, whereby 74,194 transcripts originate from 27,667 protein-coding genes. A total of 13,524 protein-coding genes have at least one alternatively spliced transcript in AtRTD2 such that about 60% of the 22,453 protein-coding, intron-containing genes in Arabidopsis undergo alternative splicing. More than 600 putative U12 introns were identified in more than 2,000 transcripts. AtRTD2 was generated from transcript assemblies of ca. 8.5 billion pairs of reads from 285 RNA-seq data sets obtained from 129 RNA-seq libraries and merged along with the previous version, AtRTD, and Araport11 transcript assemblies. AtRTD2 increases the diversity of transcripts and through application of stringent filters represents the most extensive and accurate transcript collection for Arabidopsis to date. We have demonstrated a generally good correlation of alternative splicing ratios from RNA-seq data analysed by Salmon and experimental data from high resolution RT-PCR. However, we have observed inaccurate quantification of transcript isoforms for genes with multiple transcripts which have variation in the lengths of their UTRs. This variation is not effectively corrected in RNA-seq analysis programmes and will therefore impact RNA-seq analyses generally. To address this, we have tested different genome

  20. Electric Field Driven Self-Assembly of Colloidal Rods

    Science.gov (United States)

    Juarez, Jaime; Chaudhary, Kundan; Chen, Qian; Granick, Steve; Lewis, Jennifer

    2012-02-01

    The ability to assemble anisotropic colloidal building blocks into ordered configurations is of both scientific and technological importance. We are studying how electric field-induced interactions guide the self-assembly of these blocks into well aligned microstructures. Specifically, we present observations of the assembly of colloidal silica rods (L/D ˜ 4) within planar electrode cells as a function of different electric field parameters. Results from video microscopy and image analysis demonstrate that aligned microstructures form due to the competition between equilibrium interactions of induced dipoles and non-equilibrium processes (i.e., electro-osmosis). Under the appropriate electric field conditions (˜ kHZ AC fields), aligned colloidal rod fluids form over large areas on the electrode surface. The superposition of a DC electric field to this aligned colloidal rod fluid initiates their condensation into a vertically oriented crystalline phase. Ongoing work is now focused on exploring how temporal changes to electric fields influence colloidal rod dynamics and, hence, the assembly kinetics of aligned colloidal monolayers.

  1. Aligning with new Digital Strategy

    DEFF Research Database (Denmark)

    Yeow, Adrian; Soh, Christina; Hansen, Rina

    2017-01-01

    Prior IS research has not fully addressed the aligning process in the highly dynamic context of digital strategy. To address this gap, we conduct a longitudinal analysis of a B2B company's journey to enact its B2C digital strategy, using the dynamic capabilities approach. We found...... that as an organization shifts towards a digital strategy, misalignments between the emergent strategy and resources give rise to tension. Our study resulted in the development of an aligning process model that is comprised of three phases (exploratory, building, and extending) and generalizable organizational aligning...... actions that form the organization's sensing, seizing, and transforming capacities. These aligning actions iteratively reconfigured organizational resources and refined strategy in order to respond to both changes in the environment and internal tensions. We also recognized that there are challenges...

  2. Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

    Directory of Open Access Journals (Sweden)

    Mario Gustavo Mayer

    2012-06-01

    Full Text Available The addition of a capped mini-exon [spliced leader (SL] through trans-splicing is essential for the maturation of RNA polymerase (pol II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1 in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin, we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

  3. RRM domain of Arabidopsis splicing factor SF1 is important for pre-mRNA splicing of a specific set of genes

    KAUST Repository

    Lee, Keh Chien

    2017-04-11

    The RNA recognition motif of Arabidopsis splicing factor SF1 affects the alternative splicing of FLOWERING LOCUS M pre-mRNA and a heat shock transcription factor HsfA2 pre-mRNA. Splicing factor 1 (SF1) plays a crucial role in 3\\' splice site recognition by binding directly to the intron branch point. Although plant SF1 proteins possess an RNA recognition motif (RRM) domain that is absent in its fungal and metazoan counterparts, the role of the RRM domain in SF1 function has not been characterized. Here, we show that the RRM domain differentially affects the full function of the Arabidopsis thaliana AtSF1 protein under different experimental conditions. For example, the deletion of RRM domain influences AtSF1-mediated control of flowering time, but not the abscisic acid sensitivity response during seed germination. The alternative splicing of FLOWERING LOCUS M (FLM) pre-mRNA is involved in flowering time control. We found that the RRM domain of AtSF1 protein alters the production of alternatively spliced FLM-β transcripts. We also found that the RRM domain affects the alternative splicing of a heat shock transcription factor HsfA2 pre-mRNA, thereby mediating the heat stress response. Taken together, our results suggest the importance of RRM domain for AtSF1-mediated alternative splicing of a subset of genes involved in the regulation of flowering and adaptation to heat stress.

  4. Pre-mRNA splicing in cancer: the relevance in oncogenesis, treatment and drug resistance.

    Science.gov (United States)

    Wojtuszkiewicz, Anna; Assaraf, Yehuda G; Maas, Marielle J P; Kaspers, Gertjan J L; Jansen, Gerrit; Cloos, Jacqueline

    2015-05-01

    Aberrant pre-mRNA splicing in cancer is emerging as an important determinant of oncogenesis, response to treatment and anticancer drug resistance. At the same time, the spliceosome has become a target for a novel class of pre-clinical chemotherapeutics with a potential future application in cancer treatment. Taken together, these findings offer novel opportunities for the enhancement of the efficacy of cancer therapy. This review presents a comprehensive overview of the molecular mechanisms involved in splicing and current developments regarding splicing aberrations in relation to several aspects of cancer formation and therapy. Identified mutations in the various components of the spliceosome and their implications for cancer prognosis are delineated. Moreover, the contribution of abnormal splicing patterns as well as deregulated splicing factors to chemoresistance is discussed, along with novel splicing-based therapeutic approaches. Significant progress has been made in deciphering the role of splicing factors in cancer including carcinogenesis and drug resistance. Splicing-based prognostic tools as well as therapeutic options hold great potential towards improvements in cancer therapy. However, gaining more in-depth molecular insight into the consequences of mutations in various components of the splicing machinery as well as of cellular effects of spliceosome inhibition is a prerequisite to establish the role of splicing in tumor progression and treatment options, respectively.

  5. Nonlinear Analysis of Spliced Continuous RC Girders Strengthened with (CFRP Laminates using ANSYS

    Directory of Open Access Journals (Sweden)

    Ammar Yasir Ali

    2016-03-01

    Full Text Available This paper presents the details of the finite element analysis of spliced continuous reinforced concrete girders. Five spliced continuous girders and one non-spliced continuous girder were analyzed using the ANSYS program. Each spliced girder consisted of three precast segments spliced at two cast-in-place joints at the inflection points, using splices of hooked dowels. Three spliced girders were strengthened using different schemes of the carbon fiber reinforced polymer (CFRP laminates. The concrete was modeled using (SOLID65 eight-node brick element and the steel reinforcement was modeled discretely using (LINK8 spar element. The straight parts of the spliced bars were modeled using discrete representation with interface elements using (COMBIN39 elements to represent the bond-slip behavior while the hooked part of each spliced bar was replaced by a single spring element. The CFRP laminates were modeled using (SHELL41 shell element. The interfaces between the precast concrete segments and the joints were modeled using CONTAC52 interface elements in conjunction with vertical spring elements to represent the dowel action of the steel bars that crossing the interfaces. The ANSYS model succeeded to an acceptable degree in predicting the structural behavior of the analyzed spliced girders with average of differences of about 6% between the predicted and experimental ultimate load.

  6. A Dual Reporter Splicing Assay Using HaloTag-containing Proteins.

    Science.gov (United States)

    Oshima, Koichi; Nagase, Takahiro; Imai, Kohsuke; Nonoyama, Shigeaki; Obara, Megumi; Mizukami, Tomoyuki; Nunoi, Hiroyuki; Kanegane, Hirokazu; Kuribayashi, Futoshi; Amemiya, Shin; Ohara, Osamu

    2012-01-01

    To evaluate the effects of genetic variations on mRNA splicing, we developed a minigene-based splicing assay using reporter genes encoding luciferase and the multifunctional HaloTag protein. In addition to conventional RT-PCR analysis, splicing events can be monitored in this system using two parameters: luciferase activity and signals derived from HaloTag-containing proteins bound to a fluorescent ligand following SDS-PAGE. The luciferase activity reflects the accumulated amounts of successfully spliced HaloTag-luciferase fusion products, whereas the amounts and sizes of HaloTag-containing proteins provide quantitative insights into precursor, correctly spliced, and aberrantly spliced mRNA species. Preliminary experiments confirmed that the dual reporter minigene assay can provide estimates of overall splicing efficiency based on the levels of protein products. We then used the minigene assay to analyze a case of chronic granulomatous disease that was caused by a G>C mutation at position +5 in the 5'-splice donor site of intron 5 of the CYBB gene. We found that the G>C mutation affected CYBB mRNA splicing by changing a delicate balance of splicing efficiencies of introns 4, 5, and 6.

  7. The Role of Canonical and Noncanonical Pre-mRNA Splicing in Plant Stress Responses

    Directory of Open Access Journals (Sweden)

    A. S. Dubrovina

    2013-01-01

    Full Text Available Plants are sessile organisms capable of adapting to various environmental constraints, such as high or low temperatures, drought, soil salinity, or pathogen attack. To survive the unfavorable conditions, plants actively employ pre-mRNA splicing as a mechanism to regulate expression of stress-responsive genes and reprogram intracellular regulatory networks. There is a growing evidence that various stresses strongly affect the frequency and diversity of alternative splicing events in the stress-responsive genes and lead to an increased accumulation of mRNAs containing premature stop codons, which in turn have an impact on plant stress response. A number of studies revealed that some mRNAs involved in plant stress response are spliced counter to the traditional conception of alternative splicing. Such noncanonical mRNA splicing events include trans-splicing, intraexonic deletions, or variations affecting multiple exons and often require short direct repeats to occur. The noncanonical alternative splicing, along with common splicing events, targets the spliced transcripts to degradation through nonsense-mediated mRNA decay or leads to translation of truncated proteins. Investigation of the diversity, biological consequences, and mechanisms of the canonical and noncanonical alternative splicing events will help one to identify those transcripts which are promising for using in genetic engineering and selection of stress-tolerant plants.

  8. Detecting splicing patterns in genes involved in hereditary breast and ovarian cancer.

    Science.gov (United States)

    Davy, Grégoire; Rousselin, Antoine; Goardon, Nicolas; Castéra, Laurent; Harter, Valentin; Legros, Angelina; Muller, Etienne; Fouillet, Robin; Brault, Baptiste; Smirnova, Anna S; Lemoine, Fréderic; de la Grange, Pierre; Guillaud-Bataille, Marine; Caux-Moncoutier, Virginie; Houdayer, Claude; Bonnet, Françoise; Blanc-Fournier, Cécile; Gaildrat, Pascaline; Frebourg, Thierry; Martins, Alexandra; Vaur, Dominique; Krieger, Sophie

    2017-10-01

    Interpretation of variants of unknown significance (VUS) is a major challenge for laboratories performing molecular diagnosis of hereditary breast and ovarian cancer (HBOC), especially considering that many genes are now known to be involved in this syndrome. One important way these VUS can have a functional impact is through their effects on RNA splicing. Here we present a custom RNA-Seq assay plus bioinformatics and biostatistics pipeline to analyse specifically alternative and abnormal splicing junctions in 11 targeted HBOC genes. Our pipeline identified 14 new alternative splices in BRCA1 and BRCA2 in addition to detecting the majority of known alternative spliced transcripts therein. We provide here the first global splicing pattern analysis for the other nine genes, which will enable a comprehensive interpretation of splicing defects caused by VUS in HBOC. Previously known splicing alterations were consistently detected, occasionally with a more complex splicing pattern than expected. We also found that splicing in the 11 genes is similar in blood and breast tissue, supporting the utility and simplicity of blood splicing assays. Our pipeline is ready to be integrated into standard molecular diagnosis for HBOC, but it could equally be adapted for an integrative analysis of any multigene disorder.

  9. Chemical correction of pre-mRNA splicing defects associated with sequestration of muscleblind-like 1 protein by expanded r(CAG) transcripts

    Science.gov (United States)

    Kumar, Amit; Parkesh, Raman; Sznajder, Lukasz J.; Childs-Disney, Jessica; Sobczak, Krzysztof; Disney, Matthew D.

    2012-01-01

    Recently, it was reported that expanded r(CAG) triplet repeats (r(CAG)exp) associated with untreatable neurological diseases cause pre-mRNA mis-splicing likely due to sequestration of muscleblind-like 1 (MBNL1) splicing factor. Bioactive small molecules that bind the 5’CAG/3’GAC motif found in r(CAG)exp hairpin structure were identified by using RNA binding studies and virtual screening/chemical similarity searching. Specifically, a benzylguanidine-containing small molecule was found to improve pre-mRNA alternative splicing of MBNL1-sensitive exons in cells expressing the toxic r(CAG)exp. The compound was identified by first studying the binding of RNA 1×1 nucleotide internal loops to small molecules known to have affinity for nucleic acids. Those studies identified 4',6-diamidino-2-phenylindole (DAPI) as a specific binder to RNAs with the 5’CAG/3’GAC motif. DAPI was then used as a query molecule in a shape- and chemistry alignment-based virtual screen to identify compounds with improved properties, which identified 4-guanidinophenyl 4-guanidinobenzoate as small molecule capable of improving pre-mRNA splicing defects associated with the r(CAG)exp-MBNL1 complex. This compound may facilitate the development of therapeutics to treat diseases caused by r(CAG)exp and could serve as a useful chemical tool to dissect the mechanisms of r(CAG)exp toxicity. The approach used in these studies, defining the small RNA motifs that bind known nucleic acid binders and then using virtual screening to optimize them for bioactivity, may be generally applicable for designing small molecules that target other RNAs in human genomic sequence. PMID:22252896

  10. Chemical correction of pre-mRNA splicing defects associated with sequestration of muscleblind-like 1 protein by expanded r(CAG)-containing transcripts.

    Science.gov (United States)

    Kumar, Amit; Parkesh, Raman; Sznajder, Lukasz J; Childs-Disney, Jessica L; Sobczak, Krzysztof; Disney, Matthew D

    2012-03-16

    Recently, it was reported that expanded r(CAG) triplet repeats (r(CAG)(exp)) associated with untreatable neurological diseases cause pre-mRNA mis-splicing likely due to sequestration of muscleblind-like 1 (MBNL1) splicing factor. Bioactive small molecules that bind the 5'CAG/3'GAC motif found in r(CAG)(exp) hairpin structure were identified by using RNA binding studies and virtual screening/chemical similarity searching. Specifically, a benzylguanidine-containing small molecule was found to improve pre-mRNA alternative splicing of MBNL1-sensitive exons in cells expressing the toxic r(CAG)(exp). The compound was identified by first studying the binding of RNA 1 × 1 nucleotide internal loops to small molecules known to have affinity for nucleic acids. Those studies identified 4',6-diamidino-2-phenylindole (DAPI) as a specific binder to RNAs with the 5'CAG/3'GAC motif. DAPI was then used as a query molecule in a shape- and chemistry alignment-based virtual screen to identify compounds with improved properties, which identified 4-guanidinophenyl 4-guanidinobenzoate, a small molecule that improves pre-mRNA splicing defects associated with the r(CAG)(exp)-MBNL1 complex. This compound may facilitate the development of therapeutics to treat diseases caused by r(CAG)(exp) and could serve as a useful chemical tool to dissect the mechanisms of r(CAG)(exp) toxicity. The approach used in these studies, defining the small RNA motifs that bind small molecules with known affinity for nucleic acids and then using virtual screening to optimize them for bioactivity, may be generally applicable for designing small molecules that target other RNAs in the human genomic sequence.

  11. Characterization of sequences and mechanisms through which ISE/ISS-3 regulates FGFR2 splicing.

    Science.gov (United States)

    Hovhannisyan, Ruben H; Warzecha, Claude C; Carstens, Russ P

    2006-01-01

    Alternative splicing of fibroblast growth factor receptor-2 (FGFR2) mutually exclusive exons IIIb and IIIc results in highly cell-type-specific expression of functionally distinct receptors, FGFR2-IIIb and FGFR2-IIIc. We previously identified an RNA cis-element, ISE/ISS-3, that enhanced exon IIIb splicing and silenced exon IIIc splicing. Here, we have performed comprehensive mutational analysis to define critical sequence motifs within this element that independently either enhance splicing of upstream exons or repress splicing of downstream exons. Such analysis included use of a novel fluorescence-based splicing reporter assay that allowed quantitative determination of relative functional activity of ISE/ISS-3 mutants using flow cytometric analysis of live cells. We determined that specific sequences within this element that mediate splicing enhancement also mediate splicing repression, depending on their position relative to a regulated exon. Thus, factors that bind the element are likely to be coordinately involved in mediating both aspects of splicing regulation. Exon IIIc silencing is dependent upon a suboptimal branchpoint sequence containing a guanine branchpoint nucleotide. Previous studies of exon IIIc splicing in HeLa nuclear extracts demonstrated that this guanine branchsite primarily impaired the second step of splicing suggesting that ISE/ISS-3 may block exon IIIc inclusion at this step. However, results presented here that include use of newly developed in vitro splicing assays of FGFR2 using extracts from a cell line expressing FGFR2-IIIb strongly suggest that cell-type-specific silencing of exon IIIc occurs at or prior to the first step of splicing.

  12. Magnetic field alignment of randomly oriented, high aspect ratio silicon microwires into vertically oriented arrays.

    Science.gov (United States)

    Beardslee, Joseph A; Sadtler, Bryce; Lewis, Nathan S

    2012-11-27

    External magnetic fields have been used to vertically align ensembles of silicon microwires coated with ferromagnetic nickel films. X-ray diffraction and image analysis techniques were used to quantify the degree of vertical orientation of the microwires. The degree of vertical alignment and the minimum field strength required for alignment were evaluated as a function of the wire length, coating thickness, magnetic history, and substrate surface properties. Nearly 100% of 100 μm long, 2 μm diameter, Si microwires that had been coated with 300 nm of Ni could be vertically aligned by a 300 G magnetic field. For wires ranging from 40 to 60 μm in length, as the length of the wire increased, a higher degree of alignment was observed at lower field strengths, consistent with an increase in the available magnetic torque. Microwires that had been exposed to a magnetic sweep up to 300 G remained magnetized and, therefore, aligned more readily during subsequent magnetic field alignment sweeps. Alignment of the Ni-coated Si microwires occurred at lower field strengths on hydrophilic Si substrates than on hydrophobic Si substrates. The magnetic field alignment approach provides a pathway for the directed assembly of solution-grown semiconductor wires into vertical arrays, with potential applications in solar cells as well as in other electronic devices that utilize nano- and microscale components as active elements.

  13. A Feature-Based Forensic Procedure for Splicing Forgeries Detection

    Directory of Open Access Journals (Sweden)

    Irene Amerini

    2015-01-01

    Full Text Available Nowadays, determining if an image appeared somewhere on the web or in a magazine or is authentic or not has become crucial. Image forensics methods based on features have demonstrated so far to be very effective in detecting forgeries in which a portion of an image is cloned somewhere else onto the same image. Anyway such techniques cannot be adopted to deal with splicing attack, that is, when the image portion comes from another picture that then, usually, is not available anymore for an operation of feature match. In this paper, a procedure in which these techniques could also be employed will be shown to get rid of splicing attack by resorting to the use of some repositories of images available on the Internet like Google Images or TinEye Reverse Image Search. Experimental results are presented on some real case images retrieved on the Internet to demonstrate the capacity of the proposed procedure.

  14. Splice variants of porcine PPHLN1 encoding periphilin-1

    DEFF Research Database (Denmark)

    Larsen, Knud Erik; Momeni, Jamal; Farajzadeh, Leila

    2017-01-01

    of the periphilin-1 protein. Thus, variants Sp1 and Sp1 are the result of alternative splicing. The porcine PPHLN1 gene was mapped to chromosome 5. The porcine PPHLN1 gene was found to be differentially expressed in various porcine organs and tissues. The sequence of the porcine PPHLN1 cDNA, encoding the periphilin......The periphilin-1 protein is encoded by the PPHLN1 gene. Periphilin-1 is found in the cornified cell envelope during the terminal differentiation of keratinocyte at the outer layer of epidermis. In the current study we report on the cloning and characterization of the porcine PPHLN1 cDNA and two...... splice variants hereof. RT-PCR cloning using oligonucleotide primers derived from in silico sequences resulted in three PPHLN1 transcripts: a full-length mRNA and two transcript variant resulting in shorter proteins. The longest encoded periphilin-1, consisting of 373 amino acids, displays a high...

  15. A novel splicing mutation in the V2 vasopressin receptor

    DEFF Research Database (Denmark)

    Kamperis, Konstantinos; Siggaard, C; Herlin, Troels

    2000-01-01

    of the gene in both the affected male (hemizygous) and his mother (heterozygous). This mutation is likely to cause aberrant splicing of the terminal intron of the gene, leading to a non-functional AVP receptor. The clinical studies were consistent with such a hypothesis, as the affected subject had a severe......In order to elucidate the molecular basis and the clinical characteristics of X-linked recessive nephrogenic diabetes insipidus (CNDI) in a kindred of Danish descent, we performed direct sequencing of the arginine vasopressin receptor 2 (AVPR2) gene in five members of the family, as well...... as clinical investigations comprising a fluid deprivation test and a 1-deamino-8-D-arginine-vasopressin (dDAVP) infusion test in the study subject and his mother. We found a highly unusual, novel, de novo 1447A-->C point mutation (gDNA), involving the invariable splice acceptor of the second intron...

  16. Splice variants of androgen receptor and prostate cancer

    Directory of Open Access Journals (Sweden)

    Orazio Caffo

    2016-05-01

    Full Text Available Over the last ten years, two new-generation hormonal drugs and two chemotherapeutic agents have been approved for the treatment of metastatic castration-resistant prostate cancer. Unfortunately, some patients have primary resistance to them and the others eventually develop secondary resistance. It has recently been suggested that the presence of androgen receptor splice variants plays a leading role in the primary and secondary resistance to the new hormonal drugs, whereas their presence seem to have only a partial effect on the activity of the chemotherapeutic agents. The aim of this paper is to review the published data concerning the role of androgen receptor splice variants in prostate cancer biology, and their potential use as biomarkers when making therapeutic decisions.

  17. Vertical Alignment of Single Wall Carbon Nanotubes (SWNTs) in Thin Polymer Films

    Science.gov (United States)

    Mauter, Meagan; Elimelech, Menachem; Osuji, Chinedum

    2009-03-01

    Thin polymer films (1-10 um) incorporating singly dispersed, vertically aligned carbon nanotubes have a diverse set of potential applications. Desalination membranes that use aligned SWNT as pores, for instance, are predicted to exhibit high flux and salt rejection through size exclusion of hydrated ions. Current fabrication techniques, however, are unable to realize the vertical assembly of narrow diameter SWNTs. Here, we direct the vertical alignment of SWNTs in thin films by using magnetic field aligned lyoptropic surfactant mesophases as structure directing templates. The short alkyl tails of the surfactant impart negative diamagnetic anisotropy to worm-like micelles and lead to parallel alignment of the liquid crystalline (LC) director in an applied magnetic field. The nanotubes orient preferentially with their long axis parallel to the director field of the mesophase, thus promoting their vertical alignment in the system. The LC mesophase incorporates monomers that are polymerized by UV exposure after nanotube alignment to form the polymer matrix. X-ray scattering and optical spectroscopy are used to characterize the field-guided assembly process. The present system may have additional applications for polymer reinforcement using carbon nanotubes.

  18. Characterization and alternative splicing of the complex I 19-kD subunit in Dunaliella salina: expression and mutual correlation of splice variants under diverse stresses.

    Science.gov (United States)

    Cao, Yu; Jin, Nan; Xu, Hui; Liu, Yi; Zhu, Wei Hua; Li, Xin Ran; Qiao, Dai Rong; Cao, Yi

    2010-01-01

    Complex I is the first enzyme in the mitochondrial respiratory chain. It extracts energy from NADH, which is produced by the oxidation of sugars and fats, and traps the energy by virtue of a potential difference or voltage across the mitochondrial inner membrane. Herein, the genomic sequence and four splice variants encoding the complex I 19-kD subunit were isolated from Dunaliella salina. There were four transcripts coding for the complex I 19-kD subunit due to alternative splicing in algae, and the four transcripts were translated to two protein isoforms with varying C-terminals. We report the splicing pattern in the 3'-region of the D. salina 19-kD subunit, in which three of the exons (5, 6, and 7) could be alternatively spliced. Moreover, we found that four alternatively spliced variants were subject to coordinated transcription in response to different stresses by real-time quantitative PCR.

  19. Conserved and species-specific alternative splicing in mammalian genomes

    Directory of Open Access Journals (Sweden)

    Favorov Alexander V

    2007-12-01

    Full Text Available Abstract Background Alternative splicing has been shown to be one of the major evolutionary mechanisms for protein diversification and proteome expansion, since a considerable fraction of alternative splicing events appears to be species- or lineage-specific. However, most studies were restricted to the analysis of cassette exons in pairs of genomes and did not analyze functionality of the alternative variants. Results We analyzed conservation of human alternative splice sites and cassette exons in the mouse and dog genomes. Alternative exons, especially minor-isofom ones, were shown to be less conserved than constitutive exons. Frame-shifting alternatives in the protein-coding regions are less conserved than frame-preserving ones. Similarly, the conservation of alternative sites is highest for evenly used alternatives, and higher when the distance between the sites is divisible by three. The rate of alternative-exon and site loss in mouse is slightly higher than in dog, consistent with faster evolution of the former. The evolutionary dynamics of alternative sites was shown to be consistent with the model of random activation of cryptic sites. Conclusion Consistent with other studies, our results show that minor cassette exons are less conserved than major-alternative and constitutive exons. However, our study provides evidence that this is caused not only by exon birth, but also lineage-specific loss of alternative exons and sites, and it depends on exon functionality.

  20. Diverse splicing patterns of exonized Alu elements in human tissues.

    Directory of Open Access Journals (Sweden)

    Lan Lin

    2008-10-01

    Full Text Available Exonization of Alu elements is a major mechanism for birth of new exons in primate genomes. Prior analyses of expressed sequence tags show that almost all Alu-derived exons are alternatively spliced, and the vast majority of these exons have low transcript inclusion levels. In this work, we provide genomic and experimental evidence for diverse splicing patterns of exonized Alu elements in human tissues. Using Exon array data of 330 Alu-derived exons in 11 human tissues and detailed RT-PCR analyses of 38 exons, we show that some Alu-derived exons are constitutively spliced in a broad range of human tissues, and some display strong tissue-specific switch in their transcript inclusion levels. Most of such exons are derived from ancient Alu elements in the genome. In SEPN1, mutations of which are linked to a form of congenital muscular dystrophy, the muscle-specific inclusion of an Alu-derived exon may be important for regulating SEPN1 activity in muscle. Realtime qPCR analysis of this SEPN1 exon in macaque and chimpanzee tissues indicates human-specific increase in its transcript inclusion level and muscle specificity after the divergence of humans and chimpanzees. Our results imply that some Alu exonization events may have acquired adaptive benefits during the evolution of primate transcriptomes.

  1. An Engineered Split Intein for Photoactivated Protein Trans-Splicing.

    Directory of Open Access Journals (Sweden)

    Stanley Wong

    Full Text Available Protein splicing is mediated by inteins that auto-catalytically join two separated protein fragments with a peptide bond. Here we engineered a genetically encoded synthetic photoactivatable intein (named LOVInC, by using the light-sensitive LOV2 domain from Avena sativa as a switch to modulate the splicing activity of the split DnaE intein from Nostoc punctiforme. Periodic blue light illumination of LOVInC induced protein splicing activity in mammalian cells. To demonstrate the broad applicability of LOVInC, synthetic protein systems were engineered for the light-induced reassembly of several target proteins such as fluorescent protein markers, a dominant positive mutant of RhoA, caspase-7, and the genetically encoded Ca2+ indicator GCaMP2. Spatial precision of LOVInC was demonstrated by targeting activity to specific mammalian cells. Thus, LOVInC can serve as a general platform for engineering light-based control for modulating the activity of many different proteins.

  2. Changes in RNA Splicing in Developing Soybean (Glycine max Embryos

    Directory of Open Access Journals (Sweden)

    Delasa Aghamirzaie

    2013-11-01

    Full Text Available Developing soybean seeds accumulate oils, proteins, and carbohydrates that are used as oxidizable substrates providing metabolic precursors and energy during seed germination. The accumulation of these storage compounds in developing seeds is highly regulated at multiple levels, including at transcriptional and post-transcriptional regulation. RNA sequencing was used to provide comprehensive information about transcriptional and post-transcriptional events that take place in developing soybean embryos. Bioinformatics analyses lead to the identification of different classes of alternatively spliced isoforms and corresponding changes in their levels on a global scale during soybean embryo development. Alternative splicing was associated with transcripts involved in various metabolic and developmental processes, including central carbon and nitrogen metabolism, induction of maturation and dormancy, and splicing itself. Detailed examination of selected RNA isoforms revealed alterations in individual domains that could result in changes in subcellular localization of the resulting proteins, protein-protein and enzyme-substrate interactions, and regulation of protein activities. Different isoforms may play an important role in regulating developmental and metabolic processes occurring at different stages in developing oilseed embryos.

  3. Alternative Splicing of G9a Regulates Neuronal Differentiation

    Directory of Open Access Journals (Sweden)

    Ana Fiszbein

    2016-03-01

    Full Text Available Chromatin modifications are critical for the establishment and maintenance of differentiation programs. G9a, the enzyme responsible for histone H3 lysine 9 dimethylation in mammalian euchromatin, exists as two isoforms with differential inclusion of exon 10 (E10 through alternative splicing. We find that the G9a methyltransferase is required for differentiation of the mouse neuronal cell line N2a and that E10 inclusion increases during neuronal differentiation of cultured cells, as well as in the developing mouse brain. Although E10 inclusion greatly stimulates overall H3K9me2 levels, it does not affect G9a catalytic activity. Instead, E10 increases G9a nuclear localization. We show that the G9a E10+ isoform is necessary for neuron differentiation and regulates the alternative splicing pattern of its own pre-mRNA, enhancing E10 inclusion. Overall, our findings indicate that by regulating its own alternative splicing, G9a promotes neuron differentiation and creates a positive feedback loop that reinforces cellular commitment to differentiation.

  4. Tsix defective in splicing is competent to establish Xist silencing.

    Science.gov (United States)

    Sado, Takashi; Hoki, Yuko; Sasaki, Hiroyuki

    2006-12-01

    Dosage differences of X-linked genes between male and female mammals are compensated for by a mechanism known as X-inactivation, and the noncoding Xist gene plays a crucial role in this process. The expression of Xist is regulated in cis by its noncoding antisense gene, Tsix, whose transcripts (though a fraction of them stay unspliced), are processed like common proteincoding RNAs. It has been suggested that certain classes of sense-antisense pairs of RNA are causally involved in not only gene regulation but also higher order chromatin structure in various organisms. In fact, recent studies demonstrated that Tsix modulates Xist expression through modification of the chromatin structure. It is still unknown, however, whether the RNA product is important for the function of Tsix or whether the antisense transcription is sufficient. To obtain insight into this issue, we eliminated the splicing products of Tsix in the mouse and explored the effects of this elimination on Tsix-mediated Xist silencing. To our surprise, the Xist locus was stably repressed on the X carrying the splicing-defective Tsix allele. Moreover, the repressive chromatin configuration was properly established at the Xist locus. These unexpected results indicate that the splicing products are dispensable for Tsix-mediated Xist silencing.

  5. Integrative analyses of RNA editing, alternative splicing, and expression of young genes in human brain transcriptome by deep RNA sequencing.

    Science.gov (United States)

    Wu, Dong-Dong; Ye, Ling-Qun; Li, Yan; Sun, Yan-Bo; Shao, Yi; Chen, Chunyan; Zhu, Zhu; Zhong, Li; Wang, Lu; Irwin, David M; Zhang, Yong E; Zhang, Ya-Ping

    2015-08-01

    Next-generation RNA sequencing has been successfully used for identification of transcript assembly, evaluation of gene expression levels, and detection of post-transcriptional modifications. Despite these large-scale studies, additional comprehensive RNA-seq data from different subregions of the human brain are required to fully evaluate the evolutionary patterns experienced by the human brain transcriptome. Here, we provide a total of 6.5 billion RNA-seq reads from different subregions of the human brain. A significant correlation was observed between the levels of alternative splicing and RNA editing, which might be explained by a competition between the molecular machineries responsible for the splicing and editing of RNA. Young human protein-coding genes demonstrate biased expression to the neocortical and non-neocortical regions during evolution on the lineage leading to humans. We also found that a significantly greater number of young human protein-coding genes are expressed in the putamen, a tissue that was also observed to have the highest level of RNA-editing activity. The putamen, which previously received little attention, plays an important role in cognitive ability, and our data suggest a potential contribution of the putamen to human evolution. © The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

  6. Using intron splicing trick for preferential gene expression in transduced cells: an approach for suicide gene therapy.

    Science.gov (United States)

    Pourzadegan, F; Shariati, L; Taghizadeh, R; Khanahmad, H; Mohammadi, Z; Tabatabaiefar, M A

    2016-01-01

    Suicide gene therapy is one of the most innovative approaches in which a potential toxic gene is delivered to the targeted cancer cell by different target delivery methods. We constructed a transfer vector to express green fluorescent protein (GFP) in transduced cells but not in packaging cells. We placed gfp under the control of the cytomegalovirus (CMV) promoter, which is positioned between the two long-terminal repeats in reverse direction. The intron-2 sequence of the human beta globin gene with two poly-A signals and several stop codons on the antisense strand was placed on the leading strand between the CMV promoter and gfp. For lentiviral production, the HEK293T and line were co-transfected with the PMD2G, psPAX2 and pLentiGFP-Ins2 plasmids. The HEK293T and line were transduced with this virus. PCR was performed for evaluation of intron splicing in transduced cells. The GFP expression was seen in 65% of the cells transduced. The PCR amplification of the genomic DNA of transduced cells confirmed the splicing of intron 2. The strategy is significant to accomplish our goal for preserving the packaging cells from the toxic gene expression during viral assembly and the resultant reduction in viral titration. Also it serves to address several other issues in the gene therapy.

  7. Integrated analysis of differential expression and alternative splicing of non-small cell lung cancer based on RNA sequencing.

    Science.gov (United States)

    Li, Zulei; Zhao, Kai; Tian, Hui

    2017-08-01

    Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, with high morbidity and mortality rates. Numerous diagnosis and treatment methods have been proposed, and the prognosis of NSCLC has improved to a certain extent. However, the mechanisms of NSCLC remain largely unknown, and additional studies are required. In the present study, the RNA sequencing dataset of NSCLC was downloaded from the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/). The clean reads obtained from the raw data were mapped to the University of California Santa Cruz human genome (hg19), based on TopHat, and were assembled into transcripts via Cufflink. The differential expression (DE) and differential alternative splicing (DAS) genes were screened out through Cuffdiff and rMATS, respectively. The significantly enriched gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes pathways were obtained through the Database of Annotation, Visualization and Integrated Discovery (DAVID). Different numbers of DE and DAS genes were identified in different types of NSCLC samples, but a number of common functions and pathways were obtained, including biological processes associated with abnormal immune and cell activity. GO terms and pathways associated with substance metabolism, including the insulin signaling pathway and oxidative phosphorylation, were enriched in DAS genes rather than DE genes. Integrated analysis of differential expression and alternative splicing may be helpful in understanding the mechanisms of NSCLC, in addition to its early diagnosis and treatment.

  8. The hnRNP 2H9 gene, which is involved in the splicing reaction, is a multiply spliced gene

    DEFF Research Database (Denmark)

    Honoré, B

    2000-01-01

    transcripts: hnRNPs 2H9, 2H9A, 2H9B, 2H9C, 2H9D and 2H9E predicting proteins containing 346, 331, 297, 215, 145 and 139 amino acids, respectively. The hnRNP 2H9A cDNA sequence was used to obtain a genomic BAC clone and the structure of the hnRNP 2H9 gene was revealed by sequencing two subclones together...... indicates that the alternatively spliced transcripts give rise to different sets and levels of proteins expressed among various human cells and tissues. Due to their great structural variations the different proteins may thus possess different functions in the splicing reaction. Udgivelsesdato: 2000-Jun-21...

  9. Human CWC22 escorts the helicase eIF4AIII to spliceosomes and promotes exon junction complex assembly.

    Science.gov (United States)

    Barbosa, Isabelle; Haque, Nazmul; Fiorini, Francesca; Barrandon, Charlotte; Tomasetto, Catherine; Blanchette, Marco; Le Hir, Hervé

    2012-10-01

    The exon-junction complex (EJC) functionally links splicing to subsequent mRNA localization, translation and stability. Sequence-independent binding of the EJC core to RNA is ensured by the DEAD-box helicase eIF4AIII. Here, we identified the splicing factor CWC22 as a new eIF4AIII partner in flies and humans. CWC22 coexists with eIF4AIII in large protein complexes distinct from EJCs. Recombinant CWC22 directly contacts eIF4AIII and prevents it from binding RNA. In vitro splicing assays revealed that CWC22 introduces eIF4AIII to spliceosomes before remodeling to facilitate eIF4AIII incorporation into the EJC. Finally, using knockdowns in vivo, we showed that CWC22 is essential for EJC assembly. We elucidated the initial step of EJC assembly and the duality of CWC22 function that hinders eIF4AIII from nonspecifically binding RNA and escorts it to the splicing machinery to promote EJC assembly on mature mRNAs.

  10. Alternative splicing in colon, bladder, and prostate cancer identified by exon-array analysis

    DEFF Research Database (Denmark)

    Thorsen, Kasper; Sørensen, Karina D.; Brems-Eskildsen, Anne Sofie

    2008-01-01

    from colon, urinary bladder, and prostate. We identified 2069 candidate alternative splicing events between normal tissue samples from colon, bladder, and prostate and selected 15 splicing events for RT-PCR validation, 10 of which were successfully validated by RT-PCR and sequencing. Furthermore 23, 19......, and 18 candidate tumor-specific splicing alterations in colon, bladder, and prostate, respectively, were selected for RT-PCR validation on an independent set of 81 normal and tumor tissue samples. In total, seven genes with tumor-specific splice variants were identified (ACTN1, CALD1, COL6A3, LRRFIP2....... In conclusion, we identified and validated alternative splicing between normal tissue samples from colon, bladder, and prostate in addition to cancer-specific splicing events in colon, bladder, and prostate cancer that may have diagnostic and prognostic implications....

  11. Seemingly neutral polymorphic variants may confer immunity to splicing-inactivating mutations

    DEFF Research Database (Denmark)

    Nielsen, Karsten Bork; Sørensen, Suzette; Cartegni, Luca

    2007-01-01

    The idea that point mutations in exons may affect splicing is intriguing and adds an additional layer of complexity when evaluating their possible effects. Even in the best-studied examples, the molecular mechanisms are not fully understood. Here, we use patient cells, model minigenes, and in vitro...... assays to show that a missense mutation in exon 5 of the medium-chain acyl-CoA dehydrogenase (MCAD) gene primarily causes exon skipping by inactivating a crucial exonic splicing enhancer (ESE), thus leading to loss of a functional protein and to MCAD deficiency. This ESE functions by antagonizing...... a juxtaposed exonic splicing silencer (ESS) and is necessary to define a suboptimal 3' splice site. Remarkably, a synonymous polymorphic variation in MCAD exon 5 inactivates the ESS, and, although this has no effect on splicing by itself, it makes splicing immune to deleterious mutations in the ESE...

  12. Identification of recurrent regulated alternative splicing events across human solid tumors

    Science.gov (United States)

    Danan-Gotthold, Miri; Golan-Gerstl, Regina; Eisenberg, Eli; Meir, Keren; Karni, Rotem; Levanon, Erez Y.

    2015-01-01

    Cancer is a complex disease that involves aberrant gene expression regulation. Discriminating the modified expression patterns driving tumor biology from the many that have no or little contribution is important for understanding cancer molecular basis. Recurrent deregulation patterns observed in multiple cancer types are enriched for such driver events. Here, we studied splicing alterations in hundreds of matched tumor and normal RNA-seq samples of eight solid cancer types. We found hundreds of cassette exons for which splicing was altered in multiple cancer types and identified a set of highly frequent altered splicing events. Specific splicing regulators, including RBFOX2, MBNL1/2 and QKI, appear to account for many splicing alteration events in multiple cancer types. Together, our results provide a first global analysis of regulated splicing alterations in cancer and identify common events with a potential causative role in solid tumor development. PMID:25908786

  13. Genome-wide analysis of SRSF10-regulated alternative splicing by deep sequencing of chicken transcriptome

    Directory of Open Access Journals (Sweden)

    Xuexia Zhou

    2014-12-01

    Full Text Available Splicing factor SRSF10 is known to function as a sequence-specific splicing activator that is capable of regulating alternative splicing both in vitro and in vivo. We recently used an RNA-seq approach coupled with bioinformatics analysis to identify the extensive splicing network regulated by SRSF10 in chicken cells. We found that SRSF10 promoted both exon inclusion and exclusion. Functionally, many of the SRSF10-verified alternative exons are linked to pathways of response to external stimulus. Here we describe in detail the experimental design, bioinformatics analysis and GO/pathway enrichment analysis of SRSF10-regulated genes to correspond with our data in the Gene Expression Omnibus with accession number GSE53354. Our data thus provide a resource for studying regulation of alternative splicing in vivo that underlines biological functions of splicing regulatory proteins in cells.

  14. Mammalian tissues defective in nonsense-mediated mRNA decay display highly aberrant splicing patterns

    DEFF Research Database (Denmark)

    Weischenfeldt, Joachim Lütken; Waage, Johannes Eichler; Tian, Geng

    2012-01-01

    a bioinformatic pipeline that maps RNA-seq data to a combinatorial exon database, predicts NMD-susceptibility for mRNA isoforms and calculates the distribution of major splice isoform classes. We present a catalog of NMD-regulated alternative splicing events, showing that isoforms of 30% of all expressed genes......ABSTRACT: BACKGROUND: Nonsense-mediated mRNA decay (NMD) affects the outcome of alternative splicing by degrading mRNA isoforms with premature termination codons. Splicing regulators constitute important NMD targets; however, the extent to which loss of NMD causes extensive deregulation...... of alternative splicing has not previously been assayed in a global, unbiased manner. Here, we combine mouse genetics and RNA-seq to provide the first in vivo analysis of the global impact of NMD on splicing patterns in two primary mouse tissues ablated for the NMD factor UPF2. RESULTS: We developed...

  15. Characterization of TTN Novex Splicing Variants across Species and the Role of RBM20 in Novex-Specific Exon Splicing

    Directory of Open Access Journals (Sweden)

    Zhilong Chen

    2018-02-01

    Full Text Available Titin (TTN is a major disease-causing gene in cardiac muscle. Titin (TTN contains 363 exons in human encoding various sizes of TTN protein due to alternative splicing regulated mainly by RNA binding motif 20 (RBM20. Three isoforms of TTN protein are produced by mutually exclusive exons 45 (Novex 1, 46 (Novex 2, and 48 (Novex 3. Alternatively splicing in Novex isoforms across species and whether Novex isoforms are associated with heart disease remains completely unknown. Cross-species exon comparison with the mVISTA online tool revealed that exon 45 is more highly conserved across all species than exons 46 and 48. Importantly, a conserved region between exons 47 and 48 across species was revealed for the first time. Reverse transcript polymerase chain reaction (RT-PCR and DNA sequencing confirmed a new exon named as 48′ in Novex 3. In addition, with primer pairs for Novex 1, a new truncated form preserving introns 44 and 45 was discovered. We discovered that Novex 2 is not expressed in the pig, mouse, and rat with Novex 2 primer pairs. Unexpectedly, three truncated forms were identified. One TTN variant with intron 46 retention is mainly expressed in the human and frog heart, another variant with co-expression of exons 45 and 46 exists predominantly in chicken and frog heart, and a third with retention of introns 45 and 46 is mainly expressed in pig, mouse, rat, and chicken. Using Rbm20 knockout rat heart, we revealed that RBM20 is not a splicing regulator of Novex variants. Furthermore, the expression levels of Novex variants in human hearts with cardiomyopathies suggested that Novexes 2 and 3 could be associated with dilated cardiomyopathy (DCM and/or arrhythmogenic right ventricular cardiomyopathy (ARVC. Taken together, our study reveals that splicing diversity of Novex exons across species and Novex variants might play a role in cardiomyopathy.

  16. Strategic Alignment and New Product Development

    DEFF Research Database (Denmark)

    Acur, Nuran; Kandemir, Destan; Boer, Harry

    2012-01-01

    Strategic alignment is widely accepted as a prerequisite for a firm’s success, but insight into the role of alignment in, and its impact on, the new product evelopment (NPD) process and its performance is less well developed. Most publications on this topic either focus on one form of alignment...... of NPD performance indicators. Strategic planning and innovativeness appear to affect technological, market, and NPD-marketing alignment positively. Environmental munificence is negatively associated with NPD-marketing alignment, but has no effect on the two other forms of alignment. Technological change...... has a positive effect on technological alignment, a negative effect on NPD-marketing alignment, but no effect on market alignment. These findings suggest that internal capabilities are more likely to be associated with the development of strategic alignment than environmental factors are. Furthermore...

  17. Finishing of the cold mass assembly

    CERN Multimedia

    Maximilien Brice

    2001-01-01

    Photo 1 The connection-side end of the active part assembly. This view shows the electrical connections between the poles and the curved bus ended with flanges for the connection with the protection diode. Photo 2 The connection-side end of the active part assembly. This view shows the electrical connections between the poles, the auxiliary bus bars and the instrumentation wires. Photo 3 Lyre-side end of the active part assembly. One can see the mechanical support of the corretor magnets that are to be installed around the cold bore tubes. Photo 4 General view of the finishing station showing the special supporting structures (blue and yellow structures) needed for the geometric measurements and for the alignment operations. Around the magnet, there are datum points (on the tripodes) needed to build up the coordinates system for the measurements.

  18. Tablet--next generation sequence assembly visualization.

    Science.gov (United States)

    Milne, Iain; Bayer, Micha; Cardle, Linda; Shaw, Paul; Stephen, Gordon; Wright, Frank; Marshall, David

    2010-02-01

    Tablet is a lightweight, high-performance graphical viewer for next-generation sequence assemblies and alignments. Supporting a range of input assembly formats, Tablet provides high-quality visualizations showing data in packed or stacked views, allowing instant access and navigation to any region of interest, and whole contig overviews and data summaries. Tablet is both multi-core aware and memory efficient, allowing it to handle assemblies containing millions of reads, even on a 32-bit desktop machine. Tablet is freely available for Microsoft Windows, Apple Mac OS X, Linux and Solaris. Fully bundled installers can be downloaded from http://bioinf.scri.ac.uk/tablet in 32- and 64-bit versions.

  19. Sequence Analysis of In Vivo-Expressed HIV-1 Spliced RNAs Reveals the Usage of New and Unusual Splice Sites by Viruses of Different Subtypes

    Science.gov (United States)

    Vega, Yolanda; Delgado, Elena; de la Barrera, Jorge; Carrera, Cristina; Zaballos, Ángel; Cuesta, Isabel; Mariño, Ana; Ocampo, Antonio; Miralles, Celia; Pérez-Castro, Sonia; Álvarez, Hortensia; López-Miragaya, Isabel; García-Bodas, Elena; Díez-Fuertes, Francisco; Thomson, Michael M.

    2016-01-01

    HIV-1 RNAs are generated through a complex splicing mechanism, resulting in a great diversity of transcripts, which are classified in three major categories: unspliced, singly spliced (SS), and doubly spliced (DS). Knowledge on HIV-1 RNA splicing in vivo and by non-subtype B viruses is scarce. Here we analyze HIV-1 RNA splice site usage in CD4+CD25+ lymphocytes from HIV-1-infected individuals through pyrosequencing. HIV-1 DS and SS RNAs were amplified by RT-PCR in 19 and 12 samples, respectively. 13,108 sequences from HIV-1 spliced RNAs, derived from viruses of five subtypes (A, B, C, F, G), were identified. In four samples, three of non-B subtypes, five 3’ splice sites (3’ss) mapping to unreported positions in the HIV-1 genome were identified. Two, designated A4i and A4j, were used in 22% and 25% of rev RNAs in two viruses of subtypes B and A, respectively. Given their close proximity (one or two nucleotides) to A4c and A4d, respectively, they could be viewed as variants of these sites. Three 3’ss, designated A7g, A7h, and A7i, located 20, 32, and 18 nucleotides downstream of A7, respectively, were identified in a subtype C (A7g, A7h) and a subtype G (A7i) viruses, each in around 2% of nef RNAs. The new splice sites or variants of splice sites were associated with the usual sequence features of 3’ss. Usage of unusual 3’ss A4d, A4e, A5a, A7a, and A7b was also detected. A4f, previously identified in two subtype C viruses, was preferentially used by rev RNAs of a subtype C virus. These results highlight the great diversity of in vivo splice site usage by HIV-1 RNAs. The fact that four of five newly identified splice sites or variants of splice sites were detected in non-subtype B viruses allows anticipating an even greater diversity of HIV-1 splice site usage than currently known. PMID:27355361

  20. Sequence Analysis of In Vivo-Expressed HIV-1 Spliced RNAs Reveals the Usage of New and Unusual Splice Sites by Viruses of Different Subtypes.

    Directory of Open Access Journals (Sweden)

    Yolanda Vega

    Full Text Available HIV-1 RNAs are generated through a complex splicing mechanism, resulting in a great diversity of transcripts, which are classified in three major categories: unspliced, singly spliced (SS, and doubly spliced (DS. Knowledge on HIV-1 RNA splicing in vivo and by non-subtype B viruses is scarce. Here we analyze HIV-1 RNA splice site usage in CD4+CD25+ lymphocytes from HIV-1-infected individuals through pyrosequencing. HIV-1 DS and SS RNAs were amplified by RT-PCR in 19 and 12 samples, respectively. 13,108 sequences from HIV-1 spliced RNAs, derived from viruses of five subtypes (A, B, C, F, G, were identified. In four samples, three of non-B subtypes, five 3' splice sites (3'ss mapping to unreported positions in the HIV-1 genome were identified. Two, designated A4i and A4j, were used in 22% and 25% of rev RNAs in two viruses of subtypes B and A, respectively. Given their close proximity (one or two nucleotides to A4c and A4d, respectively, they could be viewed as variants of these sites. Three 3'ss, designated A7g, A7h, and A7i, located 20, 32, and 18 nucleotides downstream of A7, respectively, were identified in a subtype C (A7g, A7h and a subtype G (A7i viruses, each in around 2% of nef RNAs. The new splice sites or variants of splice sites were associated with the usual sequence features of 3'ss. Usage of unusual 3'ss A4d, A4e, A5a, A7a, and A7b was also detected. A4f, previously identified in two subtype C viruses, was preferentially used by rev RNAs of a subtype C virus. These results highlight the great diversity of in vivo splice site usage by HIV-1 RNAs. The fact that four of five newly identified splice sites or variants of splice sites were detected in non-subtype B viruses allows anticipating an even greater diversity of HIV-1 splice site usage than currently known.

  1. An EMT-driven alternative splicing program occurs in human breast cancer and modulates cellular phenotype.

    Directory of Open Access Journals (Sweden)

    Irina M Shapiro

    2011-08-01

    Full Text Available Epithelial-mesenchymal transition (EMT, a mechanism important for embryonic development, plays a critical role during malignant transformation. While much is known about transcriptional regulation of EMT, alternative splicing of several genes has also been correlated with EMT progression, but the extent of splicing changes and their contributions to the morphological conversion accompanying EMT have not been investigated comprehensively. Using an established cell culture model and RNA-Seq analyses, we determined an alternative splicing signature for EMT. Genes encoding key drivers of EMT-dependent changes in cell phenotype, such as actin cytoskeleton remodeling, regulation of cell-cell junction formation, and regulation of cell migration, were enriched among EMT-associated alternatively splicing events. Our analysis suggested that most EMT-associated alternative splicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP, or ESRP classes of splicing factors. The EMT alternative splicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMT-associated splicing pattern. Expression of EMT-associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT-dependent splicing changes occur commonly in human tumors. The functional significance of EMT-associated alternative splicing was tested by expression of the epithelial-specific splicing factor ESRP1 or by depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT-associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression.

  2. An EMT-driven alternative splicing program occurs in human breast cancer and modulates cellular phenotype.

    Science.gov (United States)

    Shapiro, Irina M; Cheng, Albert W; Flytzanis, Nicholas C; Balsamo, Michele; Condeelis, John S; Oktay, Maja H; Burge, Christopher B; Gertler, Frank B

    2011-08-01

    Epithelial-mesenchymal transition (EMT), a mechanism important for embryonic development, plays a critical role during malignant transformation. While much is known about transcriptional regulation of EMT, alternative splicing of several genes has also been correlated with EMT progression, but the extent of splicing changes and their contributions to the morphological conversion accompanying EMT have not been investigated comprehensively. Using an established cell culture model and RNA-Seq analyses, we determined an alternative splicing signature for EMT. Genes encoding key drivers of EMT-dependent changes in cell phenotype, such as actin cytoskeleton remodeling, regulation of cell-cell junction formation, and regulation of cell migration, were enriched among EMT-associated alternatively splicing events. Our analysis suggested that most EMT-associated alternative splicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP, or ESRP classes of splicing factors. The EMT alternative splicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMT-associated splicing pattern. Expression of EMT-associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT-dependent splicing changes occur commonly in human tumors. The functional significance of EMT-associated alternative splicing was tested by expression of the epithelial-specific splicing factor ESRP1 or by depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT-associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression.

  3. Single-Molecule Analysis of Pre-mRNA Splicing with Colocalization Single-Molecule Spectroscopy (CoSMoS).

    Science.gov (United States)

    Braun, Joerg E; Serebrov, Victor

    2017-01-01

    Recent development of single-molecule techniques to study pre-mRNA splicing has provided insights into the dynamic nature of the spliceosome. Colocalization single-molecule spectroscopy (CoSMoS) allows following spliceosome assembly in real time at single-molecule resolution in the full complexity of cellular extracts. A detailed protocol of CoSMoS has been published previously (Anderson and Hoskins, Methods Mol Biol 1126:217-241, 2014). Here, we provide an update on the technical advances since the first CoSMoS studies including slide surface treatment, data processing, and representation. We describe various labeling strategies to generate RNA reporters with multiple dyes (or other moieties) at specific locations.

  4. NIF Target Assembly Metrology Methodology and Results

    Energy Technology Data Exchange (ETDEWEB)

    Alger, E. T. [General Atomics, San Diego, CA (United States); Kroll, J. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Dzenitis, E. G. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Montesanti, R. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Hughes, J. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Swisher, M. [IAP, Livermore, CA (United States); Taylor, J. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Segraves, K. [IAP, Livermore, CA (United States); Lord, D. M. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Reynolds, J. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Castro, C. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Edwards, G. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2011-01-01

    During our inertial confinement fusion (ICF) experiments at the National Ignition Facility (NIF) we require cryogenic targets at the 1-cm scale to be fabricated, assembled, and metrologized to micron-level tolerances. During assembly of these ICF targets, there are physical dimensmetrology is completed using optical coordinate measurement machines that provide repeatable measurements with micron precision, while also allowing in-process data collection for absolute accuracy in assembly. To date, 51 targets have been assembled and metrologized, and 34 targets have been successfully fielded on NIF relying on these metrology data. In the near future, ignition experiments on NIF will require tighter tolerances and more demanding target assembly and metrology capability. Metrology methods, calculations, and uncertainty estimates will be discussed. Target diagnostic port alignment, target position, and capsule location results will be reviewed for the 2009 Energetics Campaign. The information is presented via control charts showing the effect of process improvements that were made during target production. Certain parameters, including capsule position, met the 2009 campaign specifications but will have much tighter requirements in the future. Finally, in order to meet these new requirements assembly process changes and metrology capability upgrades will be necessary.

  5. Adaptive Processing for Sequence Alignment

    KAUST Repository

    Zidan, Mohammed A.

    2012-01-26

    Disclosed are various embodiments for adaptive processing for sequence alignment. In one embodiment, among others, a method includes obtaining a query sequence and a plurality of database sequences. A first portion of the plurality of database sequences is distributed to a central processing unit (CPU) and a second portion of the plurality of database sequences is distributed to a graphical processing unit (GPU) based upon a predetermined splitting ratio associated with the plurality of database sequences, where the database sequences of the first portion are shorter than the database sequences of the second portion. A first alignment score for the query sequence is determined with the CPU based upon the first portion of the plurality of database sequences and a second alignment score for the query sequence is determined with the GPU based upon the second portion of the plurality of database sequences.

  6. Newnes electronics assembly handbook

    CERN Document Server

    Brindley, Keith

    2013-01-01

    Newnes Electronics Assembly Handbook: Techniques, Standards and Quality Assurance focuses on the aspects of electronic assembling. The handbook first looks at the printed circuit board (PCB). Base materials, basic mechanical properties, cleaning of assemblies, design, and PCB manufacturing processes are then explained. The text also discusses surface mounted assemblies and packaging of electromechanical assemblies, as well as the soldering process. Requirements for the soldering process; solderability and protective coatings; cleaning of PCBs; and mass solder/component reflow soldering are des

  7. Tilt assembly for tracking solar collector assembly

    Science.gov (United States)

    Almy, Charles; Peurach, John; Sandler, Reuben

    2012-01-24

    A tilt assembly is used with a solar collector assembly of the type comprising a frame, supporting a solar collector, for movement about a tilt axis by pivoting a drive element between first and second orientations. The tilt assembly comprises a drive element coupler connected to the drive element and a driver, the driver comprising a drive frame, a drive arm and a drive arm driver. The drive arm is mounted to the drive frame for pivotal movement about a drive arm axis. Movement on the drive arm mimics movement of the drive element. Drive element couplers can extend in opposite directions from the outer portion of the drive arm, whereby the assembly can be used between adjacent solar collector assemblies in a row of solar collector assemblies.

  8. Fluorescence Reporter-Based Genome-Wide RNA Interference Screening to Identify Alternative Splicing Regulators.

    Science.gov (United States)

    Misra, Ashish; Green, Michael R

    2017-01-01

    Alternative splicing is a regulated process that leads to inclusion or exclusion of particular exons in a pre-mRNA transcript, resulting in multiple protein isoforms being encoded by a single gene. With more than 90 % of human genes known to undergo alternative splicing, it represents a major source for biological diversity inside cells. Although in vitro splicing assays have revealed insights into the mechanisms regulating individual alternative splicing events, our global understanding of alternative splicing regulation is still evolving. In recent years, genome-wide RNA interference (RNAi) screening has transformed biological research by enabling genome-scale loss-of-function screens in cultured cells and model organisms. In addition to resulting in the identification of new cellular pathways and potential drug targets, these screens have also uncovered many previously unknown mechanisms regulating alternative splicing. Here, we describe a method for the identification of alternative splicing regulators using genome-wide RNAi screening, as well as assays for further validation of the identified candidates. With modifications, this method can also be adapted to study the splicing regulation of pre-mRNAs that contain two or more splice isoforms.

  9. Analysis and recognition of 5 ' UTR intron splice sites in human pre-mRNA

    DEFF Research Database (Denmark)

    Eden, E.; Brunak, Søren

    2004-01-01

    Prediction of splice sites in non-coding regions of genes is one of the most challenging aspects of gene structure recognition. We perform a rigorous analysis of such splice sites embedded in human 5' untranslated regions (UTRs), and investigate correlations between this class of splice sites...... in the synaptic weights of the neural networks trained to identify UTR donor sites. Conventional splice site prediction methods perform poorly in UTRs because the reading frame pattern is absent. The NetUTR method presented here performs 2-.3-fold better compared with NetGene2 and GenScan in 5' UTRs. We also...

  10. Trans-Splicing Improvement by the Combined Application of Antisense Strategies

    Directory of Open Access Journals (Sweden)

    Ulrich Koller

    2015-01-01

    Full Text Available Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs, presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB. We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG, lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.

  11. Operon Conservation and the Evolution of trans-Splicing in the Phylum Nematoda

    OpenAIRE

    Guiliano, David B.; Blaxter, Mark L.

    2006-01-01

    The nematode Caenorhabditis elegans is unique among model animals in that many of its genes are cotranscribed as polycistronic pre-mRNAs from operons. The mechanism by which these operonic transcripts are resolved into mature mRNAs includes trans-splicing to a family of SL2-like spliced leader exons. SL2-like spliced leaders are distinct from SL1, the major spliced leader in C. elegans and other nematode species. We surveyed five additional nematode species, representing three of the five maj...

  12. Cephalopod eye evolution was modulated by the acquisition of Pax-6 splicing variants

    National Research Council Canada - National Science Library

    Yoshida, Masa-aki; Yura, Kei; Ogura, Atsushi

    2014-01-01

    Previous studies have reported that the developmental processes of vertebrate eyes are controlled by four Pax-6 splicing variants, each modulating different downstream genes, whereas those of insect...

  13. Genome-wide activation of latent donor splice sites in stress and disease.

    Science.gov (United States)

    Nevo, Yuval; Kamhi, Eyal; Jacob-Hirsch, Jasmine; Amariglio, Ninette; Rechavi, Gideon; Sperling, Joseph; Sperling, Ruth

    2012-11-01

    Sequences that conform to the 5' splice site (5'SS) consensus are highly abundant in mammalian introns. Most of these sequences are preceded by at least one in-frame stop codon; thus, their use for splicing would result in pre-maturely terminated aberrant mRNAs. In normally grown cells, such intronic 5'SSs appear not to be selected for splicing. However, under heat shock conditions aberrant splicing involving such latent 5'SSs occurred in a number of specific gene transcripts. Using a splicing-sensitive microarray, we show here that stress-induced (e.g. heat shock) activation of latent splicing is widespread across the human transcriptome, thus highlighting the possibility that latent splicing may underlie certain diseases. Consistent with this notion, our analyses of data from the Gene Expression Omnibus (GEO) revealed widespread activation of latent splicing in cells grown under hypoxia and in certain cancers such as breast cancer and gliomas. These changes were found in thousands of transcripts representing a wide variety of functional groups; among them are genes involved in cell proliferation and differentiation. The GEO analysis also revealed a set of gene transcripts in oligodendroglioma, in which the level of activation of latent splicing increased with the severity of the disease.

  14. Cotranscriptional recruitment of yeast TRAMP complex to intronic sequences promotes optimal pre-mRNA splicing

    OpenAIRE

    Kong, Ka-Yiu Edwin; Tang, Hei-Man Vincent; Pan, Kewu; Huang, Zhe; Lee, Tsz-Hang Jimmy; Hinnebusch, Alan G.; Jin, Dong-Yan; Wong, Chi-Ming

    2013-01-01

    Most unwanted RNA transcripts in the nucleus of eukaryotic cells, such as splicing-defective pre-mRNAs and spliced-out introns, are rapidly degraded by the nuclear exosome. In budding yeast, a number of these unwanted RNA transcripts, including spliced-out introns, are first recognized by the nuclear exosome cofactor Trf4/5p-Air1/2p-Mtr4p polyadenylation (TRAMP) complex before subsequent nuclear-exosome-mediated degradation. However, it remains unclear when spliced-out introns are recognized ...

  15. Splicing variants of ADAR2 and ADAR2-mediated RNA editing in glioma.

    Science.gov (United States)

    Fu, Yao; Zhao, Xingli; Li, Zhaohui; Wei, Jun; Tian, Yu

    2016-08-01

    The roles of alternative splicing and RNA editing in gene regulation and transcriptome diversity are well documented. Adenosine deaminases acting on RNA (ADARs) are responsible for adenosine-to-inosine (A-to-I) editing and exemplify the complex association between RNA editing and alternative splicing. The self-editing activity of ADAR2, which acts on its own pre-mRNA, leads to its alternative splicing. Alternative splicing occurs independently at nine splicing sites on ADAR2 pre-mRNA, generating numerous alternative splicing variants with various catalytic activities. A-to-I RNA editing is important in a range of physiological processes in humans and is associated with several diseases, including amyotrophic lateral sclerosis, mood disorders, epilepsy and glioma. Reduced editing at the glutamine/arginine site of the AMPA receptor subunit GluA2 in glioma, without any alteration in ADAR2 expression, is a notable phenomenon. Several studies have tried to explain this alteration in the catalytic activity of ADAR2; however, the underlying mechanism remains unclear. The present review summarizes the relevant literature and shares experimental results concerning ADAR2 alternative splicing. In particular, the present review demonstrates that shifts in the relative abundance of the active and inactive splicing variants of ADAR2 may reduce the ADAR2 editing activity in glioma. Dominant expression of ADAR2 splicing variant with low enzyme activity causes reduced RNA editing of GluA2 subunit at the glutamine/arginine site in glioma.

  16. Spatio-temporal and dynamic regulation of neurofascin alternative splicing in mouse cerebellar neurons.

    Science.gov (United States)

    Suzuki, Satoko; Ayukawa, Noriko; Okada, Chisa; Tanaka, Masami; Takekoshi, Susumu; Iijima, Yoko; Iijima, Takatoshi

    2017-09-12

    Alternative splicing is crucial for molecular diversification, which greatly contributes to the complexity and specificity of neural functions in the central nervous system (CNS). Neurofascin (NF) is a polymorphic cell surface protein that has a number of splicing isoforms. As the alternative splicing of the neurofascin gene (Nfasc) is developmentally regulated, NF isoforms have distinct functions in immature and mature brains. However, the molecular mechanisms underlying the alternative splicing of Nfasc in neurons are not yet understood. Here, we demonstrate that, alongside developmental regulation, Nfasc alternative splicing is spatially controlled in the mouse brain. We then identified distinct Nfasc splicing patterns at the cell-type level in the cerebellum, with Nfasc186 being expressed in Purkinje cells and absent from granule cells (GCs). Furthermore, we show that high K+-induced depolarization triggers a shift in splicing from Nfasc140 to Nfasc186 in cerebellar GCs. Finally, we identified a neural RNA-binding protein, Rbfox, as a key player in neural NF isoform selection, specifically controlling splicing at exons 26-29. Together, our results show that Nfasc alternative splicing is spatio-temporally and dynamically regulated in cerebellar neurons. Our findings provide profound insight into the mechanisms underlying the functional diversity of neuronal cell-adhesive proteins in the mammalian CNS.

  17. Ancient antagonism between CELF and RBFOX families tunes mRNA splicing outcomes.

    Science.gov (United States)

    Gazzara, Matthew R; Mallory, Michael J; Roytenberg, Renat; Lindberg, John P; Jha, Anupama; Lynch, Kristen W; Barash, Yoseph

    2017-08-01

    Over 95% of human multi-exon genes undergo alternative splicing, a process important in normal development and often dysregulated in disease. We sought to analyze the global splicing regulatory network of CELF2 in human T cells, a well-studied splicing regulator critical to T cell development and function. By integrating high-throughput sequencing data for binding and splicing quantification with sequence features and probabilistic splicing code models, we find evidence of splicing antagonism between CELF2 and the RBFOX family of splicing factors. We validate this functional antagonism through knockdown and overexpression experiments in human cells and find CELF2 represses RBFOX2 mRNA and protein levels. Because both families of proteins have been implicated in the development and maintenance of neuronal, muscle, and heart tissues, we analyzed publicly available data in these systems. Our analysis suggests global, antagonistic coregulation of splicing by the CELF and RBFOX proteins in mouse muscle and heart in several physiologically relevant targets, including proteins involved in calcium signaling and members of the MEF2 family of transcription factors. Importantly, a number of these coregulated events are aberrantly spliced in mouse models and human patients with diseases that affect these tissues, including heart failure, diabetes, or myotonic dystrophy. Finally, analysis of exons regulated by ancient CELF family homologs in chicken, Drosophila, and Caenorhabditis elegans suggests this antagonism is conserved throughout evolution. © 2017 Gazzara et al.; Published by Cold Spring Harbor Laboratory Press.

  18. XUV ionization of aligned molecules

    Energy Technology Data Exchange (ETDEWEB)

    Kelkensberg, F.; Siu, W.; Gademann, G. [FOM Institute AMOLF, Science Park 104, NL-1098 XG Amsterdam (Netherlands); Rouzee, A.; Vrakking, M. J. J. [FOM Institute AMOLF, Science Park 104, NL-1098 XG Amsterdam (Netherlands); Max-Born-Institut, Max-Born Strasse 2A, D-12489 Berlin (Germany); Johnsson, P. [FOM Institute AMOLF, Science Park 104, NL-1098 XG Amsterdam (Netherlands); Department of Physics, Lund University, Post Office Box 118, SE-221 00 Lund (Sweden); Lucchini, M. [Department of Physics, Politecnico di Milano, Istituto di Fotonica e Nanotecnologie CNR-IFN, Piazza Leonardo da Vinci 32, 20133 Milano (Italy); Lucchese, R. R. [Department of Chemistry, Texas A and M University, College Station, Texas 77843-3255 (United States)

    2011-11-15

    New extreme-ultraviolet (XUV) light sources such as high-order-harmonic generation (HHG) and free-electron lasers (FELs), combined with laser-induced alignment techniques, enable novel methods for making molecular movies based on measuring molecular frame photoelectron angular distributions. Experiments are presented where CO{sub 2} molecules were impulsively aligned using a near-infrared laser and ionized using femtosecond XUV pulses obtained by HHG. Measured electron angular distributions reveal contributions from four orbitals and the onset of the influence of the molecular structure.

  19. Targeted RNA-Seq profiling of splicing pattern in the DMD gene: exons are mostly constitutively spliced in human skeletal muscle.

    Science.gov (United States)

    Bougé, Anne-Laure; Murauer, Eva; Beyne, Emmanuelle; Miro, Julie; Varilh, Jessica; Taulan, Magali; Koenig, Michel; Claustres, Mireille; Tuffery-Giraud, Sylvie

    2017-01-03

    We have analysed the splicing pattern of the human Duchenne Muscular Dystrophy (DMD) NB transcript in normal skeletal muscle. To achieve depth of coverage required for the analysis of this lowly expressed gene in muscle, we designed a targeted RNA-Seq procedure that combines amplification of the full-length 11.3 kb DMD cDNA sequence and 454 sequencing technology. A high and uniform coverage of the cDNA sequence was obtained that allowed to draw up a reliable inventory of the physiological alternative splicing events in the muscular DMD transcript. In contrast to previous assumptions, we evidenced that most of the 79 DMD exons are constitutively spliced in skeletal muscle. Only a limited number of 12 alternative splicing events were identified, all present at a very low level. These include previously known exon skipping events but also newly described pseudoexon inclusions and alternative 3' splice sites, of which one is the first functional NAGNAG splice site reported in the DMD gene. This study provides the first RNA-Seq-based reference of DMD splicing pattern in skeletal muscle and reports on an experimental procedure well suited to detect condition-specific differences in this low abundance transcript that may prove useful for diagnostic, research or RNA-based therapeutic applications.

  20. Combined Cell Culture-Biosensing Platform Using Vertically Aligned Patterned Peptide Nanofibers for Cellular Studies

    DEFF Research Database (Denmark)

    Taskin, Mehmet B.; Sasso, Luigi; Dimaki, Maria

    2013-01-01

    This Article presents the development of a combined cell culture–biosensing platform using vertically aligned self-assembled peptide nanofibers. Peptide nanofibers were patterned on a microchip containing gold microelectrodes to provide the cells with a 3D environment enabling them to grow and pr...

  1. Global alignment algorithms implementations | Fatumo ...

    African Journals Online (AJOL)

    In this paper, we implemented the two routes for sequence comparison, that is; the dotplot and Needleman-wunsch algorithm for global sequence alignment. Our algorithms were implemented in python programming language and were tested on Linux platform 1.60GHz, 512 MB of RAM SUSE 9.2 and 10.1 versions.

  2. Aligning Assessments for COSMA Accreditation

    Science.gov (United States)

    Laird, Curt; Johnson, Dennis A.; Alderman, Heather

    2015-01-01

    Many higher education sport management programs are currently in the process of seeking accreditation from the Commission on Sport Management Accreditation (COSMA). This article provides a best-practice method for aligning student learning outcomes with a sport management program's mission and goals. Formative and summative assessment procedures…

  3. Position list word aligned hybrid

    DEFF Research Database (Denmark)

    Deliege, Francois; Pedersen, Torben Bach

    2010-01-01

    of storage space. This paper presents the Position List Word Aligned Hybrid (PLWAH) compression scheme that improves significantly over WAH compression by better utilizing the available bits and new CPU instructions. For typical bit distributions, PLWAH compressed bitmaps are often half the size of WAH...

  4. Alignment of diabetic feet images

    NARCIS (Netherlands)

    Klein, Almar; van der Heijden, Ferdinand; Slump, Cornelis H.; Uyl, M.J.; Philips, W.

    2007-01-01

    This paper addresses the problem of aligning the images of feet taken at different instances in time. We propose to use SIFT keypoints to find the geometric deformation between two photo’s. We then have a set of landmarks for each image. By finding the corresponding landmarks (i.e. matching the

  5. Genome assembly quality: Assessment and improvement using the neutral indel model

    Science.gov (United States)

    Meader, Stephen; Hillier, LaDeana W.; Locke, Devin; Ponting, Chris P.; Lunter, Gerton

    2010-01-01

    We describe a statistical and comparative-genomic approach for quantifying error rates of genome sequence assemblies. The method exploits not substitutions but the pattern of insertions and deletions (indels) in genome-scale alignments for closely related species. Using two- or three-way alignments, the approach estimates the amount of aligned sequence containing clusters of nucleotides that were wrongly inserted or deleted during sequencing or assembly. Thus, the method is well-suited to assessing fine-scale sequence quality within single assemblies, between different assemblies of a single set of reads, and between genome assemblies for different species. When applying this approach to four primate genome assemblies, we found that average gap error rates per base varied considerably, by up to sixfold. As expected, bacterial artificial chromosome (BAC) sequences contained lower, but still substantial, predicted numbers of errors, arguing for caution in regarding BACs as the epitome of genome fidelity. We then mapped short reads, at approximately 10-fold statistical coverage, from a Bornean orangutan onto the Sumatran orangutan genome assembly originally constructed from capillary reads. This resulted in a reduced gap error rate and a separation of error-prone from high-fidelity sequence. Over 5000 predicted indel errors in protein-coding sequence were corrected in a hybrid assembly. Our approach contributes a new fine-scale quality metric for assemblies that should facilitate development of improved genome sequencing and assembly strategies. PMID:20305016

  6. Progressive multiple sequence alignments from triplets

    Directory of Open Access Journals (Sweden)

    Stadler Peter F

    2007-07-01

    Full Text Available Abstract Background The quality of progressive sequence alignments strongly depends on the accuracy of the individual pairwise alignment steps since gaps that are introduced at one step cannot be removed at later aggregation steps. Adjacent insertions and deletions necessarily appear in arbitrary order in pairwise alignments and hence form an unavoidable source of errors. Research Here we present a modified variant of progressive sequence alignments that addresses both issues. Instead of pairwise alignments we use exact dynamic programming to align sequence or profile triples. This avoids a large fractions of the ambiguities arising in pairwise alignments. In the subsequent aggregation steps we follow the logic of the Neighbor-Net algorithm, which constructs a phylogenetic network by step-wisely replacing triples by pairs instead of combining pairs to singletons. To this end the three-way alignments are subdivided into two partial alignments, at which stage all-gap columns are naturally removed. This alleviates the "once a gap, always a gap" problem of progressive alignment procedures. Conclusion The three-way Neighbor-Net based alignment program aln3nn is shown to compare favorably on both protein sequences and nucleic acids sequences to other progressive alignment tools. In the latter case one easily can include scoring terms that consider secondary structure features. Overall, the quality of resulting alignments in general exceeds that of clustalw or other multiple alignments tools even though our software does not included heuristics for context dependent (mismatch scores.

  7. Tissue-specific alternative splicing of Tak1 is conserved in deuterostomes.

    Science.gov (United States)

    Venables, Julian P; Vignal, Emmanuel; Baghdiguian, Stephen; Fort, Philippe; Tazi, Jamal

    2012-01-01

    Alternative splicing allows organisms to rapidly modulate protein functions to physiological changes and therefore represents a highly versatile adaptive process. We investigated the conservation of the evolutionary history of the "Fox" family of RNA-binding splicing factors (RBFOX) as well as the conservation of regulated alternative splicing of the genes they control. We found that the RBFOX proteins are conserved in all metazoans examined. In humans, Fox proteins control muscle-specific alternative splicing of many genes but despite the conservation of splicing factors, conservation of regulation of alternative splicing has never been demonstrated between man and nonvertebrate species. Therefore, we studied 40 known Fox-regulated human exons and found that 22 had a tissue-specific splicing pattern in muscle and heart. Of these, 11 were spliced in the same tissue-specific manner in mouse tissues and 4 were tissue-specifically spliced in muscle and heart of the frog Xenopus laevis. The inclusion of two of these alternative exons was also downregulated during tadpole development. Of the 40 in the starting set, the most conserved alternative splicing event was in the transforming growth factor (TGF) beta-activated kinase Tak1 (MAP3K7) as this was also muscle specific in urochordates and in Ambulacraria, the most ancient deuterostome clade. We found exclusion of the muscle-specific exon of Tak1 was itself under control of TGF beta in cell culture and consistently that TGF beta caused an upregulation of Fox2 (RBFOX2) expression. The alternative exon, which codes for an in-frame 27 amino acids between the kinase and known regulatory domain of TAK1, contains conserved features in all organisms including potential phosphorylation sites and likely has an important conserved function in TGF beta signaling and development. This study establishes that deuterostomes share a remarkable conserved physiological process that involves a splicing factor and expression of tissue

  8. Autonomous electrochromic assembly

    Energy Technology Data Exchange (ETDEWEB)

    Berland, Brian Spencer; Lanning, Bruce Roy; Stowell, Jr., Michael Wayne

    2015-03-10

    This disclosure describes system and methods for creating an autonomous electrochromic assembly, and systems and methods for use of the autonomous electrochromic assembly in combination with a window. Embodiments described herein include an electrochromic assembly that has an electrochromic device, an energy storage device, an energy collection device, and an electrochromic controller device. These devices may be combined into a unitary electrochromic insert assembly. The electrochromic assembly may have the capability of generating power sufficient to operate and control an electrochromic device. This control may occur through the application of a voltage to an electrochromic device to change its opacity state. The electrochromic assembly may be used in combination with a window.

  9. Firearm trigger assembly

    Science.gov (United States)

    Crandall, David L.; Watson, Richard W.

    2010-02-16

    A firearm trigger assembly for use with a firearm includes a trigger mounted to a forestock of the firearm so that the trigger is movable between a rest position and a triggering position by a forwardly placed support hand of a user. An elongated trigger member operatively associated with the trigger operates a sear assembly of the firearm when the trigger is moved to the triggering position. An action release assembly operatively associated with the firearm trigger assembly and a movable assembly of the firearm prevents the trigger from being moved to the triggering position when the movable assembly is not in the locked position.

  10. Remote alignment of Low beta quadrupoles with micrometric resolution

    CERN Document Server

    Acar, M; Herty, A; Mainaud-Durand, H; Marin, A; Quesnel, J P

    2008-01-01

    Considering their location in a high radiation environment and the alignment tolerancesrequested, the low beta quadrupoles of LHC will be positioned remotely (controlling 5 degrees of freedom), with a displacement resolution of few microns in horizontal and vertical. Stepping motor gearbox assemblies are plugged into the jacks which support the cryomagnets in order to move them to the desired position regarding the quality of the beam collisions in the detectors. This displacement will be monitored in real time by the sensors located on the magnets. This paper describes the positioning strategy implemented as well as the software tools used to manage it.

  11. Performance of Grouted Splice Sleeve Connector under Tensile Load

    Directory of Open Access Journals (Sweden)

    A. Alias

    2016-05-01

    Full Text Available The grouted splice sleeve connector system takes advantage of the bond-slip resistance of the grout and the mechanical gripping of reinforcement bars to provide resistance to tensile force. In this system, grout acts as a load-transferring medium and bonding material between the bars and sleeve. This study adopted the end-to-end rebars connection method to investigate the effect of development length and sleeve diameter on the bonding performance of the sleeve connector. The end-to-end method refers to the condition where reinforcement bars are inserted into the sleeve from both ends and meet at the centre before grout is filled. Eight specimens of grouted splice sleeve connector were tested under tensile load to determine their performance. The sleeve connector was designed using 5 mm thick circular hollow section (CHS steel pipe and consisted of one external and two internal sleeves. The tensile test results show that connectors with a smaller external and internal sleeve diameter appear to provide better bonding performance. Three types of failure were observed in this research, which are bar fracture (outside the sleeve, bar pullout, and internal sleeve pullout. With reference to these failure types, the development length of 200 mm is the optimum value due to its bar fracture type, which indicates that the tensile capacity of the connector is higher than the reinforcement bar. It is found that the performance of the grouted splice sleeve connector is influenced by the development length of the reinforcement bar and the diameter of the sleeve.

  12. Alternative splicing of CNOT7 diversifies CCR4-NOT functions.

    Science.gov (United States)

    Chapat, Clément; Chettab, Kamel; Simonet, Pierre; Wang, Peng; De La Grange, Pierre; Le Romancer, Muriel; Corbo, Laura

    2017-08-21

    The CCR4-associated factor CAF1, also called CNOT7, is a catalytic subunit of the CCR4-NOT complex, which has been implicated in all aspects of the mRNA life cycle, from mRNA synthesis in the nucleus to degradation in the cytoplasm. In human cells, alternative splicing of the CNOT7 gene yields a second CNOT7 transcript leading to the formation of a shorter protein, CNOT7 variant 2 (CNOT7v2). Biochemical characterization indicates that CNOT7v2 interacts with CCR4-NOT subunits, although it does not bind to BTG proteins. We report that CNOT7v2 displays a distinct expression profile in human tissues, as well as a nuclear sub-cellular localization compared to CNOT7v1. Despite a conserved DEDD nuclease domain, CNOT7v2 is unable to degrade a poly(A) tail in vitro and preferentially associates with the protein arginine methyltransferase PRMT1 to regulate its activity. Using both in vitro and in cellulo systems, we have also demonstrated that CNOT7v2 regulates the inclusion of CD44 variable exons. Altogether, our findings suggest a preferential involvement of CNOT7v2 in nuclear processes, such as arginine methylation and alternative splicing, rather than mRNA turnover. These observations illustrate how the integration of a splicing variant inside CCR4-NOT can diversify its cell- and tissue-specific functions. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Altered splicing in prelamin A-associated premature aging phenotypes.

    Science.gov (United States)

    De Sandre-Giovannoli, Annachiara; Lévy, Nicolas

    2006-01-01

    Hutchinson-Gilford progeria (HGPS), a rare and severe developmental disorder characterized by features recalling premature aging, and restrictive dermopathy (RD), a neonatal lethal genodermatosis, have recently been identified as being primary or secondary "laminopathies." These are heterogeneous disorders due to altered function of lamins A/C or related proteins. In physiological conditions, mature lamin A is obtained through a series of post-translational processing steps performed on a protein precursor, prelamin A. The major pathophysiological mechanism involved in progeria is an aberrant splicing of pre-mRNAs issued from the LMNA gene, due to a de novo heterozygous point mutation, leading to the production and accumulation of truncated lamin A precursors. Aberrant splicing of prelamin A pre-mRNAs causing the production of more extensively truncated precursors is involved in the allelic disease restrictive dermopathy. Other restrictive dermopathy cases are due to the inactivation of a key enzyme involved in the maturation of lamin A precursors (ZMPSTE24). In functional terms, all these conditions share the same pathophysiological basis: intranuclear accumulation of lamin A precursors, which cannot be fully processed (due to primary or secondary events) and exert toxic, dominant negative effects on nuclear homeostasis. Most other laminopathies are due to autosomal dominant LMNA point mutations inferred to cause single amino acid substitutions. In any case, the impact of these mutations on pre-mRNA splicing has rarely been assessed. These disorders affect different tissues and organs, mainly including bone, skin, striated muscles, adipose tissue, vessels, and peripheral nerves in isolated or combined fashions, giving rise to syndromes whose severity ranges from mild to perinatally lethal. In this chapter we review the structure and functions of lamins A/C in physiological and pathological conditions, describe their known or putative roles, namely, in the

  14. Periostin shows increased evolutionary plasticity in its alternatively spliced region

    Directory of Open Access Journals (Sweden)

    Hoersch Sebastian

    2010-01-01

    Full Text Available Abstract Background Periostin (POSTN is a secreted extracellular matrix protein of poorly defined function that has been related to bone and heart development as well as to cancer. In human and mouse, it is known to undergo alternative splicing in its C-terminal region, which is devoid of known protein domains. Differential expression of periostin, sometimes of specific splicing isoforms, is observed in a broad range of human cancers, including breast, pancreatic, and colon cancer. Here, we combine genomic and transcriptomic sequence data from vertebrate organisms to study the evolution of periostin and particularly of its C-terminal region. Results We found that the C-terminal part of periostin is markedly more variable among vertebrates than the rest of periostin in terms of exon count, length, and splicing pattern, which we interpret as a consequence of neofunctionalization after the split between periostin and its paralog transforming growth factor, beta-induced (TGFBI. We also defined periostin's sequential 13-amino acid repeat units - well conserved in teleost fish, but more obscure in higher vertebrates - whose secondary structure is predicted to be consecutive beta strands. We suggest that these beta strands may mediate binding interactions with other proteins through an extended beta-zipper in a manner similar to the way repeat units in bacterial cell wall proteins have been reported to bind human fibronectin. Conclusions Our results, obtained with the help of the increasingly large collection of complete vertebrate genomes, document the evolutionary plasticity of periostin's C-terminal region, and for the first time suggest a basis for its functional role.

  15. NoFold: RNA structure clustering without folding or alignment.

    Science.gov (United States)

    Middleton, Sarah A; Kim, Junhyong

    2014-11-01

    Structures that recur across multiple different transcripts, called structure motifs, often perform a similar function-for example, recruiting a specific RNA-binding protein that then regulates translation, splicing, or subcellular localization. Identifying common motifs between coregulated transcripts may therefore yield significant insight into their binding partners and mechanism of regulation. However, as most methods for clustering structures are based on folding individual sequences or doing many pairwise alignments, this results in a tradeoff between speed and accuracy that can be problematic for large-scale data sets. Here we describe a novel method for comparing and characterizing RNA secondary structures that does not require folding or pairwise alignment of the input sequences. Our method uses the idea of constructing a distance function between two objects by their respective distances to a collection of empirical examples or models, which in our case consists of 1973 Rfam family covariance models. Using this as a basis for measuring structural similarity, we developed a clustering pipeline called NoFold to automatically identify and annotate structure motifs within large sequence data sets. We demonstrate that NoFold can simultaneously identify multiple structure motifs with an average sensitivity of 0.80 and precision of 0.98 and generally exceeds the performance of existing methods. We also perform a cross-validation analysis of the entire set of Rfam families, achieving an average sensitivity of 0.57. We apply NoFold to identify motifs enriched in dendritically localized transcripts and report 213 enriched motifs, including both known and novel structures. © 2014 Middleton and Kim; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  16. Underwater Splicing of SD Coaxial Cable--FY78 Progress.

    Science.gov (United States)

    1978-12-01

    u i d s , casto r oil and Marcol 70 minera l oil were chosen for testing because of their low water absorption characteristics and good dielectric...the gelled castor oil was tested in the splice configuration. Al so Marcol 70 minera l oil was gel led and tested on the spl ice. Experiments...determined that approximately 9.4% by weight of Cab-0-Sil added to castor oil produced a gel which had the consistency of soft grease. For Marcol 70 minera l

  17. LHC interaction region quadrupole cryostat production, alignment, and performance summary

    CERN Document Server

    Nicol, T H; Kerby, J S; Page, Thomas M; Peterson, Thomas J; Rabehl, Roger Jon

    2004-01-01

    The cryostat of a Large Hadron Collider (LHC) Interaction Region (IR) quadrupole magnet consists of all components of the inner triplet except the magnet assembly itself. It serves to support the magnet accurately and reliably within the vacuum vessel, to provide all required cryogenic piping, and to insulate the cold mass from heat radiated and conducted from the environment. The major components of the cryostat are the vacuum vessel, thermal shield, multi-layer insulation system, cryogenic piping, interconnections, and suspension system. While responsibility for the design and manufacture of the main quadrupole elements is divided between Fermilab and KEK, Fermilab alone is responsible for the design and final assembly of the cryostat for the LHC inner triplets. This paper describes the experience gained during fabrication of the first complete Q2 magnets, the alignment operation and results, and the cryogenic performance of the magnet on the test stand at Fermilab. 4 Refs.

  18. Microtubule alignment and manipulation using AC electrokinetics.

    Science.gov (United States)

    Uppalapati, Maruti; Huang, Ying-Ming; Jackson, Thomas N; Hancock, William O

    2008-09-01

    The kinesin-microtubule system plays an important role in intracellular transport and is a model system for integrating biomotor-driven transport into microengineered devices. AC electrokinetics provides a novel tool for manipulating and organizing microtubules in solution, enabling new experimental geometries for investigating and controlling the interactions of microtubules and microtubule motors in vitro. By fabricating microelectrodes on glass substrates and generating AC electric fields across solutions of microtubules in low-ionic-strength buffers, bundles of microtubules are collected and aligned and the electrical properties of microtubules in solution are measured. The AC electric fields result in electro-osmotic flow, electrothermal flow, and dielectrophoresis of microtubules, which can be controlled by varying the solution conductivity, AC frequency, and electrode geometry. By mapping the solution conductivity and AC frequency over which positive dielectrophoresis occurs, the apparent conductivity of taxol-stabilized bovine-brain microtubules in PIPES buffer is measured to be 250 mS m(-1). By maximizing dielectrophoretic forces and minimizing electro-osmotic and electrothermal flow, microtubules are assembled into opposed asters. These experiments demonstrate that AC electrokinetics provides a powerful new tool for kinesin-driven transport applications and for investigating the role of microtubule motors in development and maintenance of the mitotic spindle.

  19. Alternative splicing events are a late feature of pathology in a mouse model of spinal muscular atrophy

    National Research Council Canada - National Science Library

    Bäumer, Dirk; Lee, Sheena; Nicholson, George; Davies, Joanna L; Parkinson, Nicholas J; Murray, Lyndsay M; Gillingwater, Thomas H; Ansorge, Olaf; Davies, Kay E; Talbot, Kevin

    2009-01-01

    .... Widespread splicing abnormalities have recently been reported at end-stage in a mouse model of SMA, leading to the proposition that disruption of efficient splicing is the primary mechanism of motor neuron death...

  20. Tiling Assembly: a new tool for reference annotation-independent transcript assembly and novel gene identification by RNA-sequencing

    Science.gov (United States)

    Watanabe, Kenneth A.; Homayouni, Arielle; Tufano, Tara; Lopez, Jennifer; Ringler, Patricia; Rushton, Paul; Shen, Qingxi J.

    2015-01-01

    Annotation of the rice (Oryza sativa) genome has evolved significantly since release of its draft sequence, but it is far from complete. Several published transcript assembly programmes were tested on RNA-sequencing (RNA-seq) data to determine their effectiveness in identifying novel genes to improve the rice genome annotation. Cufflinks, a popular assembly software, did not identify all transcripts suggested by the RNA-seq data. Other assembly software was CPU intensive, lacked documentation, or lacked software updates. To overcome these shortcomings, a heuristic ab initio transcript assembly algorithm, Tiling Assembly, was developed to identify genes based on short read and junction alignment. Tiling Assembly was compared with Cufflinks to evaluate its gene-finding capabilities. Additionally, a pipeline was developed to eliminate false-positive gene identification due to noise or repetitive regions in the genome. By combining Tiling Assembly and Cufflinks, 767 unannotated genes were identified in the rice genome, demonstrating that combining both programmes proved highly efficient for novel gene identification. We also demonstrated that Tiling Assembly can accurately determine transcription start sites by comparing the Tiling Assembly genes with their corresponding full-length cDNA. We applied our pipeline to additional organisms and identified numerous unannotated genes, demonstrating that Tiling Assembly is an organism-independent tool for genome annotation. PMID:26341416

  1. Functional diversity of human protein kinase splice variants marks significant expansion of human kinome

    Directory of Open Access Journals (Sweden)

    Anamika Krishanpal

    2009-12-01

    Full Text Available Abstract Background Protein kinases are involved in diverse spectrum of cellular processes. Availability of draft version of the human genomic data in the year 2001 enabled recognition of repertoire of protein kinases. However, over the years the human genomic data is being refined and the current release of human genomic data has helped us to recognize a larger repertoire of over 900 human protein kinases represented mainly by splice variants. Results Many of these identified protein kinases are alternatively spliced products. Interestingly, some of the human kinase splice variants appear to be significantly diverged in terms of their functional properties as represented by incorporation or absence of one or more domains. Many sets of protein kinase splice variants have substantially different domain organization and in a few sets of splice variants kinase domains belong to different subfamilies of kinases suggesting potential participation in different signal transduction pathways. Conclusions Addition or deletion of a domain between splice variants of multi-domain kinases appears to be a means of generating differences in the functional features of otherwise similar kinases. It is intriguing that marked sequence diversity within the catalytic regions of some of the splice variant kinases result in kinases belonging to different subfamilies. These human kinase splice variants with different functions might contribute to diversity of eukaryotic cellular signaling.

  2. Decreased alternative splicing of estrogen receptor-α mRNA in the Alzheimer's disease brain

    NARCIS (Netherlands)

    Ishunina, Tatjana A.; Swaab, Dick F.

    2012-01-01

    In this study we identified 62 estrogen receptor alpha (ERα) mRNA splice variants in different human brain areas of Alzheimer's disease (AD) and control cases and classified them into 12 groups. Forty-eight of these splice forms were identified for the first time. The distribution of alternatively

  3. Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing

    Directory of Open Access Journals (Sweden)

    Delphine Trochet

    2016-01-01

    Full Text Available Dynamin 2 (DNM2 is a large GTPase, ubiquitously expressed, involved in membrane trafficking and regulation of actin and microtubule cytoskeletons. DNM2 mutations cause autosomal dominant centronuclear myopathy which is a rare congenital myopathy characterized by skeletal muscle weakness and histopathological features including nuclear centralization in absence of regeneration. No curative treatment is currently available for the DNM2-related autosomal dominant centronuclear myopathy. In order to develop therapeutic strategy, we evaluated here the potential of Spliceosome-Mediated RNA Trans-splicing technology to reprogram the Dnm2-mRNA in vitro and in vivo in mice. We show that classical 3′-trans-splicing strategy cannot be considered as accurate therapeutic strategy regarding toxicity of the pre-trans-splicing molecules leading to low rate of trans-splicing in vivo. Thus, we tested alternative strategies devoted to prevent this toxicity and enhance frequency of trans-splicing events. We succeeded to overcome the toxicity through a 5′-trans-splicing strategy which also allows detection of trans-splicing events at mRNA and protein levels in vitro and in vivo. These results suggest that the Spliceosome-Mediated RNA Trans-splicing strategy may be used to reprogram mutated Dnm2-mRNA but highlight the potential toxicity linked to the molecular tools which have to be carefully investigated during preclinical development.

  4. Identification of a novel function of CX-4945 as a splicing regulator.

    Directory of Open Access Journals (Sweden)

    Hyeongki Kim

    Full Text Available Alternative splicing is a nearly ubiquitous versatile process that controls gene expression and creates numerous protein isoforms with different functions from a single gene. The significance of alternative splicing has been confirmed by the increasing number of human diseases that are caused by misregulation of splicing events. Very few compounds, however, have been reported to act as inhibitors of alternative splicing, and their potential clinical use needs to be evaluated. Here, we report that CX-4945, a previously well-characterized inhibitor of casein kinase 2 (CK2 and a molecule currently in clinical trials (Phase II for cancer treatment, regulates splicing in mammalian cells in a CK2-independent manner. Transcriptome-wide analysis using exon array also showed a widespread alteration in alternative splicing of numerous genes. We found that CX-4945 potently inhibits the Cdc2-like kinases (Clks in vitro and in turn, leads to suppression of the phosphorylation of serine/arginine-rich (SR proteins in mammalian cells. Surprisingly, the overall efficacy of CX-4945 on Clks (IC50 = 3-90 nM was stronger than that of TG-003, the strongest inhibitor reported to date. Of the Clks, Clk2 was most strongly inhibited by CX-4945 in an ATP-competitive manner. Our research revealed an unexpected activity of the drug candidate CX-4945 as a potent splicing modulator and also suggested a potential application for therapy of diseases caused by abnormal splicing.

  5. Neuronal fast activating and meningeal silent modulatory BK channel splice variants cloned from rat

    DEFF Research Database (Denmark)

    Poulsen, Asser Nyander; Jansen-Olesen, Inger; Olesen, Jes

    2011-01-01

    The big conductance calcium-activated K(+) channel (BK) is involved in regulating neuron and smooth muscle cell excitability. Functional diversity of BK is generated by alpha-subunit splice variation and co-expression with beta subunits. Here, we present six different splice combinations cloned f...

  6. Unusual intron conservation near tissue-regulated exons found by splicing microarrays.

    Directory of Open Access Journals (Sweden)

    Charles W Sugnet

    2006-01-01

    Full Text Available Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe differential expression of RNA containing at least two alternative splice junctions for about 40% of the 6,216 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif that is strikingly similar to the branchpoint consensus but is located downstream of the 5' splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase genes reveals that each contains a paralogous tissue-regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families.

  7. Operon conservation and the evolution of trans-splicing in the phylum Nematoda.

    Science.gov (United States)

    Guiliano, David B; Blaxter, Mark L

    2006-11-24

    The nematode Caenorhabditis elegans is unique among model animals in that many of its genes are cotranscribed as polycistronic pre-mRNAs from operons. The mechanism by which these operonic transcripts are resolved into mature mRNAs includes trans-splicing to a family of SL2-like spliced leader exons. SL2-like spliced leaders are distinct from SL1, the major spliced leader in C. elegans and other nematode species. We surveyed five additional nematode species, representing three of the five major clades of the phylum Nematoda, for the presence of operons and the use of trans-spliced leaders in resolution of polycistronic pre-mRNAs. Conserved operons were found in Pristionchus pacificus, Nippostrongylus brasiliensis, Strongyloides ratti, Brugia malayi, and Ascaris suum. In nematodes closely related to the rhabditine C. elegans, a related family of SL2-like spliced leaders is used for operonic transcript resolution. However, in the tylenchine S. ratti operonic transcripts are resolved using a family of spliced leaders related to SL1. Non-operonic genes in S. ratti may also receive these SL1 variants. In the spirurine nematodes B. malayi and A. suum operonic transcripts are resolved using SL1. Mapping these phenotypes onto the robust molecular phylogeny for the Nematoda suggests that operons evolved before SL2-like spliced leaders, which are an evolutionary invention of the rhabditine lineage.

  8. Operon conservation and the evolution of trans-splicing in the phylum Nematoda.

    Directory of Open Access Journals (Sweden)

    David B Guiliano

    2006-11-01

    Full Text Available The nematode Caenorhabditis elegans is unique among model animals in that many of its genes are cotranscribed as polycistronic pre-mRNAs from operons. The mechanism by which these operonic transcripts are resolved into mature mRNAs includes trans-splicing to a family of SL2-like spliced leader exons. SL2-like spliced leaders are distinct from SL1, the major spliced leader in C. elegans and other nematode species. We surveyed five additional nematode species, representing three of the five major clades of the phylum Nematoda, for the presence of operons and the use of trans-spliced leaders in resolution of polycistronic pre-mRNAs. Conserved operons were found in Pristionchus pacificus, Nippostrongylus brasiliensis, Strongyloides ratti, Brugia malayi, and Ascaris suum. In nematodes closely related to the rhabditine C. elegans, a related family of SL2-like spliced leaders is used for operonic transcript resolution. However, in the tylenchine S. ratti operonic transcripts are resolved using a family of spliced leaders related to SL1. Non-operonic genes in S. ratti may also receive these SL1 variants. In the spirurine nematodes B. malayi and A. suum operonic transcripts are resolved using SL1. Mapping these phenotypes onto the robust molecular phylogeny for the Nematoda suggests that operons evolved before SL2-like spliced leaders, which are an evolutionary invention of the rhabditine lineage.

  9. Features of 5'-splice-site efficiency derived from disease-causing mutations and comparative genomics

    DEFF Research Database (Denmark)

    Roca, Xavier; Olson, Andrew J; Rao, Atmakuri R

    2007-01-01

    Many human diseases, including Fanconi anemia, hemophilia B, neurofibromatosis, and phenylketonuria, can be caused by 5'-splice-site (5'ss) mutations that are not predicted to disrupt splicing, according to position weight matrices. By using comparative genomics, we identify pairwise dependencies...

  10. Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene

    Energy Technology Data Exchange (ETDEWEB)

    Conboy, John G.; Parra, Marilyn K.; Tan, Jeff S.; Mohandas, Narla; Conboy, John G.

    2008-11-07

    In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branch points for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism whereby alternative promoters can be coordinated with downstream alternative splicing.

  11. Conservation and sex-specific splicing of the doublesex gene in the ...

    Indian Academy of Sciences (India)

    Genetic control of sex determination in insects has been best characterized in Drosophila melanogaster, where the master gene Sxl codes for RNA that is sex specifically spliced to produce a functional protein only in females. SXL regulates the sex-specific splicing of transformer (tra) RNA which, in turn, regulates the ...

  12. A new view of transcriptome complexity and regulation through the lens of local splicing variations

    National Research Council Canada - National Science Library

    Vaquero-Garcia, Jorge; Barrera, Alejandro; Gazzara, Matthew R; González-Vallinas, Juan; Lahens, Nicholas F; Hogenesch, John B; Lynch, Kristen W; Barash, Yoseph

    2016-01-01

    .... Amongst those are novel isoforms in the Camk2 family and a novel poison exon in Ptbp1, a key splice factor in neurogenesis. We anticipate the approach presented here will advance the ability to relate tissue-specific splice variation to genetic variation, phenotype, and disease.

  13. Effect of splice-site polymorphisms of the TMPRSS4, NPHP4 and ...

    Indian Academy of Sciences (India)

    Unknown

    Next, the effect of these polymorphisms on the mode of pre-mRNA splicing was investigated by ... [Yamada H., Shinmura K., Tsuneyoshi T. and Sugimura H. 2005 Effect of splice-site polymorphisms of the TMPRSS4, NPHP4 and. ORCTL4 genes on their ..... 2A>C polymorphism lack major parts of the sugar trans- porter and ...

  14. Differential recruitment of nuclear receptor coactivators may determine alternative RNA splice site choice in target genes

    Science.gov (United States)

    Auboeuf, Didier; Dowhan, Dennis H.; Kang, Yun Kyoung; Larkin, Kimberly; Lee, Jae Woon; Berget, Susan M.; O'Malley, Bert W.

    2004-01-01

    The biological consequences of steroid hormone-mediated transcriptional activation of target genes might be difficult to predict because alternative splicing of a single neosynthesized precursor RNA can result in production of different protein isoforms with opposite biological activities. Therefore, an important question to address is the manner in which steroid hormones affect the splicing of their target gene transcripts. In this report, we demonstrate that individual steroid hormones had different and opposite effects on alternative splicing decisions, stimulating the production of different spliced variants produced from genes driven by steroid hormone-dependent promoters. Steroid hormone transcriptional effects are mediated by steroid hormone receptor coregulators that also modify alternative splicing decisions. Our data suggest that activated steroid hormone receptors recruit coregulators to the target promoter that participate in both the production and the splicing of the target gene transcripts. Because different coregulators activating transcription can have opposite effects on alternative splicing decisions, we conclude that the precise nature of the transcriptional coregulators recruited by activated steroid receptors, depending on the promoter and cellular contexts, may play a major role in regulating the nature of the spliced variants produced from certain target genes in response to steroid hormones. PMID:14982999

  15. The role of CD44 splice variants in human metastatic cancer

    NARCIS (Netherlands)

    Sleeman, J.; Moll, J.; Sherman, L.; Dall, P.; Pals, S. T.; Ponta, H.; Herrlich, P.

    1995-01-01

    The large family of CD44 splice variants are likely to serve multiple functions in the embryo and in the adult organism. This is reflected in their complex patterns of expression. In molecular terms these functions are largely unknown. Certain splice variants (CD44v) can promote the metastatic

  16. Effect of tension lap splice on the behavior of high strength concrete (HSC beams

    Directory of Open Access Journals (Sweden)

    Ahmed El-Azab

    2014-12-01

    Full Text Available In the recent years, many research efforts have been carried out on the bond strength between normal strength concrete (NSC and reinforcing bars spliced in tension zones in beams. Many codes gave a minimum splice length for tension and compression reinforcement as a factor of the bar diameter depending on many parameters such as concrete strength, steel yield stress, shape of bar end, shape of bar surface and also bar location. Also, codes gave another restriction about the percentage of total reinforcement to be spliced at the same time. Comparatively limited attention has been directed toward the bond between high strength concrete (HSC and reinforcing bars spliced in tension zones in beams. HSC has high modulus of elasticity, high density and long-term durability. This research presents an experimental study on the bond between high strength concrete (HSC and reinforcing bars spliced in tension zones in beams. It reports the influence of several parameters on bond in splices. The parameters covered are casting position, splice length as a factor of bar diameter, bar diameter and reinforcement ratio. The research involved tests on sixteen simply-supported beams of 1800 mm span, 200 mm width and 400 mm thickness made of HSC. In each beam, the total tensile steel bars were spliced in the constant moment zone. Crack pattern, crack propagation, cracking load, failure load and mi span deflection were recorded and analyzed to study the mentioned parameters effect.

  17. Global analysis of aberrant pre-mRNA splicing in glioblastoma using exon expression arrays

    Directory of Open Access Journals (Sweden)

    Nixon Tamara J

    2008-05-01

    Full Text Available Abstract Background Tumor-predominant splice isoforms were identified during comparative in silico sequence analysis of EST clones, suggesting that global aberrant alternative pre-mRNA splicing may be an epigenetic phenomenon in cancer. We used an exon expression array to perform an objective, genome-wide survey of glioma-specific splicing in 24 GBM and 12 nontumor brain samples. Validation studies were performed using RT-PCR on glioma cell lines, patient tumor and nontumor brain samples. Results In total, we confirmed 14 genes with glioma-specific splicing; seven were novel events identified by the exon expression array (A2BP1, BCAS1, CACNA1G, CLTA, KCNC2, SNCB, and TPD52L2. Our data indicate that large changes (> 5-fold in alternative splicing are infrequent in gliomagenesis ( Conclusion While we observed some tumor-specific alternative splicing, the number of genes showing exclusive tumor-specific isoforms was on the order of tens, rather than the hundreds suggested previously by in silico mining. Given the important role of alternative splicing in neural differentiation, there may be selective pressure to maintain a majority of splicing events in order to retain glial-like characteristics of the tumor cells.

  18. Suppression of an atypically spliced rice CACTA transposon transcript in transgenic plants

    NARCIS (Netherlands)

    Greco, R.; Ouwerkerk, P.B.F.; Pereira, A.B.

    2005-01-01

    OsES1, a rice homolog of the maize En/Spm transposon, is transcribed to produce TnpA-like and TnpD-like transcripts. However, an alternatively spliced form of the TnpA-like transcript., which was found to be suppressed in transgenic plants, was revealed to be clue to atypical splicing of a Hipa-like

  19. Measurement of Resistance and Strength of Conductor Splices in the MICE Coupling Magnets

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Feng Yu; Pan, Heng; Wu, Hong; Lui, X. K.; Li, E.; Dietderich, Dan; Higley, Hugh; Tam, D. G.; Trillaud, Fredric; Wang, Li; Green, M.A.

    2009-08-19

    The superconducting magnets for the Muon Ionization Cooling Experiment [1] (MICE) use a copper based Nb-Ti conductor with un-insulated dimensions of 0.95 by 1.60 mm. There may be as many as twelve splices in one MICE superconducting coupling coil. These splices are to be wound in the coil. The conductor splices produce Joule heating, which may cause the magnet to quench. A technique of making conductor splices was developed by ICST. Two types of 1-meter long of soldered lap-joints have been tested. Side-by-side splices and up-down one splices were studied theoretically and experimentally using two types of soft solder made of eutectic tin-lead solder and tin-silver solder. The resistances of the splices made by ICST were tested at LBNL at liquid helium temperatures over a range of magnetic fields up to 5 T. The breaking strength of 250 mm long splices was also measured at room temperature and liquid nitrogen temperature.

  20. The chromatin remodeling complex Swi/Snf regulates splicing of meiotic transcripts in Saccharomyces cerevisiae.

    Science.gov (United States)

    Venkataramanan, Srivats; Douglass, Stephen; Galivanche, Anoop R; Johnson, Tracy L

    2017-07-27

    Despite its relatively streamlined genome, there are important examples of regulated RNA splicing in Saccharomyces cerevisiae, such as splicing of meiotic transcripts. Like other eukaryotes, S. cerevisiae undergoes a dramatic reprogramming of gene expression during meiosis, including regulated splicing of a number of crucial meiosis-specific RNAs. Splicing of a subset of these is dependent upon the splicing activator Mer1. Here we show a crucial role for the chromatin remodeler Swi/Snf in regulation of splicing of meiotic genes and find that the complex affects meiotic splicing in two ways. First, we show that Swi/Snf regulates nutrient-dependent downregulation of ribosomal protein encoding RNAs, leading to the redistribution of spliceosomes from this abundant class of intron-containing RNAs (the ribosomal protein genes) to Mer1-regulated transcripts. We also demonstrate that Mer1 expression is dependent on Snf2, its acetylation state and histone H3 lysine 9 acetylation at the MER1 locus. Hence, Snf2 exerts systems level control of meiotic gene expression through two temporally distinct mechanisms, demonstrating that it is a key regulator of meiotic splicing in S. cerevisiae. We also reveal an evolutionarily conserved mechanism whereby the cell redirects its energy from maintaining its translational capacity to the process of meiosis. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.