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Sample records for alu rna editing

  1. Alu sequences in undifferentiated human embryonic stem cells display high levels of A-to-I RNA editing.

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    Sivan Osenberg

    Full Text Available Adenosine to Inosine (A-to-I RNA editing is a site-specific modification of RNA transcripts, catalyzed by members of the ADAR (Adenosine Deaminase Acting on RNA protein family. RNA editing occurs in human RNA in thousands of different sites. Some of the sites are located in protein-coding regions but the majority is found in non-coding regions, such as 3'UTRs, 5'UTRs and introns - mainly in Alu elements. While editing is found in all tissues, the highest levels of editing are found in the brain. It was shown that editing levels within protein-coding regions are increased during embryogenesis and after birth and that RNA editing is crucial for organism viability as well as for normal development. In this study we characterized the A-to-I RNA editing phenomenon during neuronal and spontaneous differentiation of human embryonic stem cells (hESCs. We identified high editing levels of Alu repetitive elements in hESCs and demonstrated a global decrease in editing levels of non-coding Alu sites when hESCs are differentiating, particularly into the neural lineage. Using RNA interference, we showed that the elevated editing levels of Alu elements in undifferentiated hESCs are highly dependent on ADAR1. DNA microarray analysis showed that ADAR1 knockdown has a global effect on gene expression in hESCs and leads to a significant increase in RNA expression levels of genes involved in differentiation and development processes, including neurogenesis. Taken together, we speculate that A-to-I editing of Alu sequences plays a role in the regulation of hESC early differentiation decisions.

  2. Alu Sequences in Undifferentiated Human Embryonic Stem Cells Display High Levels of A-to-I RNA Editing

    Science.gov (United States)

    Osenberg, Sivan; Paz Yaacov, Nurit; Safran, Michal; Moshkovitz, Sharon; Shtrichman, Ronit; Sherf, Ofra; Jacob-Hirsch, Jasmine; Keshet, Gilmor; Amariglio, Ninette; Itskovitz-Eldor, Joseph; Rechavi, Gideon

    2010-01-01

    Adenosine to Inosine (A-to-I) RNA editing is a site-specific modification of RNA transcripts, catalyzed by members of the ADAR (Adenosine Deaminase Acting on RNA) protein family. RNA editing occurs in human RNA in thousands of different sites. Some of the sites are located in protein-coding regions but the majority is found in non-coding regions, such as 3′UTRs, 5′UTRs and introns - mainly in Alu elements. While editing is found in all tissues, the highest levels of editing are found in the brain. It was shown that editing levels within protein-coding regions are increased during embryogenesis and after birth and that RNA editing is crucial for organism viability as well as for normal development. In this study we characterized the A-to-I RNA editing phenomenon during neuronal and spontaneous differentiation of human embryonic stem cells (hESCs). We identified high editing levels of Alu repetitive elements in hESCs and demonstrated a global decrease in editing levels of non-coding Alu sites when hESCs are differentiating, particularly into the neural lineage. Using RNA interference, we showed that the elevated editing levels of Alu elements in undifferentiated hESCs are highly dependent on ADAR1. DNA microarray analysis showed that ADAR1 knockdown has a global effect on gene expression in hESCs and leads to a significant increase in RNA expression levels of genes involved in differentiation and development processes, including neurogenesis. Taken together, we speculate that A-to-I editing of Alu sequences plays a role in the regulation of hESC early differentiation decisions. PMID:20574523

  3. Consistent levels of A-to-I RNA editing across individuals in coding sequences and non-conserved Alu repeats

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    Osenberg Sivan

    2010-10-01

    Full Text Available Abstract Background Adenosine to inosine (A-to-I RNA-editing is an essential post-transcriptional mechanism that occurs in numerous sites in the human transcriptome, mainly within Alu repeats. It has been shown to have consistent levels of editing across individuals in a few targets in the human brain and altered in several human pathologies. However, the variability across human individuals of editing levels in other tissues has not been studied so far. Results Here, we analyzed 32 skin samples, looking at A-to-I editing level in three genes within coding sequences and in the Alu repeats of six different genes. We observed highly consistent editing levels across different individuals as well as across tissues, not only in coding targets but, surprisingly, also in the non evolutionary conserved Alu repeats. Conclusions Our findings suggest that A-to-I RNA-editing of Alu elements is a tightly regulated process and, as such, might have been recruited in the course of primate evolution for post-transcriptional regulatory mechanisms.

  4. Large-scale analysis of structural, sequence and thermodynamic characteristics of A-to-I RNA editing sites in human Alu repeats

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    Eisenberg Eli

    2010-07-01

    Full Text Available Abstract Background Alu repeats in the human transcriptome undergo massive adenosine to inosine RNA editing. This process is selective, as editing efficiency varies greatly among different adenosines. Several studies have identified weak sequence motifs characterizing the editing sites, but these alone do not account for the large diversity observed. Results Here we build a dataset of 29,971 editing sites and use it to characterize editing preferences. We focus on structural aspects, studying the double-stranded RNA structure of the Alu repeats, and show the editing frequency of a given site to depend strongly on the micro-structure it resides in. Surprisingly, we find that interior loops, and especially the nucleotides at their edges, are more likely to be edited than helices. In addition, the sequence motifs characterizing editing sites vary with the micro-structure. Finally, we show that thermodynamic stability of the site is important for its editing. Conclusions Analysis of a large dataset of editing events reveals more information on sequence and structural motifs characterizing the A-to-I editing process

  5. Selective stimulation of translational expression by Alu RNA

    OpenAIRE

    Rubin, Carol M.; Kimura, Richard H.; Schmid, Carl W.

    2002-01-01

    Human Alu and adenovirus VA1 RNAs each stimulate the translational expression of reporter genes in co-transient transfection assays without affecting either the rate of global protein synthesis or the abundance of the reporter mRNA. This selective, post-transcriptional stimulation of expression, which is observed in human and mouse cell lines and for three reporters, acts through a PKR- independent mechanism. The activity of Alu and VA1 RNAs in this assay is transient, causing a reduction in ...

  6. REDIdb: the RNA editing database.

    Science.gov (United States)

    Picardi, Ernesto; Regina, Teresa Maria Rosaria; Brennicke, Axel; Quagliariello, Carla

    2007-01-01

    The RNA Editing Database (REDIdb) is an interactive, web-based database created and designed with the aim to allocate RNA editing events such as substitutions, insertions and deletions occurring in a wide range of organisms. The database contains both fully and partially sequenced DNA molecules for which editing information is available either by experimental inspection (in vitro) or by computational detection (in silico). Each record of REDIdb is organized in a specific flat-file containing a description of the main characteristics of the entry, a feature table with the editing events and related details and a sequence zone with both the genomic sequence and the corresponding edited transcript. REDIdb is a relational database in which the browsing and identification of editing sites has been simplified by means of two facilities to either graphically display genomic or cDNA sequences or to show the corresponding alignment. In both cases, all editing sites are highlighted in colour and their relative positions are detailed by mousing over. New editing positions can be directly submitted to REDIdb after a user-specific registration to obtain authorized secure access. This first version of REDIdb database stores 9964 editing events and can be freely queried at http://biologia.unical.it/py_script/search.html.

  7. Alu-miRNA interactions modulate transcript isoform diversity in stress response and reveal signatures of positive selection.

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    Pandey, Rajesh; Bhattacharya, Aniket; Bhardwaj, Vivek; Jha, Vineet; Mandal, Amit K; Mukerji, Mitali

    2016-01-01

    Primate-specific Alus harbor different regulatory features, including miRNA targets. In this study, we provide evidence for miRNA-mediated modulation of transcript isoform levels during heat-shock response through exaptation of Alu-miRNA sites in mature mRNA. We performed genome-wide expression profiling coupled with functional validation of miRNA target sites within exonized Alus, and analyzed conservation of these targets across primates. We observed that two miRNAs (miR-15a-3p and miR-302d-3p) elevated in stress response, target RAD1, GTSE1, NR2C1, FKBP9 and UBE2I exclusively within Alu. These genes map onto the p53 regulatory network. Ectopic overexpression of miR-15a-3p downregulates GTSE1 and RAD1 at the protein level and enhances cell survival. This Alu-mediated fine-tuning seems to be unique to humans as evident from the absence of orthologous sites in other primate lineages. We further analyzed signatures of selection on Alu-miRNA targets in the genome, using 1000 Genomes Phase-I data. We found that 198 out of 3177 Alu-exonized genes exhibit signatures of selection within Alu-miRNA sites, with 60 of them containing SNPs supported by multiple evidences (global-FST > 0.3, pair-wise-FST > 0.5, Fay-Wu's H  2.0, high ΔDAF) and implicated in p53 network. We propose that by affecting multiple genes, Alu-miRNA interactions have the potential to facilitate population-level adaptations in response to environmental challenges. PMID:27586304

  8. Investigating RNA editing factors from trypanosome mitochondria

    Science.gov (United States)

    Aphasizheva, Inna; Zhang, Liye; Aphasizhev, Ruslan

    2016-01-01

    Mitochondrial U-insertion/deletion mRNA editing is carried out by two principal multiprotein assemblies, enzymatic RNA editing core (RECC) and RNA editing substrate binding (RESC) complexes, and a plethora of auxiliary factors. An integral part of mitochondrial gene expression, editing receives inputs from primary mRNA and gRNA precursor processing pathways, and generates substrates for mRNA polyadenylation and translation. Although nearly all RECC-embedded enzymes have been implicated in specific editing reactions, the majority of proteins that populate the RESC are also essential for generating edited mRNAs. However, lack of recognizable motifs in RESC subunits limits the prowess of bioinformatics in guiding biochemical experiments and elucidating their specific biological functions. In this chapter, we describe a generic workflow for investigating mitochondrial mRNA editing in Trypanosoma brucei and focus on several methods that proved instrumental is assigning definitive functions to editing factors lacking known signature sequences. PMID:27020893

  9. A structural determinant required for RNA editing

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    Tian, Nan; Yang, Yun; Sachsenmaier, Nora; Muggenhumer, Dominik; Bi, Jingpei; Waldsich, Christina; Jantsch, Michael F.; Jin, Yongfeng

    2011-01-01

    RNA editing by adenosine deaminases acting on RNAs (ADARs) can be both specific and non-specific, depending on the substrate. Specific editing of particular adenosines may depend on the overall sequence and structural context. However, the detailed mechanisms underlying these preferences are not fully understood. Here, we show that duplex structures mimicking an editing site in the Gabra3 pre-mRNA unexpectedly fail to support RNA editing at the Gabra3 I/M site, although phylogenetic analysis suggest an evolutionarily conserved duplex structure essential for efficient RNA editing. These unusual results led us to revisit the structural requirement for this editing by mutagenesis analysis. In vivo nuclear injection experiments of mutated editing substrates demonstrate that a non-conserved structure is a determinant for editing. This structure contains bulges either on the same or the strand opposing the edited adenosine. The position of these bulges and the distance to the edited base regulate editing. Moreover, elevated folding temperature can lead to a switch in RNA editing suggesting an RNA structural change. Our results indicate the importance of RNA tertiary structure in determining RNA editing. PMID:21427087

  10. Altered A-to-I RNA Editing in Human Embryogenesis

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    Mandel, Rachel; Ziskind, Anna; Nahor, Irit; Safran, Michal; Osenberg, Sivan; Sherf, Ofra; Rechavi, Gideon; Itskovitz-Eldor, Joseph

    2012-01-01

    Post-transcriptional events play an important role in human development. The question arises as to whether Adenosine to Inosine RNA editing, catalyzed by the ADAR (Adenosine Deaminase acting on RNA) enzymes, differs in human embryogenesis and in adulthood. We tested the editing of various target genes in coding (FLNA, BLCAP, CYFIP2) and non-coding sequences at their Alu elements (BRCA1, CARD11, RBBP9, MDM4, FNACC), as well as the transcriptional levels of the ADAR1 enzymes. This analysis was performed on five fetal and adult human tissues: brain, heart, liver, kidney, and spleen, as well as on human embryonic stem cells (hESCs), which represent the blastocyst stage in early human development. Our results show substantially greater editing activity for most adult tissue samples relative to fetal ones, in six of the eight genes tested. To test the effect of reduced A-to-I RNA editing activity in early human development we used human embryonic stem cells (hESCs) as a model and tried to generate hESC clones that overexpress the ADAR1–p110 isoform. We were unable to achieve overexpression of ADAR1–p110 by either transfection or lentiviral infection, though we easily generated hESC clones that expressed the GFP transgene and overexpressed ADAR1-p110 in 293T cells and in primary human foreskin fibroblast (HFF) cells. Moreover, in contrast to the expected overexpression of ADAR1-p110 protein following its introduction into hESCs, the expression levels of this protein decreased dramatically 24–48 hr post infection. Similar results were obtained when we tried to overexpress ADAR1-p110 in pluripotent embryonal carcinoma cells. This suggests that ADAR1 protein is substantially regulated in undifferentiated pluripotent hESCs. Overall, our data suggest that A-to-I RNA editing plays a critical role during early human development. PMID:22859999

  11. Altered A-to-I RNA editing in human embryogenesis.

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    Ronit Shtrichman

    Full Text Available Post-transcriptional events play an important role in human development. The question arises as to whether Adenosine to Inosine RNA editing, catalyzed by the ADAR (Adenosine Deaminase acting on RNA enzymes, differs in human embryogenesis and in adulthood. We tested the editing of various target genes in coding (FLNA, BLCAP, CYFIP2 and non-coding sequences at their Alu elements (BRCA1, CARD11, RBBP9, MDM4, FNACC, as well as the transcriptional levels of the ADAR1 enzymes. This analysis was performed on five fetal and adult human tissues: brain, heart, liver, kidney, and spleen, as well as on human embryonic stem cells (hESCs, which represent the blastocyst stage in early human development. Our results show substantially greater editing activity for most adult tissue samples relative to fetal ones, in six of the eight genes tested. To test the effect of reduced A-to-I RNA editing activity in early human development we used human embryonic stem cells (hESCs as a model and tried to generate hESC clones that overexpress the ADAR1-p110 isoform. We were unable to achieve overexpression of ADAR1-p110 by either transfection or lentiviral infection, though we easily generated hESC clones that expressed the GFP transgene and overexpressed ADAR1-p110 in 293T cells and in primary human foreskin fibroblast (HFF cells. Moreover, in contrast to the expected overexpression of ADAR1-p110 protein following its introduction into hESCs, the expression levels of this protein decreased dramatically 24-48 hr post infection. Similar results were obtained when we tried to overexpress ADAR1-p110 in pluripotent embryonal carcinoma cells. This suggests that ADAR1 protein is substantially regulated in undifferentiated pluripotent hESCs. Overall, our data suggest that A-to-I RNA editing plays a critical role during early human development.

  12. Iron Toxicity in the Retina Requires Alu RNA and the NLRP3 Inflammasome

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    Bradley D. Gelfand

    2015-06-01

    Full Text Available Excess iron induces tissue damage and is implicated in age-related macular degeneration (AMD. Iron toxicity is widely attributed to hydroxyl radical formation through Fenton’s reaction. We report that excess iron, but not other Fenton catalytic metals, induces activation of the NLRP3 inflammasome, a pathway also implicated in AMD. Additionally, iron-induced degeneration of the retinal pigmented epithelium (RPE is suppressed in mice lacking inflammasome components caspase-1/11 or Nlrp3 or by inhibition of caspase-1. Iron overload increases abundance of RNAs transcribed from short interspersed nuclear elements (SINEs: Alu RNAs and the rodent equivalent B1 and B2 RNAs, which are inflammasome agonists. Targeting Alu or B2 RNA prevents iron-induced inflammasome activation and RPE degeneration. Iron-induced SINE RNA accumulation is due to suppression of DICER1 via sequestration of the co-factor poly(C-binding protein 2 (PCBP2. These findings reveal an unexpected mechanism of iron toxicity, with implications for AMD and neurodegenerative diseases associated with excess iron.

  13. RNA Editing Dynamically Rewrites the Cancer Code

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    Rayon-Estrada, Violeta; Papavasiliou, F. Nina; Harjanto, Dewi

    2016-01-01

    Global analyses of cancer transcriptomes demonstrate that ADAR (adenosine deaminase, RNA-specific)-mediated RNA editing dynamically contributes to genetic alterations in cancer, and directly correlates with progression and prognosis. RNA editing is abundant and frequently elevated in cancer, and affects functionally and clinically relevant sites in both coding and non-coding regions of the transcriptome. Therefore, ADAR and differentially edited transcripts may be promising biomarkers or targets for therapy.

  14. Exploring the RNA editing potential of RNA-Seq data by ExpEdit.

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    D'Antonio, Mattia; Picardi, Ernesto; Castrignanò, Tiziana; D'Erchia, Anna Maria; Pesole, Graziano

    2015-01-01

    Revealing the impact of A-to-I RNA editing in RNA-Seq experiments is relevant in humans because RNA editing can influence gene expression. In addition, its deregulation has been linked to a variety of human diseases. Exploiting the RNA editing potential in complete RNA-Seq datasets, however, is a challenging task. Indeed, no dedicated software is available, and sometimes deep computational skills and appropriate hardware resources are required. To explore the impact of known RNA editing events in massive transcriptome sequencing experiments, we developed the ExpEdit web service application. In the present work, we provide an overview of ExpEdit as well as methodologies to investigate known RNA editing in human RNA-Seq datasets.

  15. Statistical Physics Approaches to RNA Editing

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    Bundschuh, Ralf

    2012-02-01

    The central dogma of molecular Biology states that DNA is transcribed base by base into RNA which is in turn translated into proteins. However, some organisms edit their RNA before translation by inserting, deleting, or substituting individual or short stretches of bases. In many instances the mechanisms by which an organism recognizes the positions at which to edit or by which it performs the actual editing are unknown. One model system that stands out by its very high rate of on average one out of 25 bases being edited are the Myxomycetes, a class of slime molds. In this talk we will show how the computational methods and concepts from statistical Physics can be used to analyze DNA and protein sequence data to predict editing sites in these slime molds and to guide experiments that identified previously unknown types of editing as well as the complete set of editing events in the slime mold Physarum polycephalum.

  16. Some aspects of RNA repair and editing

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    Kovalchuk M. V.

    2010-11-01

    Full Text Available All cellular RNA molecules are damaged at the scale of DNA molecules, or even more. In the present review the RNA damaging agents, some mechanisms of RNA repair and editing, their difference from DNA repair mechanisms have been discussed.

  17. RNA Editing and Drug Discovery for Cancer Therapy

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    Wei-Hsuan Huang

    2013-01-01

    Full Text Available RNA editing is vital to provide the RNA and protein complexity to regulate the gene expression. Correct RNA editing maintains the cell function and organism development. Imbalance of the RNA editing machinery may lead to diseases and cancers. Recently, RNA editing has been recognized as a target for drug discovery although few studies targeting RNA editing for disease and cancer therapy were reported in the field of natural products. Therefore, RNA editing may be a potential target for therapeutic natural products. In this review, we provide a literature overview of the biological functions of RNA editing on gene expression, diseases, cancers, and drugs. The bioinformatics resources of RNA editing were also summarized.

  18. Genetic Architectures of Quantitative Variation in RNA Editing Pathways.

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    Gu, Tongjun; Gatti, Daniel M; Srivastava, Anuj; Snyder, Elizabeth M; Raghupathy, Narayanan; Simecek, Petr; Svenson, Karen L; Dotu, Ivan; Chuang, Jeffrey H; Keller, Mark P; Attie, Alan D; Braun, Robert E; Churchill, Gary A

    2016-02-01

    RNA editing refers to post-transcriptional processes that alter the base sequence of RNA. Recently, hundreds of new RNA editing targets have been reported. However, the mechanisms that determine the specificity and degree of editing are not well understood. We examined quantitative variation of site-specific editing in a genetically diverse multiparent population, Diversity Outbred mice, and mapped polymorphic loci that alter editing ratios globally for C-to-U editing and at specific sites for A-to-I editing. An allelic series in the C-to-U editing enzyme Apobec1 influences the editing efficiency of Apob and 58 additional C-to-U editing targets. We identified 49 A-to-I editing sites with polymorphisms in the edited transcript that alter editing efficiency. In contrast to the shared genetic control of C-to-U editing, most of the variable A-to-I editing sites were determined by local nucleotide polymorphisms in proximity to the editing site in the RNA secondary structure. Our results indicate that RNA editing is a quantitative trait subject to genetic variation and that evolutionary constraints have given rise to distinct genetic architectures in the two canonical types of RNA editing.

  19. HEXIM1蛋白与Alu SINE RNA相互作用的研究%Study of the interaction between HEXIM1 protein and Alu SINE RNA

    Institute of Scientific and Technical Information of China (English)

    田平平; 吴传芳; 欧阳劲; 秦岭

    2014-01-01

    为了探讨Alu SINE RNA和HEXIM1蛋白之间是否存在相互作用.本实验构建了带有FLAG标签的HEXIM1真核表达载体,转染人胚肾293(HEK293)细胞,做anti FLAG 的RNA免疫共沉淀(RIP)后运用免疫印记和逆转录PCR (reverse transcription PCR,RT PCR)等方法检测.证明了Alu SINE RNA和HEXIM1蛋白之间存在相互作用,表明AluSINE RNA和HEXIM1蛋白共同影响基因转录调控,以及细胞在应对外界刺激时能及时在基因水平做出有效反应的可能.

  20. Gene regulation by mRNA editing

    Energy Technology Data Exchange (ETDEWEB)

    Ashkenas, J. [Univ. of Washington, Seattle, WA (United States)

    1997-02-01

    The commonly cited figure of 10{sup 5} genes in the human genome represents a tremendous underestimate of our capacity to generate distinct gene products with unique functions. Our cells possess an impressive collection of tools for altering the products of a single gene to create a variety of proteins. The different gene products may have related but distinct functions, allowing cells of different types or at different developmental stages to fine-tune their patterns of gene expression. These tools may act in the cytoplasm, as when proteins undergo post-translational modifications, or in the nucleus, in the processing of pre-mRNA. Two forms of intranuclear fine-tuning are well established and widely studied: alternative splicing of pre-mRNAs and alternative polyadenylation site selection. In recent years it has become clear that cells possess yet another tool to create RNA sequence diversity, mRNA editing. The term {open_quotes}editing{close_quotes} is applied to posttranscriptional modifications of a purine or pyrimidine, which alter an mRNA sequence as it is read, for example, by ribosomes. Covalent changes to the structure of nucleotide bases are well known to occur on tRNA and rRNA molecules, but such changes in mRNA sequence are novel in that they have the capacity to change specific protein sequences. 43 refs., 1 fig.

  1. Pentatricopeptide repeat proteins involved in plant organellar RNA editing

    OpenAIRE

    Yagi, Yusuke; Tachikawa, Makoto; Noguchi, Hisayo; Satoh, Soichirou; Obokata, Junichi; Nakamura, Takahiro

    2013-01-01

    C-to-U RNA editing has been widely observed in organellar RNAs in terrestrial plants. Recent research has revealed the significance of a large, plant-specific family of pentatricopeptide repeat (PPR) proteins for RNA editing and other RNA processing events in plant mitochondria and chloroplasts. PPR protein is a sequence-specific RNA-binding protein that identifies specific C residues for editing. Discovery of the RNA recognition code for PPR motifs, including verification and prediction of t...

  2. REDIdb: an upgraded bioinformatics resource for organellar RNA editing sites.

    Science.gov (United States)

    Picardi, Ernesto; Regina, Teresa M R; Verbitskiy, Daniil; Brennicke, Axel; Quagliariello, Carla

    2011-03-01

    RNA editing is a post-transcriptional molecular process whereby the information in a genetic message is modified from that in the corresponding DNA template by means of nucleotide substitutions, insertions and/or deletions. It occurs mostly in organelles by clade-specific diverse and unrelated biochemical mechanisms. RNA editing events have been annotated in primary databases as GenBank and at more sophisticated level in the specialized databases REDIdb, dbRES and EdRNA. At present, REDIdb is the only freely available database that focuses on the organellar RNA editing process and annotates each editing modification in its biological context. Here we present an updated and upgraded release of REDIdb with a web-interface refurbished with graphical and computational facilities that improve RNA editing investigations. Details of the REDIdb features and novelties are illustrated and compared to other RNA editing databases. REDIdb is freely queried at http://biologia.unical.it/py_script/REDIdb/.

  3. Alu element-containing RNAs maintain nucleolar structure and function.

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    Caudron-Herger, Maïwen; Pankert, Teresa; Seiler, Jeanette; Németh, Attila; Voit, Renate; Grummt, Ingrid; Rippe, Karsten

    2015-11-12

    Non-coding RNAs play a key role in organizing the nucleus into functional subcompartments. By combining fluorescence microscopy and RNA deep-sequencing-based analysis, we found that RNA polymerase II transcripts originating from intronic Alu elements (aluRNAs) were enriched in the nucleolus. Antisense-oligo-mediated depletion of aluRNAs or drug-induced inhibition of RNA polymerase II activity disrupted nucleolar structure and impaired RNA polymerase I-dependent transcription of rRNA genes. In contrast, overexpression of a prototypic aluRNA sequence increased both nucleolus size and levels of pre-rRNA, suggesting a functional link between aluRNA, nucleolus integrity and pre-rRNA synthesis. Furthermore, we show that aluRNAs interact with nucleolin and target ectopic genomic loci to the nucleolus. Our study suggests an aluRNA-based mechanism that links RNA polymerase I and II activities and modulates nucleolar structure and rRNA production.

  4. An RNA editing fingerprint of cancer stem cell reprogramming

    OpenAIRE

    Crews, Leslie A; Jiang, Qingfei; Zipeto, Maria A; de Lazzari, Elisa; Court, Angela C.; Ali, Shawn; Barrett, Christian L.; Frazer, Kelly A; Jamieson, Catriona HM

    2015-01-01

    Background Deregulation of RNA editing by adenosine deaminases acting on dsRNA (ADARs) has been implicated in the progression of diverse human cancers including hematopoietic malignancies such as chronic myeloid leukemia (CML). Inflammation-associated activation of ADAR1 occurs in leukemia stem cells specifically in the advanced, often drug-resistant stage of CML known as blast crisis. However, detection of cancer stem cell-associated RNA editing by RNA sequencing in these rare cell populatio...

  5. Alu elements in ANRIL non-coding RNA at chromosome 9p21 modulate atherogenic cell functions through trans-regulation of gene networks.

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    Lesca M Holdt

    Full Text Available The chromosome 9p21 (Chr9p21 locus of coronary artery disease has been identified in the first surge of genome-wide association and is the strongest genetic factor of atherosclerosis known today. Chr9p21 encodes the long non-coding RNA (ncRNA antisense non-coding RNA in the INK4 locus (ANRIL. ANRIL expression is associated with the Chr9p21 genotype and correlated with atherosclerosis severity. Here, we report on the molecular mechanisms through which ANRIL regulates target-genes in trans, leading to increased cell proliferation, increased cell adhesion and decreased apoptosis, which are all essential mechanisms of atherogenesis. Importantly, trans-regulation was dependent on Alu motifs, which marked the promoters of ANRIL target genes and were mirrored in ANRIL RNA transcripts. ANRIL bound Polycomb group proteins that were highly enriched in the proximity of Alu motifs across the genome and were recruited to promoters of target genes upon ANRIL over-expression. The functional relevance of Alu motifs in ANRIL was confirmed by deletion and mutagenesis, reversing trans-regulation and atherogenic cell functions. ANRIL-regulated networks were confirmed in 2280 individuals with and without coronary artery disease and functionally validated in primary cells from patients carrying the Chr9p21 risk allele. Our study provides a molecular mechanism for pro-atherogenic effects of ANRIL at Chr9p21 and suggests a novel role for Alu elements in epigenetic gene regulation by long ncRNAs.

  6. Deletions in cox2 mRNA result in loss of splicing and RNA editing and gain of novel RNA editing sites.

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    Stefanie Grüttner

    Full Text Available As previously demonstrated, the maize cox2 RNA is fully edited in cauliflower mitochondria. Use of constructs with a deleted cox2 intron, however, led to a loss of RNA editing at almost all editing sites, with only a few sites still partially edited. Likewise, one deletion in exon 1 and three in exon 2 abolish RNA editing at all cox2 sites analyzed. Furthermore, intron splicing is abolished using these deletions. Mutation of a cytosine residue, which is normally edited and localized directly adjacent to the intron, to thymidine did not result in restoration of splicing, indicating that the loss of splicing was not due to loss of RNA editing. One deletion in exon 2 did not lead to loss of splicing. Instead, most editing sites were found to be edited, only three were not edited. Unexpectedly, we observed additional RNA editing events at new sites. Thus it appears that deletions in the cox2 RNA sequence can have a strong effect on RNA processing, leading to loss of splicing, loss of editing at all sites, or even to a gain of new editing sites. As these effects are not limited to the vicinity of the respective deletions, but appear to be widespread or even affect all editing sites, they may not be explained by the loss of PPR binding sites. Instead, it appears that several parts of the cox2 transcript are required for proper RNA processing. This indicates the roles of the RNA sequence and structural elements in the recognition of the editing sites.

  7. A minimum principle in mRNA editing of Physarum ?

    CERN Document Server

    Frappat, L; Sorba, Paul

    2000-01-01

    mRNA editing in three sequences of Physarum polycephalum is analyzed. Once fixed the edited peptide chain, the nature of the inserted nucleotides and the position of the insertion sites are explained by introducing a minimum principle in the framework of the crystal basis model of the genetic code introduced by the authors.

  8. A model for codon position bias in RNA editing

    CERN Document Server

    Liu, T; Liu, Tsunglin; Bundschuh, Ralf

    2005-01-01

    RNA editing can be crucial for the expression of genetic information via inserting, deleting, or substituting a few nucleotides at specific positions in an RNA sequence. Within coding regions in an RNA sequence, editing usually occurs with a certain bias in choosing the positions of the editing sites. In the mitochondrial genes of {\\it Physarum polycephalum}, many more editing events have been observed at the third codon position than at the first and second, while in some plant mitochondria the second codon position dominates. Here we propose an evolutionary model that explains this bias as the basis of selection at the protein level. The model predicts a distribution of the three positions rather close to the experimental observation in {\\it Physarum}. This suggests that the codon position bias in {\\it Physarum} is mainly a consequence of selection at the protein level.

  9. Comprehensive analysis of RNA-Seq data reveals extensive RNA editing in a human transcriptome

    DEFF Research Database (Denmark)

    Peng, Zhiyu; Cheng, Yanbing; Tan, Bertrand Chin-Ming;

    2012-01-01

    a computational pipeline that carefully controls for false positives while calling RNA editing events from genome and whole-transcriptome data of the same individual. We identified 22,688 RNA editing events in noncoding genes and introns, untranslated regions and coding sequences of protein-coding genes. Most...... changes (∼93%) converted A to I(G), consistent with known editing mechanisms based on adenosine deaminase acting on RNA (ADAR). We also found evidence of other types of nucleotide changes; however, these were validated at lower rates. We found 44 editing sites in microRNAs (miRNAs), suggesting a potential...

  10. RNA Editing in Chloroplasts of Spirodela polyrhiza, an Aquatic Monocotelydonous Species.

    Science.gov (United States)

    Wang, Wenqin; Zhang, Wei; Wu, Yongrui; Maliga, Pal; Messing, Joachim

    2015-01-01

    RNA editing is the post-transcriptional conversion from C to U before translation, providing a unique feature in the regulation of gene expression. Here, we used a robust and efficient method based on RNA-seq from non-ribosomal total RNA to simultaneously measure chloroplast-gene expression and RNA editing efficiency in the Greater Duckweed, Spirodela polyrhiza, a species that provides a new reference for the phylogenetic studies of monocotyledonous plants. We identified 66 editing sites at the genome-wide level, with an average editing efficiency of 76%. We found that the expression levels of chloroplast genes were relatively constant, but 11 RNA editing sites show significant changes in editing efficiency, when fronds turn into turions. Thus, RNA editing efficiency contributes more to the yield of translatable transcripts than steady state mRNA levels. Comparison of RNA editing sites in coconut, Spirodela, maize, and rice suggests that RNA editing originated from a common ancestor. PMID:26517707

  11. Using REDItools to Detect RNA Editing Events in NGS Datasets.

    Science.gov (United States)

    Picardi, Ernesto; D'Erchia, Anna Maria; Montalvo, Antonio; Pesole, Graziano

    2015-03-09

    RNA editing is a post-transcriptional/co-transcriptional molecular phenomenon whereby a genetic message is modified from the corresponding DNA template by means of substitutions, insertions, and/or deletions. It occurs in a variety of organisms and different cellular locations through evolutionally and biochemically unrelated proteins. RNA editing has a plethora of biological effects including the modulation of alternative splicing and fine-tuning of gene expression. RNA editing events by base substitutions can be detected on a genomic scale by NGS technologies through the REDItools package, an ad hoc suite of Python scripts to study RNA editing using RNA-Seq and DNA-Seq data or RNA-Seq data alone. REDItools implement effective filters to minimize biases due to sequencing errors, mapping errors, and SNPs. The package is freely available at Google Code repository (http://code.google.com/p/reditools/) and released under the MIT license. In the present unit we show three basic protocols corresponding to three main REDItools scripts.

  12. The art of editing RNA structural alignments

    DEFF Research Database (Denmark)

    Andersen, Ebbe Sloth

    2014-01-01

    , it is rewarded by great insight into the evolution of structure and function of your favorite RNA molecule. In this chapter I will review the methods and considerations that go into constructing RNA structural alignments at the secondary and tertiary structure level; introduce software, databases, and algorithms...

  13. Organelle RNA recognition motif-containing (ORRM) proteins are plastid and mitochondrial editing factors in Arabidopsis.

    Science.gov (United States)

    Shi, Xiaowen; Bentolila, Stephane; Hanson, Maureen R

    2016-05-01

    Post-transcriptional C-to-U RNA editing occurs at specific sites in plastid and plant mitochondrial transcripts. Members of the Arabidopsis pentatricopeptide repeat (PPR) motif-containing protein family and RNA-editing factor Interacting Protein (RIP, also known as MORF) family have been characterized as essential components of the RNA editing apparatus. Recent studies reveal that several organelle-targeted RNA recognition motif (RRM)-containing proteins are involved in either plastid or mitochondrial RNA editing. ORRM1 (Organelle RRM protein 1) is essential for plastid editing, whereas ORRM2, ORRM3 and ORRM4 are involved in mitochondrial RNA editing. The RRM domain of ORRM1, ORRM3 and ORRM4 is required for editing activity, whereas the auxiliary RIP and Glycine-Rich (GR) domains mediate the ORRM proteins' interactions with other editing factors. The identification of the ORRM proteins as RNA editing factors further expands our knowledge of the composition of the editosome. PMID:27082488

  14. Rapid and dynamic transcriptome regulation by RNA editing and RNA modifications.

    Science.gov (United States)

    Licht, Konstantin; Jantsch, Michael F

    2016-04-11

    Advances in next-generation sequencing and mass spectrometry have revealed widespread messenger RNA modifications and RNA editing, with dramatic effects on mammalian transcriptomes. Factors introducing, deleting, or interpreting specific modifications have been identified, and analogous with epigenetic terminology, have been designated "writers," "erasers," and "readers." Such modifications in the transcriptome are referred to as epitranscriptomic changes and represent a fascinating new layer of gene expression regulation that has only recently been appreciated. Here, we outline how RNA editing and RNA modification can rapidly affect gene expression, making both processes as well suited to respond to cellular stress and to regulate the transcriptome during development or circadian periods. PMID:27044895

  15. Comprehensive high-resolution analysis of the role of an Arabidopsis gene family in RNA editing.

    Science.gov (United States)

    Bentolila, Stéphane; Oh, Julyun; Hanson, Maureen R; Bukowski, Robert

    2013-06-01

    In flowering plants, mitochondrial and chloroplast mRNAs are edited by C-to-U base modification. In plant organelles, RNA editing appears to be generally a correcting mechanism that restores the proper function of the encoded product. Members of the Arabidopsis RNA editing-Interacting Protein (RIP) family have been recently shown to be essential components of the plant editing machinery. We report the use of a strand- and transcript-specific RNA-seq method (STS-PCRseq) to explore the effect of mutation or silencing of every RIP gene on plant organelle editing. We confirm RIP1 to be a major editing factor that controls the editing extent of 75% of the mitochondrial sites and 20% of the plastid C targets of editing. The quantitative nature of RNA sequencing allows the precise determination of overlapping effects of RIP factors on RNA editing. Over 85% of the sites under the influence of RIP3 and RIP8, two moderately important mitochondrial factors, are also controlled by RIP1. Previously uncharacterized RIP family members were found to have only a slight effect on RNA editing. The preferential location of editing sites controlled by RIP7 on some transcripts suggests an RNA metabolism function for this factor other than editing. In addition to a complete characterization of the RIP factors for their effect on RNA editing, our study highlights the potential of RNA-seq for studying plant organelle editing. Unlike previous attempts to use RNA-seq to analyze RNA editing extent, our methodology focuses on sequencing of organelle cDNAs corresponding to known transcripts. As a result, the depth of coverage of each editing site reaches unprecedented values, assuring a reliable measurement of editing extent and the detection of numerous new sites. This strategy can be applied to the study of RNA editing in any organism.

  16. Comprehensive high-resolution analysis of the role of an Arabidopsis gene family in RNA editing.

    Directory of Open Access Journals (Sweden)

    Stéphane Bentolila

    2013-06-01

    Full Text Available In flowering plants, mitochondrial and chloroplast mRNAs are edited by C-to-U base modification. In plant organelles, RNA editing appears to be generally a correcting mechanism that restores the proper function of the encoded product. Members of the Arabidopsis RNA editing-Interacting Protein (RIP family have been recently shown to be essential components of the plant editing machinery. We report the use of a strand- and transcript-specific RNA-seq method (STS-PCRseq to explore the effect of mutation or silencing of every RIP gene on plant organelle editing. We confirm RIP1 to be a major editing factor that controls the editing extent of 75% of the mitochondrial sites and 20% of the plastid C targets of editing. The quantitative nature of RNA sequencing allows the precise determination of overlapping effects of RIP factors on RNA editing. Over 85% of the sites under the influence of RIP3 and RIP8, two moderately important mitochondrial factors, are also controlled by RIP1. Previously uncharacterized RIP family members were found to have only a slight effect on RNA editing. The preferential location of editing sites controlled by RIP7 on some transcripts suggests an RNA metabolism function for this factor other than editing. In addition to a complete characterization of the RIP factors for their effect on RNA editing, our study highlights the potential of RNA-seq for studying plant organelle editing. Unlike previous attempts to use RNA-seq to analyze RNA editing extent, our methodology focuses on sequencing of organelle cDNAs corresponding to known transcripts. As a result, the depth of coverage of each editing site reaches unprecedented values, assuring a reliable measurement of editing extent and the detection of numerous new sites. This strategy can be applied to the study of RNA editing in any organism.

  17. Genes (including RNA editing information) - RMG | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available [ Credits ] BLAST Search Image Search Home About Archive Update History Contact us RMG Genes... (including RNA editing information) Data detail Data name Genes (including RNA editing information...ase Site Policy | Contact Us Genes (including RNA editing information) - RMG | LSDB Archive ...

  18. Variable frequency of plastid RNA editing among ferns and repeated loss of uridine-to-cytidine editing from vascular plants.

    Science.gov (United States)

    Guo, Wenhu; Grewe, Felix; Mower, Jeffrey P

    2015-01-01

    The distinct distribution and abundance of C-to-U and U-to-C RNA editing among land plants suggest that these two processes originated and evolve independently, but the paucity of information from several key lineages limits our understanding of their evolution. To examine the evolutionary diversity of RNA editing among ferns, we sequenced the plastid transcriptomes from two early diverging species, Ophioglossum californicum and Psilotum nudum. Using a relaxed automated approach to minimize false negatives combined with manual inspection to eliminate false positives, we identified 297 C-to-U and three U-to-C edit sites in the O. californicum plastid transcriptome but only 27 C-to-U and no U-to-C edit sites in the P. nudum plastid transcriptome. A broader comparison of editing content with the leptosporangiate fern Adiantum capillus-veneris and the hornwort Anthoceros formosae uncovered large variance in the abundance of plastid editing, indicating that the frequency and type of RNA editing is highly labile in ferns. Edit sites that increase protein conservation among species are more abundant and more efficiently edited than silent and non-conservative sites, suggesting that selection maintains functionally important editing. The absence of U-to-C editing from P. nudum plastid transcripts and other vascular plants demonstrates that U-to-C editing loss is a recurrent phenomenon in vascular plant evolution.

  19. Alu element-containing RNAs maintain nucleolar structure and function.

    Science.gov (United States)

    Caudron-Herger, Maïwen; Pankert, Teresa; Seiler, Jeanette; Németh, Attila; Voit, Renate; Grummt, Ingrid; Rippe, Karsten

    2015-11-12

    Non-coding RNAs play a key role in organizing the nucleus into functional subcompartments. By combining fluorescence microscopy and RNA deep-sequencing-based analysis, we found that RNA polymerase II transcripts originating from intronic Alu elements (aluRNAs) were enriched in the nucleolus. Antisense-oligo-mediated depletion of aluRNAs or drug-induced inhibition of RNA polymerase II activity disrupted nucleolar structure and impaired RNA polymerase I-dependent transcription of rRNA genes. In contrast, overexpression of a prototypic aluRNA sequence increased both nucleolus size and levels of pre-rRNA, suggesting a functional link between aluRNA, nucleolus integrity and pre-rRNA synthesis. Furthermore, we show that aluRNAs interact with nucleolin and target ectopic genomic loci to the nucleolus. Our study suggests an aluRNA-based mechanism that links RNA polymerase I and II activities and modulates nucleolar structure and rRNA production. PMID:26464461

  20. Identification of RNA editing sites in the SNP database

    OpenAIRE

    Eisenberg, Eli; Adamsky, Konstantin; Cohen, Lital; Amariglio, Ninette; Hirshberg, Abraham; Rechavi, Gideon; Levanon, Erez Y.

    2005-01-01

    The relationship between human inherited genomic variations and phenotypic differences has been the focus of much research effort in recent years. These studies benefit from millions of single-nucleotide polymorphism (SNP) records available in public databases, such as dbSNP. The importance of identifying false dbSNP records increases with the growing role played by SNPs in linkage analysis for disease traits. In particular, the emerging understanding of the abundance of DNA and RNA editing c...

  1. A-to-I editing of protein coding and noncoding RNAs.

    Science.gov (United States)

    Mallela, Arka; Nishikura, Kazuko

    2012-01-01

    Adenosine deaminase acting on RNA (ADAR) catalyzes the hydrolytic deamination of adenosine to inosine in double-stranded RNA (dsRNA) substrates. Inosine pairs preferentially with cytidine, as opposed to uridine; therefore, ADAR editing alters the sequence and base pairing properties of both protein-coding and non-coding RNA. Editing can directly alter the sequence of protein-coding transcripts and modify splicing, or affect a variety of non-coding targets, including microRNA, small interfering RNA, viral transcripts, and repeat elements such as Alu and LINE. Such editing has a wide range of physiological effects, including modification of targets in the brain and in disease states.

  2. Widespread establishment and regulatory impact of Alu exons in human genes.

    Science.gov (United States)

    Shen, Shihao; Lin, Lan; Cai, James J; Jiang, Peng; Kenkel, Elizabeth J; Stroik, Mallory R; Sato, Seiko; Davidson, Beverly L; Xing, Yi

    2011-02-15

    The Alu element has been a major source of new exons during primate evolution. Thousands of human genes contain spliced exons derived from Alu elements. However, identifying Alu exons that have acquired genuine biological functions remains a major challenge. We investigated the creation and establishment of Alu exons in human genes, using transcriptome profiles of human tissues generated by high-throughput RNA sequencing (RNA-Seq) combined with extensive RT-PCR analysis. More than 25% of Alu exons analyzed by RNA-Seq have estimated transcript inclusion levels of at least 50% in the human cerebellum, indicating widespread establishment of Alu exons in human genes. Genes encoding zinc finger transcription factors have significantly higher levels of Alu exonization. Importantly, Alu exons with high splicing activities are strongly enriched in the 5'-UTR, and two-thirds (10/15) of 5'-UTR Alu exons tested by luciferase reporter assays significantly alter mRNA translational efficiency. Mutational analysis reveals the specific molecular mechanisms by which newly created 5'-UTR Alu exons modulate translational efficiency, such as the creation or elongation of upstream ORFs that repress the translation of the primary ORFs. This study presents genomic evidence that a major functional consequence of Alu exonization is the lineage-specific evolution of translational regulation. Moreover, the preferential creation and establishment of Alu exons in zinc finger genes suggest that Alu exonization may have globally affected the evolution of primate and human transcriptomes by regulating the protein production of master transcriptional regulators in specific lineages.

  3. The moss Physcomitrella patens, a model plant for the study of RNA editing in plant organelles

    OpenAIRE

    Tasaki, Eiji; Sugita, Mamoru

    2010-01-01

    RNA editing is an enigmatic phenomenon in which specific cytidines (C) in the transcripts are changed to uridines (U). In flowering plants, over 500 editing sites have been identified in the mitochondria and plastids. By contrast, in a moss Physcomitrella patens, only 12 editing sites are found in both organelles. Recent extensive genetics studies have revealed involvement of the pentatricopeptide repeat (PPR) proteins with a C-terminal DYW domain (PPR-DYW) in site-specific RNA editing events...

  4. Diversity of Endonuclease V: From DNA Repair to RNA Editing

    Directory of Open Access Journals (Sweden)

    Isao Kuraoka

    2015-09-01

    Full Text Available Deamination of adenine occurs in DNA, RNA, and their precursors via a hydrolytic reaction and a nitrosative reaction. The generated deaminated products are potentially mutagenic because of their structural similarity to natural bases, which in turn leads to erroneous nucleotide pairing and subsequent disruption of cellular metabolism. Incorporation of deaminated precursors into the nucleic acid strand occurs during nucleotide synthesis by DNA and RNA polymerases or base modification by DNA- and/or RNA-editing enzymes during cellular functions. In such cases, removal of deaminated products from DNA and RNA by a nuclease might be required depending on the cellular function. One such enzyme, endonuclease V, recognizes deoxyinosine and cleaves 3' end of the damaged base in double-stranded DNA through an alternative excision repair mechanism in Escherichia coli, whereas in Homo sapiens, it recognizes and cleaves inosine in single-stranded RNA. However, to explore the role of endonuclease V in vivo, a detailed analysis of cell biology is required. Based on recent reports and developments on endonuclease V, we discuss the potential functions of endonuclease V in DNA repair and RNA metabolism.

  5. RNA editing in Drosophila melanogaster: new targets and functionalconsequences

    Energy Technology Data Exchange (ETDEWEB)

    Stapleton, Mark; Carlson, Joseph W.; Celniker, Susan E.

    2006-09-05

    Adenosine deaminases that act on RNA (ADARs) catalyze the site-specific conversion of adenosine to inosine in primary mRNA transcripts. These re-coding events affect coding potential, splice-sites, and stability of mature mRNAs. ADAR is an essential gene and studies in mouse, C. elegans, and Drosophila suggest its primary function is to modify adult behavior by altering signaling components in the nervous system. By comparing the sequence of isogenic cDNAs to genomic DNA, we have identified and experimentally verified 27 new targets of Drosophila ADAR. Our analyses lead us to identify new classes of genes whose transcripts are targets of ADAR including components of the actin cytoskeleton, and genes involved in ion homeostasis and signal transduction. Our results indicate that editing in Drosophila increases the diversity of the proteome, and does so in a manner that has direct functional consequences on protein function.

  6. Mitochondrial tRNA 5'-editing in Dictyostelium discoideum and Polysphondylium pallidum.

    Science.gov (United States)

    Abad, Maria G; Long, Yicheng; Kinchen, R Dimitri; Schindel, Elinor T; Gray, Michael W; Jackman, Jane E

    2014-05-30

    Mitochondrial tRNA (mt-tRNA) 5'-editing was first described more than 20 years ago; however, the first candidates for 5'-editing enzymes were only recently identified in a eukaryotic microbe (protist), the slime mold Dictyostelium discoideum. In this organism, eight of 18 mt-tRNAs are predicted to be edited based on the presence of genomically encoded mismatched nucleotides in their aminoacyl-acceptor stem sequences. Here, we demonstrate that mt-tRNA 5'-editing occurs at all predicted sites in D. discoideum as evidenced by changes in the sequences of isolated mt-tRNAs compared with the expected sequences encoded by the mitochondrial genome. We also identify two previously unpredicted editing events in which G-U base pairs are edited in the absence of any other genomically encoded mismatches. A comparison of 5'-editing in D. discoideum with 5'-editing in another slime mold, Polysphondylium pallidum, suggests organism-specific idiosyncrasies in the treatment of U-G/G-U pairs. In vitro activities of putative D. discoideum editing enzymes are consistent with the observed editing reactions and suggest an overall lack of tRNA substrate specificity exhibited by the repair component of the editing enzyme. Although the presence of terminal mismatches in mt-tRNA sequences is highly predictive of the occurrence of mt-tRNA 5'-editing, the variability in treatment of U-G/G-U base pairs observed here indicates that direct experimental evidence of 5'-editing must be obtained to understand the complete spectrum of mt-tRNA editing events in any species. PMID:24737330

  7. In vitro RNA-binding assay for studying trans-factors for RNA editing in chloroplasts.

    Science.gov (United States)

    Shikanai, Toshiharu; Okuda, Kenji

    2011-01-01

    In plant organelles, specific C residues are modified to U by RNA editing. Short RNA sequences surrounding the target site (i.e., cis-elements) are recognized by trans-factors, which were recently shown to be pentatricopeptide repeat (PPR) proteins. PPR proteins consist of tandem arrays of a highly degenerate unit of 35 (pentatrico) amino acids, and PPR motifs are believed to recognize specific RNA sequences. In Arabidopsis thaliana, more than 450 sites are edited in mitochondria and plastids, and a similar number of PPR proteins are encoded in the nuclear genome. To study how the tandem array of a PPR motif facilitates the recognition of RNA sequences, an efficient biochemical strategy is an in vitro binding assay of recombinant PPR proteins with target RNA. This analysis is especially powerful with a combination of in vivo analyses based on the phenotypes of mutants and transgenic plants. In this chapter, we describe methods for the expression of recombinant PPR proteins in Escherichia coli, preparation of probe RNAs, and RNA gel shift assays. These methods can also be utilized for other RNA-binding proteins.

  8. Parallel Evolution and Lineage-Specific Expansion of RNA Editing in Ctenophores.

    Science.gov (United States)

    Kohn, Andrea B; Sanford, Rachel S; Yoshida, Masa-aki; Moroz, Leonid L

    2015-12-01

    RNA editing is a process of targeted alterations of nucleotides in all types of RNA molecules (e.g., rRNA, tRNA, mRNA, and miRNA). As a result, the transcriptional output differs from its genomic DNA template. RNA editing can be defined both by biochemical mechanisms and by enzymes that perform these reactions. There are high levels of RNA editing detected in the mammalian nervous system, suggesting that nervous systems use this mechanism to increase protein diversity, because the post-transcription modifications lead to new gene products with novel functions. By re-annotating the ctenophore genomes, we found that the number of predicted RNA-editing enzymes is comparable to the numbers in mammals, but much greater than in other non-bilaterian basal metazoans. However, the overall molecular diversity of RNA-editing enzymes in ctenophores is lower, suggesting a possible "compensation" by an expansion of the ADAT1-like subfamily in this lineage. In two genera of ctenophores, Pleurobrachia and Mnemiopsis, there are high levels of expression for RNA-editing enzymes in their aboral organs, the integrative center involved in control of locomotion and geotaxis. This finding supports the hypothesis that RNA editing is correlated with the complexity of tissues and behaviors. Smaller numbers of RNA-editing enzymes in Porifera and Placozoa also correlates with the primary absence of neural and muscular systems in these lineages. In ctenophores, the expansion of the RNA-editing machinery can also provide mechanisms that support the remarkable capacity for regeneration in these animals. In summary, despite their compact genomes, a wide variety of epigenomic mechanisms employed by ctenophores and other non-bilaterian basal metazoans can provide novel insights into the evolutionary origins of biological novelties. PMID:26089435

  9. Parallel Evolution and Lineage-Specific Expansion of RNA Editing in Ctenophores.

    Science.gov (United States)

    Kohn, Andrea B; Sanford, Rachel S; Yoshida, Masa-aki; Moroz, Leonid L

    2015-12-01

    RNA editing is a process of targeted alterations of nucleotides in all types of RNA molecules (e.g., rRNA, tRNA, mRNA, and miRNA). As a result, the transcriptional output differs from its genomic DNA template. RNA editing can be defined both by biochemical mechanisms and by enzymes that perform these reactions. There are high levels of RNA editing detected in the mammalian nervous system, suggesting that nervous systems use this mechanism to increase protein diversity, because the post-transcription modifications lead to new gene products with novel functions. By re-annotating the ctenophore genomes, we found that the number of predicted RNA-editing enzymes is comparable to the numbers in mammals, but much greater than in other non-bilaterian basal metazoans. However, the overall molecular diversity of RNA-editing enzymes in ctenophores is lower, suggesting a possible "compensation" by an expansion of the ADAT1-like subfamily in this lineage. In two genera of ctenophores, Pleurobrachia and Mnemiopsis, there are high levels of expression for RNA-editing enzymes in their aboral organs, the integrative center involved in control of locomotion and geotaxis. This finding supports the hypothesis that RNA editing is correlated with the complexity of tissues and behaviors. Smaller numbers of RNA-editing enzymes in Porifera and Placozoa also correlates with the primary absence of neural and muscular systems in these lineages. In ctenophores, the expansion of the RNA-editing machinery can also provide mechanisms that support the remarkable capacity for regeneration in these animals. In summary, despite their compact genomes, a wide variety of epigenomic mechanisms employed by ctenophores and other non-bilaterian basal metazoans can provide novel insights into the evolutionary origins of biological novelties.

  10. Canonical A-to-I and C-to-U RNA editing is enriched at 3'UTRs and microRNA target sites in multiple mouse tissues.

    Directory of Open Access Journals (Sweden)

    Tongjun Gu

    Full Text Available RNA editing is a process that modifies RNA nucleotides and changes the efficiency and fidelity of the central dogma. Enzymes that catalyze RNA editing are required for life, and defects in RNA editing are associated with many diseases. Recent advances in sequencing have enabled the genome-wide identification of RNA editing sites in mammalian transcriptomes. Here, we demonstrate that canonical RNA editing (A-to-I and C-to-U occurs in liver, white adipose, and bone tissues of the laboratory mouse, and we show that apparent non-canonical editing (all other possible base substitutions is an artifact of current high-throughput sequencing technology. Further, we report that high-confidence canonical RNA editing sites can cause non-synonymous amino acid changes and are significantly enriched in 3' UTRs, specifically at microRNA target sites, suggesting both regulatory and functional consequences for RNA editing.

  11. TbRGG2 facilitates kinetoplastid RNA editing initiation and progression past intrinsic pause sites.

    Science.gov (United States)

    Ammerman, Michelle L; Presnyak, Vladimir; Fisk, John C; Foda, Bardees M; Read, Laurie K

    2010-11-01

    TbRGG2 is an essential kinetoplastid RNA editing accessory factor that acts specifically on pan-edited RNAs. To understand the mechanism of TbRGG2 action, we undertook an in-depth analysis of edited RNA populations in TbRGG2 knockdown cells and an in vitro examination of the biochemical activities of the protein. We demonstrate that TbRGG2 down-regulation more severely impacts editing at the 5' ends of pan-edited RNAs than at their 3' ends. The initiation of editing is reduced to some extent in TbRGG2 knockdown cells. In addition, TbRGG2 plays a post-initiation role as editing becomes stalled in TbRGG2-depleted cells, resulting in an overall decrease in the 3' to 5' progression of editing. Detailed analyses of edited RNAs from wild-type and TbRGG2-depleted cells reveal that TbRGG2 facilitates progression of editing past intrinsic pause sites that often correspond to the 3' ends of cognate guide RNAs (gRNAs). In addition, noncanonically edited junction regions are either absent or significantly shortened in TbRGG2-depleted cells, consistent with impaired gRNA transitions. Sequence analysis further suggests that TbRGG2 facilitates complete utilization of certain gRNAs. In vitro RNA annealing and in vivo RNA unwinding assays demonstrate that TbRGG2 can modulate RNA-RNA interactions. Collectively, these data are consistent with a model in which TbRGG2 facilitates initiation and 3' to 5' progression of editing through its ability to affect gRNA utilization, both during the transition between specific gRNAs and during usage of certain gRNAs. PMID:20855539

  12. RNA Editing Sites Exist in Protein-coding Genes in the Chloroplast Genome of Cycas taitungensis

    Institute of Scientific and Technical Information of China (English)

    Haiyan Chen; Likun Deng; Yuan Jiang; Ping Lu; Jianing Yu

    2011-01-01

    RNA editing is a post-transcriptional process that results in modifications of ribonucleotides at specific locations.In land plants editing can occur in both mitochondria and chloroplasts and most commonly involves C-to-U changes,especially in seed plants.Using prediction and experimental determination,we investigated RNA editing in 40 protein-coding genes from the chloroplast genome of Cycas taitungensis.A total of 85 editing sites were identified in 25 transcripts.Comparison analysis of the published editotypes of these 25 transcripts in eight species showed that RNA editing events gradually disappear during plant evolution.The editing in the first and third codon position disappeared quicker than that in the second codon position,ndh genes have the highest editing frequency while serine and proline codons were more frequently edited than the codons of other amino acids.These results imply that retained RNA editing sites have imbalanced distribution in genes and most of them may function by changing protein structure or interaction.Mitochondrion protein-coding genes have three times the editing sites compared with chloroplast genes of Cycas,most likely due to slower evolution speed.

  13. Reprogramming, Circular Reasoning and Self versus Non-self: One-Stop Shopping with RNA Editing

    Science.gov (United States)

    Savva, Yiannis A.; Rezaei, Ali; St. Laurent, Georges; Reenan, Robert A.

    2016-01-01

    Transcription of genetic information from archival DNA into RNA molecule working copies is vital for proper cellular function and is highly accurate. In turn, RNAs serve structural, enzymatic, and regulatory roles, as well as being informational templates for the ribosomal translation of proteins. Following RNA synthesis, maturing of RNA molecules occurs through various RNA processing events. One component of the collection of processes involving RNA species, broadly defined as RNA metabolism, is the RNA-editing pathway and is found in all animals. Acting specifically on RNA substrates with double-stranded character, RNA editing has been shown to regulate a plethora of genomic outputs, including gene recoding, RNA splicing, biogenesis and targeting actions of microRNAs and small interfering RNAs, and global gene expression. Recent evidence suggests that RNA modifications mediated via RNA editing influence the biogenesis of circular RNAs and safeguard against aberrant innate immune responses generated to endogenous RNA sources. These novel roles have the potential to contribute new insights into molecular mechanisms underlying pathogenesis mediated by mishandling of double-stranded RNA. Here, we discuss recent advances in the field, which highlight novel roles associated with the RNA-editing process and emphasize their importance during cellular RNA metabolism. In addition, we highlight the relevance of these newly discovered roles in the context of neurological disorders and the more general concept of innate recognition of self versus non-self. PMID:27458478

  14. RNA聚合酶Ⅱ转录的ALU序列对HEK293细胞凋亡的影响%Effect of RNA PolⅡ Driven ALU Transcripts on Apoptosis of HEK293 Cells

    Institute of Scientific and Technical Information of China (English)

    李璇; 唐开福; 高建; 杨梅; 胡文艳; 田绿; 彭湃澜; 王峰; 高昌益; 任红

    2011-01-01

    目的 探讨RNA聚合酶Ⅱ转录的ALU序列对人胚肾293(HEK293)细胞凋亡的影响以及干扰素(IFN)在此机制中的作用.方法 取对数生长期的HEK293细胞,分为6组,ALU-293组(瞬时转染重组质粒pcDNA3.1-ALU)、peDNA3.1-293组[瞬时转染空质粒peDNA3.1(-),作为阴性对照],Poly Ⅰ:C-293组[瞬时转染dsRNA的多聚肌苷胞苷酸(Poly Ⅰ:C),作为阳性对照]、IFNβ-293组(加入1.65×104U IFNβ,作为阳性对照)、空白对照组(未经处理的HEK293细胞)和HBs-293组(瞬时转染重组质粒pcDNA3.1-HBs),转染后48 h,采用MTT法检测细胞的增殖活性;Cellular DNA Fragmentation ELISA和DNA Ladder 法检测细胞的凋亡情况;Real-time PCR检测细胞中IFNβ基因mRNA的水平.结果 瞬时转染重组质粒pcDNA3.1-ALU能够抑制HEK293细胞增殖,并促使其凋亡,且细胞中IFNβ mRNA的水平显著上调.结论 RNA聚合酶Ⅱ转录的ALU序列能够通过激活干扰素系统来诱导细胞凋亡.%Objective To investigate the effect of RNA Pol Ⅱ driven ALU transcripts on the apoptosis of human embryonic kidney 293 (HEK293) cells as well as the role of IFN in this mechanism.Methods The HEK293 cells at logarithmic growth phase were divided into six groups.The cells in ALU-293, pcDNA3.1-293, Poly Ⅰ: C-293 and HBs-293 groups were transiently transfected with recombinant plasmid pcDNA3.1-ALU, empty vector pcDNA3.1 (-) (negative control ), Poly Ⅰ: C ( positive control ), recombinant plasmid pcDNA3.1-HBs respectively, while those in IFNβ-293 group was added with 1.65 × 104 U IFNβ (positive control ).However, the cells in blank control group were untreated.The cells in various groups 48 h after transfection were determined for proliferative activity by MTT method, for apoptosis by Cellular DNA Fragmentation ELISA and DNA ladder, and for IFNβ mRNA level by real-time PCR.Results Transient transfection with recombinant plasmid pcDNA3.1-ALU inhibited the proliferation and promoted the apoptosis of HEK293

  15. Role of tRNAPro in pretransfer editing of alanine by prolyl-tRNA synthetase

    Directory of Open Access Journals (Sweden)

    Boyarshin K. S.

    2013-09-01

    Full Text Available Aim. To characterize the process of tRNA-dependent pretransfer edi- ting of alanine by prolyl-tRNA synthetase of bacteria Enterococcus faecalis (ProRSEf. Methods. Velocity of the editing processes in vitro was determined by ATP hydrolysis by ProRSEf. Pretransfer and posttransfer editing were experimentally separated by site-directed mutagenesis. Results. tRNA-dependent pretransfer editing is characterized by three-fold larger velocity then tRNA-independent editing. Effectivity of the process depends on the presence of 2'-hydroxyle group of A76 tRNAPro. In the absence of tRNAPro selective release of alanyl-AMP occurs simultaneously with tRNA-independent pretransfer editing. Released alanyl-AMP can be re-bound and hydrolyzed. Conclusions. tRNA-dependent pretransfer editing of alanine by ProRSEf is the catalytic mechanism, mediated by 2'-hydroxyl group of A76 tRNAPro. In the absence of tRNAPro tRNA-independent pretransfer editing and selective release of alanyl-AMP occur.

  16. Improved Computational Target Site Prediction for Pentatricopeptide Repeat RNA Editing Factors

    OpenAIRE

    TAKENAKA, MIZUKI; Zehrmann, Anja; Brennicke, Axel; Graichen, Knut

    2013-01-01

    Pentatricopeptide repeat (PPR) proteins with an E domain have been identified as specific factors for C to U RNA editing in plant organelles. These PPR proteins bind to a unique sequence motif 5′ of their target editing sites. Recently, involvement of a combinatorial amino acid code in the P (normal length) and S type (short) PPR domains in sequence specific RNA binding was reported. PPR proteins involved in RNA editing, however, contain not only P and S motifs but also their long variants L ...

  17. A-to-I RNA editing: A new mechanism of genomic information modification

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A-to-I RNA editing, the important event of gene modification, which takes place at post-transcriptional level, was firstly reported in 1991. The molecular mechanism of A-to-I RNA editing involves site-selective deamination of adenosine to inosine in pre-mRNA, which leads to altering translation codons and splicing in nuclear transcripts, thereby functionally distinct proteins can be produced from a single gene. The mammalian editing enzymes ADARs (adenosine deaminases acting on RNA) are widely expressed in brain and other tissues, however, up to date their substrates are mainly found in the central nervous system. It has recently been noticed that imperfect editing of these RNA substrates play critical roles in corresponding diseases, indicating that A-to-I RNA editing may be quite important in physiological or pathophysiological processes. Finding more new substrates of ADARs, especially in peripheral tissues, and performing functional research on new genes will be helpful to elucidate the biological significance of A-to-I RNA editing.

  18. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system

    OpenAIRE

    Xie, Kabin; Minkenberg, Bastian; Yang, Yinong

    2015-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system has recently emerged as an efficient and versatile tool for genome editing in various organisms. However, its targeting capability and multiplex editing efficiency are often limited by the guide RNA (gRNA)-expressing device. This study demonstrates a general strategy and platform for precise processing and efficient production of numerous gRNAs in vivo from a synthetic poly...

  19. Genome-Wide Characterization of RNA Editing in Chicken Embryos Reveals Common Features among Vertebrates

    Science.gov (United States)

    Frésard, Laure; Leroux, Sophie; Roux, Pierre-François; Klopp, Christophe; Fabre, Stéphane; Esquerré, Diane; Dehais, Patrice; Djari, Anis; Gourichon, David

    2015-01-01

    RNA editing results in a post-transcriptional nucleotide change in the RNA sequence that creates an alternative nucleotide not present in the DNA sequence. This leads to a diversification of transcription products with potential functional consequences. Two nucleotide substitutions are mainly described in animals, from adenosine to inosine (A-to-I) and from cytidine to uridine (C-to-U). This phenomenon is described in more details in mammals, notably since the availability of next generation sequencing technologies allowing whole genome screening of RNA-DNA differences. The number of studies recording RNA editing in other vertebrates like chicken is still limited. We chose to use high throughput sequencing technologies to search for RNA editing in chicken, and to extend the knowledge of its conservation among vertebrates. We performed sequencing of RNA and DNA from 8 embryos. Being aware of common pitfalls inherent to sequence analyses that lead to false positive discovery, we stringently filtered our datasets and found fewer than 40 reliable candidates. Conservation of particular sites of RNA editing was attested by the presence of 3 edited sites previously detected in mammals. We then characterized editing levels for selected candidates in several tissues and at different time points, from 4.5 days of embryonic development to adults, and observed a clear tissue-specificity and a gradual increase of editing level with time. By characterizing the RNA editing landscape in chicken, our results highlight the extent of evolutionary conservation of this phenomenon within vertebrates, attest to its tissue and stage specificity and provide support of the absence of non A-to-I events from the chicken transcriptome. PMID:26024316

  20. Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

    Science.gov (United States)

    Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

    2009-01-01

    Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4+ T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5′ untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1. PMID:19651874

  1. A distant cis acting intronic element induces site-selective RNA editing.

    Science.gov (United States)

    Daniel, Chammiran; Venø, Morten T; Ekdahl, Ylva; Kjems, Jørgen; Öhman, Marie

    2012-10-01

    Transcripts have been found to be site selectively edited from adenosine-to-inosine (A-to-I) in the mammalian brain, mostly in genes involved in neurotransmission. While A-to-I editing occurs at double-stranded structures, other structural requirements are largely unknown. We have investigated the requirements for editing at the I/M site in the Gabra-3 transcript of the GABA(A) receptor. We identify an evolutionarily conserved intronic duplex, 150 nt downstream of the exonic hairpin where the I/M site resides, which is required for its editing. This is the first time a distant RNA structure has been shown to be important for A-to-I editing. We demonstrate that the element also can induce editing in related but normally not edited RNA sequences. In human, thousands of genes are edited in duplexes formed by inverted repeats in non-coding regions. It is likely that numerous such duplexes can induce editing of coding regions throughout the transcriptome. PMID:22848101

  2. Did RNA editing in plant organellar genomes originate under natural selection or through genetic drift?

    Directory of Open Access Journals (Sweden)

    Jobson Richard W

    2008-10-01

    Full Text Available Abstract Background The C↔U substitution types of RNA editing have been observed frequently in organellar genomes of land plants. Although various attempts have been made to explain why such a seemingly inefficient genetic mechanism would have evolved, no satisfactory explanation exists in our view. In this study, we examined editing patterns in chloroplast genomes of the hornwort Anthoceros formosae and the fern Adiantum capillus-veneris and in mitochondrial genomes of the angiosperms Arabidopsis thaliana, Beta vulgaris and Oryza sativa, to gain an understanding of the question of how RNA editing originated. Results We found that 1 most editing sites were distributed at the 2nd and 1st codon positions, 2 editing affected codons that resulted in larger hydrophobicity and molecular size changes much more frequently than those with little change involved, 3 editing uniformly increased protein hydrophobicity, 4 editing occurred more frequently in ancestrally T-rich sequences, which were more abundant in genes encoding membrane-bound proteins with many hydrophobic amino acids than in genes encoding soluble proteins, and 5 editing occurred most often in genes found to be under strong selective constraint. Conclusion These analyses show that editing mostly affects functionally important and evolutionarily conserved codon positions, codons and genes encoding membrane-bound proteins. In particular, abundance of RNA editing in plant organellar genomes may be associated with disproportionately large percentages of genes in these two genomes that encode membrane-bound proteins, which are rich in hydrophobic amino acids and selectively constrained. These data support a hypothesis that natural selection imposed by protein functional constraints has contributed to selective fixation of certain editing sites and maintenance of the editing activity in plant organelles over a period of more than four hundred millions years. The retention of genes encoding RNA

  3. Glycine receptors caught between genome and proteome - functional implications of RNA editing and splicing

    Directory of Open Access Journals (Sweden)

    Pascal Legendre

    2009-11-01

    Full Text Available Information processing in the brain requires a delicate balance between excitation and inhibition. Glycine receptors (GlyR are involved in inhibitory mechanisms mainly at a synaptic level, but potential novel roles for these receptors recently emerged due to the discovery of posttranscriptional processing. GLR transcripts are edited through enzymatic modification of a single nucleotide leading to amino acid substitution within the neurotransmitter binding domain. RNA editing produces gain-of-function receptors well suited for generation and maintenance of tonic inhibition of neuronal excitability. As neuronal activity deprivation in early stages of development or in epileptic tissue is detrimental to neurons and because RNA editing of GlyR is up-regulated in temporal lobe epilepsy patients with a severe course of disease a pathophysiological role of these receptors emerges. This review contains a state-of-the-art discussion of (pathophysiological implications of GlyR RNA editing.

  4. Distinct role of Arabidopsis mitochondrial P-type pentatricopeptide repeat protein-modulating editing protein, PPME, in nad1 RNA editing.

    Science.gov (United States)

    Leu, Kuan-Chieh; Hsieh, Ming-Hsiun; Wang, Huei-Jing; Hsieh, Hsu-Liang; Jauh, Guang-Yuh

    2016-06-01

    The mitochondrion is an important power generator in most eukaryotic cells. To preserve its function, many essential nuclear-encoded factors play specific roles in mitochondrial RNA metabolic processes, including RNA editing. RNA editing consists of post-transcriptional deamination, which alters specific nucleotides in transcripts to mediate gene expression. In plant cells, many pentatricopeptide repeat proteins (PPRs) participate in diverse organellar RNA metabolic processes, but only PLS-type PPRs are involved in RNA editing. Here, we report a P-type PPR protein from Arabidopsis thaliana, P-type PPR-Modulating Editing (PPME), which has a distinct role in mitochondrial nad1 RNA editing via RNA binding activity. In the homozygous ppme mutant, cytosine (C)-to-uracil (U) conversions at both the nad1-898 and 937 sites were abolished, disrupting Arg(300)-to-Trp(300) and Pro(313)-to-Ser(313) amino acid changes in the mitochondrial NAD1 protein. NAD1 is a critical component of mitochondrial respiration complex I; its activity is severely reduced in the homozygous ppme mutant, resulting in significantly altered growth and development. Both abolished RNA editing and defective complex I activity were completely rescued by CaMV 35S promoter- and PPME native promoter-driven PPME genomic fragments tagged with GFP in a homozygous ppme background. Our experimental results demonstrate a distinct role of a P-type PPR protein, PPME, in RNA editing in plant organelles. PMID:27149614

  5. Cas9 gRNA engineering for genome editing, activation and repression

    OpenAIRE

    Kiani, Samira; Chavez, Alejandro; Tuttle, Marcelle; Hall, Richard N; Chari, Raj; Ter-Ovanesyan, Dmitry; Qian, Jason; Pruitt, Benjamin W.; Beal, Jacob; Vora, Suhani; Buchthal, Joanna; Kowal, Emma J K; Ebrahimkhani, Mohammad R.; James J Collins; Weiss, Ron

    2015-01-01

    We demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.

  6. Splice-mediated insertion of an Alu sequence inactivates ornithine δ-aminotransferase: A role for Alu elements in human mutation

    International Nuclear Information System (INIS)

    In studies of mutations causing deficiency of ornithine δ-aminotransferase the authors found an allele whose mature mRNA has a 142-nucleotide insertion at the junction of sequences from exons 3 and 4. The insert derives from an Alu element in ornithine δ-aminotransferase intron 3 oriented in the direction opposite to transcription (an antisense Alu). A guanine → cytosine transversion creates a donor splice site in this Alu, activating a cryptic acceptor splice site at its 5' end and causing splice-mediated insertion of an Alu fragment into the mature ornithine-δ-aminotransferase mRNA. The authors note that the complement of the Alu consensus sequence has at least two cryptic acceptor sites and several potential donor sequences and predict that similar mutations will be found in other genes

  7. Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

    OpenAIRE

    Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

    2009-01-01

    Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent ...

  8. Synthetic CRISPR RNA-Cas9-guided genome editing in human cells.

    Science.gov (United States)

    Rahdar, Meghdad; McMahon, Moira A; Prakash, Thazha P; Swayze, Eric E; Bennett, C Frank; Cleveland, Don W

    2015-12-22

    Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA-RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA. Taken together, these findings provide a platform for therapeutic applications, especially for nervous system disease, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 nuclease activity.

  9. LINE-1 ORF1 protein enhances Alu SINE retrotransposition.

    Science.gov (United States)

    Wallace, Nicholas; Wagstaff, Bradley J; Deininger, Prescott L; Roy-Engel, Astrid M

    2008-08-01

    Retroelements have contributed over one third of the human genome mass. The currently active LINE-1 (L1) codes for two proteins (ORF1p and ORF2p), both strictly required for retrotransposition. In contrast, the non-coding parasitic SINE (Alu) only appears to need the L1 ORF2p for its own amplification. This requirement was previously determined using a tissue culture assay system in human cells (HeLa). Because HeLa are likely to express functional L1 proteins, it is possible that low levels of endogenous ORF1p are necessary for the observed tagged Alu mobilization. By individually expressing ORF1 and ORF2 proteins from both human (L1RP and LRE3) and rodent (L1A102 and L1spa) L1 sources, we demonstrate that increasing amounts of ORF1 expressing vector enhances tagged Alu mobilization in HeLa cells. In addition, using chicken fibroblast cells as an alternate cell culture source, we confirmed that ORF1p is not strictly required for Alu mobilization in our assay. Supporting our observations in HeLa cells, we find that tagged Alu retrotransposition is improved by supplementation of ORF1p in the cultured chicken cells. We postulate that L1 ORF1p plays either a direct or indirect role in enhancing the interaction between the Alu RNA and the required factors needed for its retrotransposition.

  10. The expression of apoB mRNA editing factors is not the sole determinant for the induction of editing in differentiating Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Galloway, Chad A. [Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642 (United States); Smith, Harold C., E-mail: harold.smith@rochester.edu [Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642 (United States)

    2010-01-01

    Apolipoprotein B mRNA is edited at cytidine 6666 in the enterocytes lining the small intestine of all mammals; converting a CAA codon to a UAA stop codon. The conversion is {approx}80% efficient in this tissue and leads to the expression of the truncated protein, ApoB48, essential for secretion of dietary lipid as chylomicrons. Caco-2 cell raft cultures have been used as an in vitro model for the induction of editing activity during human small intestinal cell differentiation. This induction of apoB mRNA editing has been ascribed to the expression of APOBEC-1. In agreement our data demonstrated differentiation-dependent induction of expression of the editing enzyme APOBEC-1 and in addition we show alternative splicing of the essential auxiliary factor ACF. However, transfection of these editing factors in undifferentiated proliferating Caco-2 cells was not sufficient to induce robust apoB mRNA editing activity. Only differentiation of Caco-2 cells could induce more physiological like levels of apoB mRNA editing. The data suggested that additional regulatory mechanism(s) were induced by differentiation that controlled the functional activity of editing factors.

  11. The expression of apoB mRNA editing factors is not the sole determinant for the induction of editing in differentiating Caco-2 cells

    International Nuclear Information System (INIS)

    Apolipoprotein B mRNA is edited at cytidine 6666 in the enterocytes lining the small intestine of all mammals; converting a CAA codon to a UAA stop codon. The conversion is ∼80% efficient in this tissue and leads to the expression of the truncated protein, ApoB48, essential for secretion of dietary lipid as chylomicrons. Caco-2 cell raft cultures have been used as an in vitro model for the induction of editing activity during human small intestinal cell differentiation. This induction of apoB mRNA editing has been ascribed to the expression of APOBEC-1. In agreement our data demonstrated differentiation-dependent induction of expression of the editing enzyme APOBEC-1 and in addition we show alternative splicing of the essential auxiliary factor ACF. However, transfection of these editing factors in undifferentiated proliferating Caco-2 cells was not sufficient to induce robust apoB mRNA editing activity. Only differentiation of Caco-2 cells could induce more physiological like levels of apoB mRNA editing. The data suggested that additional regulatory mechanism(s) were induced by differentiation that controlled the functional activity of editing factors.

  12. A Novel Computational Strategy to Identify A-to-I RNA Editing Sites by RNA-Seq Data: De Novo Detection in Human Spinal Cord Tissue

    Science.gov (United States)

    Picardi, Ernesto; Gallo, Angela; Galeano, Federica; Tomaselli, Sara; Pesole, Graziano

    2012-01-01

    RNA editing is a post-transcriptional process occurring in a wide range of organisms. In human brain, the A-to-I RNA editing, in which individual adenosine (A) bases in pre-mRNA are modified to yield inosine (I), is the most frequent event. Modulating gene expression, RNA editing is essential for cellular homeostasis. Indeed, its deregulation has been linked to several neurological and neurodegenerative diseases. To date, many RNA editing sites have been identified by next generation sequencing technologies employing massive transcriptome sequencing together with whole genome or exome sequencing. While genome and transcriptome reads are not always available for single individuals, RNA-Seq data are widespread through public databases and represent a relevant source of yet unexplored RNA editing sites. In this context, we propose a simple computational strategy to identify genomic positions enriched in novel hypothetical RNA editing events by means of a new two-steps mapping procedure requiring only RNA-Seq data and no a priori knowledge of RNA editing characteristics and genomic reads. We assessed the suitability of our procedure by confirming A-to-I candidates using conventional Sanger sequencing and performing RNA-Seq as well as whole exome sequencing of human spinal cord tissue from a single individual. PMID:22957051

  13. A potential role for NF1 mRNA editing in the pathogenesis of NF1 tumors

    Energy Technology Data Exchange (ETDEWEB)

    Cappione, A.J.; French, B.L.; Skuse, G.R. [Univ. of Rochester School of Medicine and Dentistry, NY (United States)

    1997-02-01

    Neurofibromatosis type I (NF1) is a common disorder that predisposes to neoplasia in tissues derived from the embryonic neural crest. The NF1 gene encodes a tumor suppressor that most likely acts through the interaction of its GTPase-activating protein (GAP)-related domain (GRD) with the product of the ras protooncogene. We have previously identified a site in the NF1 mRNA, within the first half of the NF1 GRD, which undergoes base-modification editing. Editing at that site changes a C to a U, thereby introducing an in-frame stop codon. NF1 RNA editing has been detected in all cell types studied, to date. In order to investigate the role played by editing in NF1 tumorigenesis, we analyzed RNA from 19 NF1 and 4 non-NF1 tumors. We observed varying levels of NF1 mRNA editing in different tumors, with a higher range of editing levels in more malignant tumors (e.g., neurofibrosarcomas) compared to benign tumors (cutaneous neurofibromas). Plexiform neurofibromas have an intermediate range of levels of NF1 mRNA editing. We also compared tumor and nontumor tissues from several NF1 individuals, to determine the extent of variability present in the constitutional levels of NF1 mRNA editing and to determine whether higher levels are present in tumors. The constitutional levels of NF1 mRNA editing varied slightly but were consistent with the levels observed in non-NF1 individuals. In every case, there was a greater level of NF1 mRNA editing in the tumor than in the nontumor tissue from the same patient. These results suggest that inappropriately high levels of NF1 mRNA editing does play a role in NF1 tumorigenesis and that editing may result in the functional equivalent of biallelic inactivation of the NF1 tumor suppressor. 24 refs., 4 figs., 2 tabs.

  14. Perturbing A-to-I RNA editing using genetics and homologous recombination.

    Science.gov (United States)

    Staber, Cynthia J; Gell, Selena; Jepson, James E C; Reenan, Robert A

    2011-01-01

    Evidence for the chemical conversion of adenosine-to-inosine (A-to-I) in messenger RNA (mRNA) has been detected in numerous metazoans, especially those "most successful" phyla: Arthropoda, Mollusca, and Chordata. The requisite enzymes for A-to-I editing, ADARs (adenosine deaminases acting on RNA) are highly conserved and are present in every higher metazoan genome sequenced to date. The fruit fly, Drosophila melanogaster, represents an ideal model organism for studying A-to-I editing, both in terms of fundamental biochemistry and in relation to determining adaptive downstream effects on physiology and behavior. The Drosophila genome contains a single structural gene for ADAR (dAdar), yet the fruit fly transcriptome has the widest range of conserved and validated ADAR targets in coding mRNAs of any known organism. In addition, many of the genes targeted by dADAR have been genetically identified as playing a role in nervous system function, providing a rich source of material to investigate the biological relevance of this intriguing process. Here, we discuss how recent advances in the use of ends-out homologous recombination (HR) in Drosophila make possible both the precise control of the editing status for defined adenosine residues and the engineering of flies with globally altered RNA editing of the fly transcriptome. These new approaches promise to significantly improve our understanding of how mRNA modification contributes to insect physiology and ethology.

  15. The Genomic Landscape and Clinical Relevance of A-to-I RNA Editing in Human Cancers | Office of Cancer Genomics

    Science.gov (United States)

    Adenosine-to-inosine (A-to-I) RNA editing is a widespread post-transcriptional mechanism, but its genomic landscape and clinical relevance in cancer have not been investigated systematically. We characterized the global A-to-I RNA editing profiles of 6,236 patient samples of 17 cancer types from The Cancer Genome Atlas and revealed a striking diversity of altered RNA-editing patterns in tumors relative to normal tissues. We identified an appreciable number of clinically relevant editing events, many of which are in noncoding regions.

  16. New Insights into the Biological Role of Mammalian ADARs; the RNA Editing Proteins

    Directory of Open Access Journals (Sweden)

    Niamh Mannion

    2015-09-01

    Full Text Available The ADAR proteins deaminate adenosine to inosine in double-stranded RNA which is one of the most abundant modifications present in mammalian RNA. Inosine can have a profound effect on the RNAs that are edited, not only changing the base-pairing properties, but can also result in recoding, as inosine behaves as if it were guanosine. In mammals there are three ADAR proteins and two ADAR-related proteins (ADAD expressed. All have a very similar modular structure; however, both their expression and biological function differ significantly. Only two of the ADAR proteins have enzymatic activity. However, both ADAR and ADAD proteins possess the ability to bind double-strand RNA. Mutations in ADARs have been associated with many diseases ranging from cancer, innate immunity to neurological disorders. Here, we will discuss in detail the domain structure of mammalian ADARs, the effects of RNA editing, and the role of ADARs in human diseases.

  17. Targeted Genome Editing of Sweet Orange Using Cas9/sgRNA

    OpenAIRE

    Hongge Jia; Nian Wang

    2014-01-01

    Genetic modification, including plant breeding, has been widely used to improve crop yield and quality, as well as to increase disease resistance. Targeted genome engineering is expected to contribute significantly to future varietal improvement, and genome editing technologies using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9/single guide RNA (sgRNA) have already been succes...

  18. Epigenome Editing of Potato by Grafting Using Transgenic Tobacco as siRNA Donor.

    Science.gov (United States)

    Kasai, Atsushi; Bai, Songling; Hojo, Hatsune; Harada, Takeo

    2016-01-01

    In plants, it is possible to induce heritable transcriptional gene silencing (TGS) via RNA-directed DNA methylation (RdDM) using artificially synthesized small RNA (siRNA) homologous to the 5'-flanking region of the target gene. As the siRNA signal with a specific RNA determinant moves through plasmodesmata and sieve elements, we attempted to induce TGS of a transgene and an endogenous gene of potato (Solanum tuberosum) rootstock by grafting using siRNA produced in a tobacco (Nicotiana benthamiana) scion. Our results provide evidence that this system can induce TGS of target genes in tubers formed on potato rootstock. The TGS is maintained in the progeny tubers lacking the transported siRNAs. Our findings reveal that epigenome editing using mobile RNA has the potential to allow breeding of artificial sport cultivars in vegetative propagation crops. PMID:27564864

  19. In vitro substrate specificities of 3'-5' polymerases correlate with biological outcomes of tRNA 5'-editing reactions.

    Science.gov (United States)

    Long, Yicheng; Jackman, Jane E

    2015-07-22

    Protozoan mitochondrial tRNAs (mt-tRNAs) are repaired by a process known as 5'-editing. Mt-tRNA sequencing revealed organism-specific patterns of editing G-U base pairs, wherein some species remove G-U base pairs during 5'-editing, while others retain G-U pairs in the edited tRNA. We tested whether 3'-5' polymerases that catalyze the repair step of 5'-editing exhibit organism-specific preferences that explain the treatment of G-U base pairs. Biochemical and kinetic approaches revealed that a 3'-5' polymerase from Acanthamoeba castellanii tolerates G-U wobble pairs in editing substrates much more readily than several other enzymes, consistent with its biological pattern of editing.

  20. Messenger RNA editing in mammals: new members of the APOBEC family seeking roles in the family business.

    Science.gov (United States)

    Wedekind, Joseph E; Dance, Geoffrey S C; Sowden, Mark P; Smith, Harold C

    2003-04-01

    Alteration of mRNA sequence through base modification mRNA editing frequently generates protein diversity. Several proteins have been identified as being similar to C-to-U mRNA editing enzymes based on their structural domains and the occurrence of a catalytic domain characteristic of cytidine deaminases. In light of the hypothesis that these proteins might represent novel mRNA editing systems that could affect proteome diversity, we consider their structure, expression and relevance to biomedically significant processes or pathologies. PMID:12683974

  1. Identification and Characterization of Two Novel RNA Editing Sites in grin1b Transcripts of Embryonic Danio rerio

    Directory of Open Access Journals (Sweden)

    Pedro Pozo

    2012-01-01

    Full Text Available Discovering RNA editing sites in model organisms provides an insight into their adaptations in addition to finding potential sites for the regulation of neural activity and the basis of integrated models of metazoan editing with a variety of applications, including potential clinical treatments of neural dysregulation. The zebrafish, Danio rerio, is an important vertebrate model system. We focused on the grin1b gene of zebrafish due to its important function in the nervous tissue as a glutamate receptor. Using a comparative sequence-based approach, we located possible RNA editing events within the grin1b transcript. Surprisingly, sequence analysis also revealed a new editing site which was not predicted by the comparative approach. We here report the discovery of two novel RNA editing events in grin1b transcripts of embryonic zebrafish. The frequency of these editing events and their locations within the grin1b transcript are also described.

  2. RNA editing of hepatitis B virus transcripts by activation-induced cytidine deaminase.

    Science.gov (United States)

    Liang, Guoxin; Kitamura, Kouichi; Wang, Zhe; Liu, Guangyan; Chowdhury, Sajeda; Fu, Weixin; Koura, Miki; Wakae, Kousho; Honjo, Tasuku; Muramatsu, Masamichi

    2013-02-01

    Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. The mechanism by which AID triggers SHM and CSR has been explained by two distinct models. In the DNA deamination model, AID converts cytidine bases in DNA into uridine. The uridine is recognized by the DNA repair system, which produces DNA strand breakages and point mutations. In the alternative model, RNA edited by AID is responsible for triggering CSR and SHM. However, RNA deamination by AID has not been demonstrated. Here we found that C-to-T and G-to-A mutations accumulated in hepatitis B virus (HBV) nucleocapsid DNA when AID was expressed in HBV-replicating hepatic cell lines. AID expression caused C-to-T mutations in the nucleocapsid DNA of RNase H-defective HBV, which does not produce plus-strand viral DNA. Furthermore, the RT-PCR products of nucleocapsid viral RNA from AID-expressing cells exhibited significant C-to-T mutations, whereas viral RNAs outside the nucleocapsid did not accumulate C-to-U mutations. Moreover, AID was packaged within the nucleocapsid by forming a ribonucleoprotein complex with HBV RNA and the HBV polymerase protein. The encapsidation of the AID protein with viral RNA and DNA provides an efficient environment for evaluating AID's RNA and DNA deamination activities. A bona fide RNA-editing enzyme, apolipoprotein B mRNA editing catalytic polypeptide 1, induced a similar level of C-to-U mutations in nucleocapsid RNA as AID. Taken together, the results indicate that AID can deaminate the nucleocapsid RNA of HBV.

  3. The SLO1 PPR protein is required for RNA editing at multiple sites with similar upstream sequences in Arabidopsis mitochondria.

    Science.gov (United States)

    Sung, Tzu-Ying; Tseng, Ching-Chih; Hsieh, Ming-Hsiun

    2010-08-01

    In Arabidopsis, RNA editing changes more than 500 cytidines to uridines in mitochondrial transcripts. The editing enzyme and co-factors involved in these processes are largely unknown. We have identified a nuclear gene SLOW GROWTH1 (SLO1) encoding an E motif-containing pentatricopeptide repeat protein that is required for RNA editing of nad4 and nad9 in Arabidopsis mitochondria. The SLO1 protein is localized to the mitochondrion, and its absence gives rise to small plants with slow growth and delayed development. A survey of approximately 500 mitochondrial RNA editing sites in Arabidopsis reveals that the editing of two sites, nad4-449 and nad9-328, is abolished in the slo1 mutants. Sequence comparison in the upstream (from -1 to -15 bp) of nad4-449 and nad9-328 editing sites shows that nine of the 15 nucleotides are identical. In addition to RNA editing, we used RNA gel blot analysis to compare the abundance and banding patterns of mitochondrial transcripts between the wild type and slo1 mutants. Of the 79 genes and open reading frames examined, steady-state levels of 56 mitochondrial transcripts are increased in the slo1 mutants. These results suggest that the SLO1 protein may indirectly regulate plant growth and development via affecting mitochondrial RNA editing and gene expression.

  4. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing

    OpenAIRE

    Tsai, Shengdar Q.; Wyvekens, Nicolas; Khayter, Cyd; Foden, Jennifer A.; Thapar, Vishal; Reyon, Deepak; Goodwin, Mathew J.; Aryee, Martin J; Joung, J. Keith

    2014-01-01

    Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI Nucleases (RFNs) that recognize extended sequences and can edit endogenous genes with high efficiencies in human cells. The cleavage activity of an RFN depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation and therefore show improved specificities relative to wi...

  5. Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design

    OpenAIRE

    Farboud, B; Meyer, BJ

    2015-01-01

    Success with genome editing by the RNA-programmed nuclease Cas9 has been limited by the inability to predict effective guide RNAs and DNA target sites. Not all guide RNAs have been successful, and even those that were, varied widely in their efficacy. Here we describe and validate a strategy for Caenorhabditis elegans that reliably achieved a high frequency of genome editing for all targets tested in vivo. The key innovation was to design guide RNAs with a GG motif at the 3′ end of their targ...

  6. The mobile genetic element Alu in the human genome

    Energy Technology Data Exchange (ETDEWEB)

    Novick, G.E. [Florida International Univ., Miami, FL (United States); Batzer, M.A.; Deininger, P.L. [Louisiana State Univ. Medical Center, New Orleans, LA (United States)] [and others

    1996-01-01

    Genetic material has been traditionally envisioned as relatively static with the exception of occasional, often deleterious mutations. The sequence DNA-to-RNA-to-protein represented for many years the central dogma relating gene structure and function. Recently, the field of molecular genetics has provided revolutionary information on the dynamic role of repetitive elements in the function of the genetic material and the evolution of humans and other organisms. Alu sequences represent the largest family of short interspersed repetitive elements (SINEs) in humans, being present in an excess of 500,000 copies per haploid genome. Alu elements, as well as the other repetitive elements, were once considered to be useless. Today, the biology of Alu transposable elements is being widely examined in order to determine the molecular basis of a growing number of identified diseases and to provide new directions in genome mapping and biomedical research. 66 refs., 5 figs.

  7. Redefining the structural motifs that determine RNA binding and RNA editing by pentatricopeptide repeat proteins in land plants.

    Science.gov (United States)

    Cheng, Shifeng; Gutmann, Bernard; Zhong, Xiao; Ye, Yongtao; Fisher, Mark F; Bai, Fengqi; Castleden, Ian; Song, Yue; Song, Bo; Huang, Jiaying; Liu, Xin; Xu, Xun; Lim, Boon L; Bond, Charles S; Yiu, Siu-Ming; Small, Ian

    2016-02-01

    The pentatricopeptide repeat (PPR) proteins form one of the largest protein families in land plants. They are characterised by tandem 30-40 amino acid motifs that form an extended binding surface capable of sequence-specific recognition of RNA strands. Almost all of them are post-translationally targeted to plastids and mitochondria, where they play important roles in post-transcriptional processes including splicing, RNA editing and the initiation of translation. A code describing how PPR proteins recognise their RNA targets promises to accelerate research on these proteins, but making use of this code requires accurate definition and annotation of all of the various nucleotide-binding motifs in each protein. We have used a structural modelling approach to define 10 different variants of the PPR motif found in plant proteins, in addition to the putative deaminase motif that is found at the C-terminus of many RNA-editing factors. We show that the super-helical RNA-binding surface of RNA-editing factors is potentially longer than previously recognised. We used the redefined motifs to develop accurate and consistent annotations of PPR sequences from 109 genomes. We report a high error rate in PPR gene models in many public plant proteomes, due to gene fusions and insertions of spurious introns. These consistently annotated datasets across a wide range of species are valuable resources for future comparative genomics studies, and an essential pre-requisite for accurate large-scale computational predictions of PPR targets. We have created a web portal (http://www.plantppr.com) that provides open access to these resources for the community. PMID:26764122

  8. Regulation of gene expression in neuronal tissue by RNA interference and editing

    DEFF Research Database (Denmark)

    Venø, Morten Trillingsgaard

    No tissue in the mammalian organism is more complex than the brain. This complexity is in part the result of precise timing and interplay of a large number mechanisms modulating gene expression post-transcriptionally. Fine-tuning mechanisms such as A-to-I editing of RNA transcripts and regulation...... mediated by microRNAs are crucial for the correct function of the mammalian brain. We are addressing A-to-I editing and regulation by microRNAs with spatio-temporal resolution in the embryonic porcine brain by Solexa sequencing of microRNAs and 454 sequencing of edited neuronal messenger RNAs, resulting......RNAs, causing these transgenic mice to be less prone to cocaine addictive behavior. Another study demonstrated that abolishing the expression of histone methylases, GLP and G9a, increases the expression level of a large number of miRNAs. A possible feed-back mechanism is suggested, since a subset of these mi...

  9. Impact of Alu repeats on the evolution of human p53 binding sites

    Directory of Open Access Journals (Sweden)

    Sirotin Michael V

    2011-01-01

    Full Text Available Abstract Background The p53 tumor suppressor protein is involved in a complicated regulatory network, mediating expression of ~1000 human genes. Recent studies have shown that many p53 in vivo binding sites (BSs reside in transposable repeats. The relationship between these BSs and functional p53 response elements (REs remains unknown, however. We sought to understand whether the p53 REs also reside in transposable elements and particularly in the most-abundant Alu repeats. Results We have analyzed ~160 functional p53 REs identified so far and found that 24 of them occur in repeats. More than half of these repeat-associated REs reside in Alu elements. In addition, using a position weight matrix approach, we found ~400,000 potential p53 BSs in Alu elements genome-wide. Importantly, these putative BSs are located in the same regions of Alu repeats as the functional p53 REs - namely, in the vicinity of Boxes A/A' and B of the internal RNA polymerase III promoter. Earlier nucleosome-mapping experiments showed that the Boxes A/A' and B have a different chromatin environment, which is critical for the binding of p53 to DNA. Here, we compare the Alu-residing p53 sites with the corresponding Alu consensus sequences and conclude that the p53 sites likely evolved through two different mechanisms - the sites overlapping with the Boxes A/A' were generated by CG → TG mutations; the other sites apparently pre-existed in the progenitors of several Alu subfamilies, such as AluJo and AluSq. The binding affinity of p53 to the Alu-residing sites generally correlates with the age of Alu subfamilies, so that the strongest sites are embedded in the 'relatively young' Alu repeats. Conclusions The primate-specific Alu repeats play an important role in shaping the p53 regulatory network in the context of chromatin. One of the selective factors responsible for the frequent occurrence of Alu repeats in introns may be related to the p53-mediated regulation of Alu

  10. Small RNA and A-to-I Editing in Autism Spectrum Disorders

    Science.gov (United States)

    Eran, Alal

    One in every 88 children is diagnosed with Autism Spectrum Disorders (ASDs), a set of neurodevelopmental conditions characterized by social impairments, communication deficits, and repetitive behavior. ASDs have a substantial genetic component, but the specific cause of most cases remains unknown. Understanding gene-environment interactions underlying ASD is essential for improving early diagnosis and identifying critical targets for intervention and prevention. Towards this goal, we surveyed adenosine-to-inosine (A-to-I) RNA editing in autistic brains. A-to-I editing is an epigenetic mechanism that fine-tunes synaptic function in response to environmental stimuli, shown to modulate complex behavior in animals. We used ultradeep sequencing to quantify A-to-I receding of candidate synaptic genes in postmortem cerebella from individuals with ASD and neurotypical controls. We found unexpectedly wide distributions of human A-to-I editing levels, whose extremes were consistently populated by individuals with ASD. We correlated A-to-I editing with isoform usage, identified clusters of correlated sites, and examined differential editing patterns. Importantly, we found that individuals with ASD commonly use a dysfunctional form of the editing enzyme ADARB1. We next profiled small RNAs thought to regulate A-to-I editing, which originate from one of the most commonly altered loci in ASD, 15q11. Deep targeted sequencing of SNORD115 and SNORD116 transcripts enabled their high-resolution detection in human brains, and revealed a strong gender bias underlying their expression. The consistent 2-fold upregulation of 15q11 small RNAs in male vs. female cerebella could be important in delineating the role of this locus in ASD, a male dominant disorder. Overall, these studies provide an accurate population-level view of small RNA and A-to-I editing in human cerebella, and suggest that A-to-I editing of synaptic genes may be informative for assessing the epigenetic risk for autism

  11. Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design.

    Science.gov (United States)

    Farboud, Behnom; Meyer, Barbara J

    2015-04-01

    Success with genome editing by the RNA-programmed nuclease Cas9 has been limited by the inability to predict effective guide RNAs and DNA target sites. Not all guide RNAs have been successful, and even those that were, varied widely in their efficacy. Here we describe and validate a strategy for Caenorhabditis elegans that reliably achieved a high frequency of genome editing for all targets tested in vivo. The key innovation was to design guide RNAs with a GG motif at the 3' end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. We also combined the 3' GG guide improvement with a co-CRISPR/co-conversion approach. For this co-conversion scheme, animals were only screened for genome editing at designated targets if they exhibited a dominant phenotype caused by Cas9-dependent editing of an unrelated target. Combining the two strategies further enhanced the ease of mutant recovery, thereby providing a powerful means to obtain desired genetic changes in an otherwise unaltered genome.

  12. Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design.

    Science.gov (United States)

    Farboud, Behnom; Meyer, Barbara J

    2015-04-01

    Success with genome editing by the RNA-programmed nuclease Cas9 has been limited by the inability to predict effective guide RNAs and DNA target sites. Not all guide RNAs have been successful, and even those that were, varied widely in their efficacy. Here we describe and validate a strategy for Caenorhabditis elegans that reliably achieved a high frequency of genome editing for all targets tested in vivo. The key innovation was to design guide RNAs with a GG motif at the 3' end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. We also combined the 3' GG guide improvement with a co-CRISPR/co-conversion approach. For this co-conversion scheme, animals were only screened for genome editing at designated targets if they exhibited a dominant phenotype caused by Cas9-dependent editing of an unrelated target. Combining the two strategies further enhanced the ease of mutant recovery, thereby providing a powerful means to obtain desired genetic changes in an otherwise unaltered genome. PMID:25695951

  13. CRISPR–Cas9-mediated genome editing and guide RNA design

    OpenAIRE

    Wiles, Michael V.; Qin, Wenning; Cheng, Albert W; Wang, Haoyi

    2015-01-01

    CRISPR and CRISPR-associated (Cas) proteins, which in nature comprise the RNA-based adaptive immune system in bacteria and archaea, have emerged as particularly powerful genome editing tools owing to their unrivaled ease of use and ability to modify genomes across mammalian model systems. As such, the CRISPR–Cas9 system holds promise as a “system of choice” for functional mammalian genetic studies across biological disciplines. Here we briefly review this fast moving field, introduce the CRIS...

  14. Reciprocal regulation of A-to-I RNA editing and the vertebrate nervous system

    Directory of Open Access Journals (Sweden)

    Andrew Charles Penn

    2013-04-01

    Full Text Available The fine control of molecules mediating communication in the nervous system is key to adjusting neuronal responsiveness during development and in maintaining the stability of established networks in the face of altered sensory input. To prevent culmination of pathological recurrent network excitation or debilitating periods of quiescence, adaptive alterations occur in the signalling molecules and ion channels that control membrane excitability and synaptic transmission. However, rather than encoding (and thus ‘hardwiring’ modified gene copies, the nervous systems of metazoa have opted for expanding on post-transcriptional pre-mRNA splicing by altering key encoded amino acids using a conserved mechanism of A-to-I RNA editing: the enzymatic deamination of adenosine resulting in a change in the nucleotide to inosine. Inosine exhibits similar base-pairing properties to guanosine with respect to tRNA codon recognition, replication by polymerases and RNA secondary structure forming capacity. In addition to recoding within the open reading frame, adenosine deamination also occurs with high frequency throughout the non-coding transcriptome, where it affects multiple aspects of RNA metabolism and gene expression. We will describe here the recoding function of key RNA editing targets in the mammalian central nervous system (CNS and their potential to be regulated. We will then discuss how interactions of A-to-I editing with gene expression and alternative splicing could play a wider role in regulating the neuronal transcriptome. Finally, we will highlight the increasing complexity of this multifaceted control hub by summarising new findings from high-throughput studies.

  15. From End to End: tRNA Editing at 5'- and 3'-Terminal Positions

    Science.gov (United States)

    Betat, Heike; Long, Yicheng; Jackman, Jane E.; Mörl, Mario

    2014-01-01

    During maturation, tRNA molecules undergo a series of individual processing steps, ranging from exo- and endonucleolytic trimming reactions at their 5'- and 3'-ends, specific base modifications and intron removal to the addition of the conserved 3'-terminal CCA sequence. Especially in mitochondria, this plethora of processing steps is completed by various editing events, where base identities at internal positions are changed and/or nucleotides at 5'- and 3'-ends are replaced or incorporated. In this review, we will focus predominantly on the latter reactions, where a growing number of cases indicate that these editing events represent a rather frequent and widespread phenomenon. While the mechanistic basis for 5'- and 3'-end editing differs dramatically, both reactions represent an absolute requirement for generating a functional tRNA. Current in vivo and in vitro model systems support a scenario in which these highly specific maturation reactions might have evolved out of ancient promiscuous RNA polymerization or quality control systems. PMID:25535083

  16. From End to End: tRNA Editing at 5'- and 3'-Terminal Positions

    Directory of Open Access Journals (Sweden)

    Heike Betat

    2014-12-01

    Full Text Available During maturation, tRNA molecules undergo a series of individual processing steps, ranging from exo- and endonucleolytic trimming reactions at their 5'- and 3'-ends, specific base modifications and intron removal to the addition of the conserved 3'-terminal CCA sequence. Especially in mitochondria, this plethora of processing steps is completed by various editing events, where base identities at internal positions are changed and/or nucleotides at 5'- and 3'-ends are replaced or incorporated. In this review, we will focus predominantly on the latter reactions, where a growing number of cases indicate that these editing events represent a rather frequent and widespread phenomenon. While the mechanistic basis for 5'- and 3'-end editing differs dramatically, both reactions represent an absolute requirement for generating a functional tRNA. Current in vivo and in vitro model systems support a scenario in which these highly specific maturation reactions might have evolved out of ancient promiscuous RNA polymerization or quality control systems.

  17. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing.

    Science.gov (United States)

    Tsai, Shengdar Q; Wyvekens, Nicolas; Khayter, Cyd; Foden, Jennifer A; Thapar, Vishal; Reyon, Deepak; Goodwin, Mathew J; Aryee, Martin J; Joung, J Keith

    2014-06-01

    Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI nucleases (RFNs) that can recognize extended sequences and edit endogenous genes with high efficiencies in human cells. RFN cleavage activity depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation substantially reducing the likelihood that a suitable target site will occur more than once in the genome and therefore improving specificities relative to wild-type Cas9 monomers. RFNs guided by a single gRNA generally induce lower levels of unwanted mutations than matched monomeric Cas9 nickases. In addition, we describe a simple method for expressing multiple gRNAs bearing any 5' end nucleotide, which gives dimeric RFNs a broad targeting range. RFNs combine the ease of RNA-based targeting with the specificity enhancement inherent to dimerization and are likely to be useful in applications that require highly precise genome editing.

  18. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing.

    Science.gov (United States)

    Tsai, Shengdar Q; Wyvekens, Nicolas; Khayter, Cyd; Foden, Jennifer A; Thapar, Vishal; Reyon, Deepak; Goodwin, Mathew J; Aryee, Martin J; Joung, J Keith

    2014-06-01

    Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI nucleases (RFNs) that can recognize extended sequences and edit endogenous genes with high efficiencies in human cells. RFN cleavage activity depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation substantially reducing the likelihood that a suitable target site will occur more than once in the genome and therefore improving specificities relative to wild-type Cas9 monomers. RFNs guided by a single gRNA generally induce lower levels of unwanted mutations than matched monomeric Cas9 nickases. In addition, we describe a simple method for expressing multiple gRNAs bearing any 5' end nucleotide, which gives dimeric RFNs a broad targeting range. RFNs combine the ease of RNA-based targeting with the specificity enhancement inherent to dimerization and are likely to be useful in applications that require highly precise genome editing. PMID:24770325

  19. Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos

    Institute of Scientific and Technical Information of China (English)

    Nannan Chang; Changhong Sun; Lu Gao; Dan Zhu; Xiufei Xu; Xiaojun Zhu; Jing-Wei Xiong

    2013-01-01

    Recent advances with the type Ⅱ clustered regularly interspaced short palindromic repeats (CRISPR) system promise an improved approach to genome editing.However,the applicability and efficiency of this system in model organisms,such as zebrafish,are little studied.Here,we report that RNA-guided Cas9 nuclease efficiently facilitates genome editing in both mammalian cells and zebrafish embryos in a simple and robust manner.Over 35% of sitespecific somatic mutations were found when specific Cas/gRNA was used to target either etsrp,gata4 or gata5 in zebrafish embryos in vivo.The Cas9/gRNA efficiently induced biallelic conversion of etsrp or gata5 in the resulting somatic cells,recapitulating their respective vessel phenotypes in etsrpy11 mutant embryos or cardia bifida phenotypes in fautm236a mutant embryos.Finally,we successfully achieved site-specific insertion of mloxP sequence induced by Cas9/gRNA system in zebrafish embryos.These results demonstrate that the Cas9/gRNA system has the potential of becoming a simple,robust and efficient reverse genetic tool for zebrafish and other model organisms.Together with other genome-engineering technologies,the Cas9 system is promising for applications in biology,agriculture,environmental studies and medicine.

  20. Targeted genome editing of sweet orange using Cas9/sgRNA.

    Directory of Open Access Journals (Sweden)

    Hongge Jia

    Full Text Available Genetic modification, including plant breeding, has been widely used to improve crop yield and quality, as well as to increase disease resistance. Targeted genome engineering is expected to contribute significantly to future varietal improvement, and genome editing technologies using zinc finger nucleases (ZFNs, transcription activator-like effector nucleases (TALENs, and clustered regularly interspaced short palindromic repeat (CRISPR/Cas9/single guide RNA (sgRNA have already been successfully used to genetically modify plants. However, to date, there has been no reported use of any of the current genome editing approaches in sweet orange, an important fruit crop. In this study, we first developed a novel tool, Xcc-facilitated agroinfiltration, for enhancing transient protein expression in sweet orange leaves. We then successfully employed Xcc-facilitated agroinfiltration to deliver Cas9, along with a synthetic sgRNA targeting the CsPDS gene, into sweet orange. DNA sequencing confirmed that the CsPDS gene was mutated at the target site in treated sweet orange leaves. The mutation rate using the Cas9/sgRNA system was approximately 3.2 to 3.9%. Off-target mutagenesis was not detected for CsPDS-related DNA sequences in our study. This is the first report of targeted genome modification in citrus using the Cas9/sgRNA system-a system that holds significant promise for the study of citrus gene function and for targeted genetic modification.

  1. RNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing?

    Science.gov (United States)

    Gasiunas, Giedrius; Siksnys, Virginijus

    2013-11-01

    Tailor-made nucleases for precise genome modification, such as zinc finger or TALE nucleases, currently represent the state-of-the-art for genome editing. These nucleases combine a programmable protein module which guides the enzyme to the target site with a nuclease domain which cuts DNA at the addressed site. Reprogramming of these nucleases to cut genomes at specific locations requires major protein engineering efforts. RNA-guided DNA endonuclease Cas9 of the type II (clustered regularly interspaced short palindromic repeat) CRISPR-Cas system uses CRISPR RNA (crRNA) as a guide to locate the DNA target and the Cas9 protein to cut DNA. Easy programmability of the Cas9 endonuclease using customizable RNAs brings unprecedented flexibility and versatility for targeted genome modification. We highlight the potential of the Cas9 RNA-guided DNA endonuclease as a novel tool for genome surgery, and discuss possible constraints and future prospects.

  2. Rat prostatic steroid binding protein: characterisation of the Alu element upstream of the C3 genes.

    OpenAIRE

    Hurst, H C; Parker, M G

    1984-01-01

    We have characterised an Alu-like repetitive element found about 400 bp upstream of the gene encoding the C3 component of rat prostatic steroid binding protein and suggest, from comparisons with other published sequences, that it is an example of a third class of rodent Alu-equivalent sequences. Members of this class are 80-90 bp long, share greater than 90% sequence homology, and contain sequences resembling the RNA polymerase III bipartite promoter. The Alu type III element within the C3 ge...

  3. Impact of RNA editing on functions of the serotonin 2C receptor in vivo

    Directory of Open Access Journals (Sweden)

    Uade B Olaghere Da Silva

    2010-03-01

    Full Text Available Transcripts encoding 5-HT2C receptors are modified posttranscriptionally by RNA editing, generating up to 24 protein isoforms. In recombinant cells, the fully edited isoform, 5-HT2C-VGV, exhibits blunted G-protein coupling and reduced constitutive activity. The present studies examine the signal transduction properties of 5-HT2C-VGV receptors in brain to determine the in vivo consequences of altered editing. Using mice solely expressing the 5-HT2C-VGV receptor (VGV/Y, we demonstrate reduced G-protein coupling efficiency and high-affinity agonist binding of brain 5-HT2C-VGV receptors. However, enhanced behavioral sensitivity to a 5-HT2C receptor agonist was also seen in mice expressing 5-HT2C-VGV receptors, an unexpected finding given the blunted G-protein coupling. In addition, mice expressing 5-HT2C-VGV receptors had greater sensitivity to a 5-HT2C inverse agonist/antagonist enhancement of dopamine turnover relative to wild-type mice. These behavioral and biochemical results are most likely explained by increases in 5-HT2C receptor binding sites in the brains of mice solely expressing -5HT2C-VGV receptors. We conclude that 5-HT2C-VGV receptor signaling in brain is blunted, but this deficiency is masked by a marked increase in 5HT2C receptor binding site density in mice solely expressing the VGV isoform. These findings suggest that RNA editing may regulate the density of 5-HT2C receptor binding sites in brain. We further caution that the pattern of 5-HT2C receptor RNA isoforms may not reflect the pattern of protein isoforms, and hence the inferred overall function of the receptor.

  4. A core MRB1 complex component is indispensable for RNA editing in insect and human infective stages of Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Michelle L Ammerman

    Full Text Available Uridine insertion/deletion RNA editing is a unique and vital process in kinetoplastids, required for creation of translatable open reading frames in most mitochondrially-encoded RNAs. Emerging as a key player in this process is the mitochondrial RNA binding 1 (MRB1 complex. MRB1 comprises an RNA-independent core complex of at least six proteins, including the GAP1/2 guide RNA (gRNA binding proteins. The core interacts in an RNA-enhanced or -dependent manner with imprecisely defined TbRGG2 subcomplexes, Armadillo protein MRB10130, and additional factors that comprise the dynamic MRB1 complex. Towards understanding MRB1 complex function in RNA editing, we present here functional characterization of the pentein domain-containing MRB1 core protein, MRB11870. Inducible RNAi studies demonstrate that MRB11870 is essential for proliferation of both insect vector and human infective stage T. brucei. MRB11870 ablation causes a massive defect in RNA editing, affecting both pan-edited and minimally edited mRNAs, but does not substantially affect mitochondrial RNA stability or processing of precursor transcripts. The editing defect in MRB1-depleted cells occurs at the initiation stage of editing, as pre-edited mRNAs accumulate. However, the gRNAs that direct editing remain abundant in the knockdown cells. To examine the contribution of MRB11870 to MRB1 macromolecular interactions, we tagged core complexes and analyzed their composition and associated proteins in the presence and absence of MRB11870. These studies demonstrated that MRB11870 is essential for association of GAP1/2 with the core, as well as for interaction of the core with other proteins and subcomplexes. Together, these data support a model in which the MRB1 core mediates functional interaction of gRNAs with the editing machinery, having GAP1/2 as its gRNA binding constituents. MRB11870 is a critical component of the core, essential for its structure and function.

  5. Editing for an AMPA receptor subunit RNA in prefrontal cortex and striatum in Alzheimer's disease, Huntington's disease and schizophrenia

    Science.gov (United States)

    Akbarian, S.; Smith, M. A.; Jones, E. G.; Bloom, F. E. (Principal Investigator)

    1995-01-01

    Animal studies and cell culture experiments demonstrated that posttranscriptional editing of the transcript of the GluR-2 gene, resulting in substitution of an arginine for glutamine in the second transmembrane region (TM II) of the expressed protein, is associated with a reduction in Ca2+ permeability of the receptor channel. Thus, disturbances in GluR-2 RNA editing with alteration of intracellular Ca2+ homeostasis could lead to neuronal dysfunction and even neuronal degeneration. The present study determined the proportions of edited and unedited GluR-2 RNA in the prefrontal cortex of brains from patients with Alzheimer's disease, in the striatum of brains from patients with Huntington's disease, and in the same areas of brains from age-matched schizophrenics and controls, by using reverse transcriptase-polymerase chain reaction, restriction endonuclease digestion, gel electrophoresis and scintillation radiometry. In the prefrontal cortex of controls, 99.9% were edited; in the prefrontal cortex both of schizophrenics and of Alzheimer's patients approximately 1.0% of all GluR-2 RNA molecules were unedited and 99% were edited. In the striatum of controls and of schizophrenics, approximately 0.5% of GluR-2 RNA molecules were unedited and 99.5% were edited; in the striatum of Huntington's patients nearly 5.0% of GluR-2 RNA was unedited. In the prefrontal white matter of controls, approximately 7.0% of GluR-2 RNA was unedited. In the normal human prefrontal cortex and striatum, the large majority of GluR-2 RNA molecules contains a CGG codon for arginine in the TMII coding region; this implies that the corresponding AMPA receptors have a low Ca2+ permeability, as previously demonstrated for the rat brain. The process of GluR-2 RNA editing is compromised in a region-specific manner in schizophrenia, in Alzheimer's disease and Huntington's Chorea although in each of these disorders there is still a large excess of edited GluR-2 RNA molecules. Disturbances of GluR-2 RNA

  6. RNA聚合酶Ⅱ转录的ALU对PKR磷酸化的调节作用%Regulatory Effect of ALU Transcribed by RNA Polymerase Ⅱ on PKR Phosphorylation

    Institute of Scientific and Technical Information of China (English)

    杨梅; 沈薇; 吴进峰; 曾贵利; 王峰; 任红; 唐开福

    2010-01-01

    目的 研究ALU自身的RNA聚合酶Ⅲ启动子对RNA聚合酶Ⅱ转录的影响,探讨RNA聚合酶Ⅱ转录的ALU对PKR磷酸化的调节作用.方法 将ALU全基因序列插入pcDNA3.1(-)载体的CMV启动子(一个RNA聚合酶Ⅱ启动子)的下游,构建重组质粒pcDNA3.1-ALU;转染HEK 293细胞,提取细胞总RNA,RT-PCR筛选稳定表达ALU的细胞;用干扰素处理稳定表达ALU序列的细胞,Western blot法检测PKR的磷酸化水平.结果 ALU全基因序列插入pcDNA3.1载体的CMV启动子直接下游,能够被RNA聚合酶Ⅱ有效转录;在稳定表达ALU的HEK 293细胞中,加干扰素与未加干扰素组PKR的磷酸化水平均明显高于相应的HEK 293细胞对照组.结论 ALU自身的RNA聚合酶Ⅲ启动子对RNA聚合酶Ⅱ转录无明显影响,但与RNA聚合酶Ⅲ启动下转录的ALU不同,RNA聚合酶Ⅱ转录的ALU失去了对干扰素的拮抗作用,反而可以通过形成双链RNA的方式激活PKR的活性.

  7. Alu-directed transcriptional regulation of some novel miRNAs

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    Zhao Xi W

    2009-11-01

    Full Text Available Abstract Background Despite many studies on the biogenesis, molecular structure and biological functions of microRNAs, little is known about the transcriptional regulatory mechanisms controlling the spatiotemporal expression pattern of human miRNA gene loci. Several lines of experimental results have indicated that both polymerase II (Pol-II and polymerase III (Pol-III may be involved in transcribing miRNAs. Here, we assessed the genomic evidence for Alu-directed transcriptional regulation of some novel miRNA genes in humans. Our data demonstrate that the expression of these Alu-related miRNAs may be modulated by Pol-III. Results We present a comprehensive exploration of the Alu-directed transcriptional regulation of some new miRNAs. Using a new computational approach, a variety of Alu-related sequences from multiple sources were pooled and filtered to obtain a subset containing Alu elements and characterized miRNA genes for which there is clear evidence of full-length transcription (embedded in EST. We systematically demonstrated that 73 miRNAs including five known ones may be transcribed by Pol-III through Alu or MIR. Among the new miRNAs, 33 were determined by high-throughput Solexa sequencing. Real-time TaqMan PCR and Northern blotting verified that three newly identified miRNAs could be induced to co-express with their upstream Alu transcripts by heat shock or cycloheximide. Conclusion Through genomic analysis, Solexa sequencing and experimental validation, we have identified candidate sequences for Alu-related miRNAs, and have found that the transcription of these miRNAs could be governed by Pol-III. Thus, this study may elucidate the mechanisms by which the expression of a class of small RNAs may be regulated by their upstream repeat elements.

  8. 4SALE – A tool for synchronous RNA sequence and secondary structure alignment and editing

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    Schultz Jörg

    2006-11-01

    Full Text Available Abstract Background In sequence analysis the multiple alignment builds the fundament of all proceeding analyses. Errors in an alignment could strongly influence all succeeding analyses and therefore could lead to wrong predictions. Hand-crafted and hand-improved alignments are necessary and meanwhile good common practice. For RNA sequences often the primary sequence as well as a secondary structure consensus is well known, e.g., the cloverleaf structure of the t-RNA. Recently, some alignment editors are proposed that are able to include and model both kinds of information. However, with the advent of a large amount of reliable RNA sequences together with their solved secondary structures (available from e.g. the ITS2 Database, we are faced with the problem to handle sequences and their associated secondary structures synchronously. Results 4SALE fills this gap. The application allows a fast sequence and synchronous secondary structure alignment for large data sets and for the first time synchronous manual editing of aligned sequences and their secondary structures. This study describes an algorithm for the synchronous alignment of sequences and their associated secondary structures as well as the main features of 4SALE used for further analyses and editing. 4SALE builds an optimal and unique starting point for every RNA sequence and structure analysis. Conclusion 4SALE, which provides an user-friendly and intuitive interface, is a comprehensive toolbox for RNA analysis based on sequence and secondary structure information. The program connects sequence and structure databases like the ITS2 Database to phylogeny programs as for example the CBCAnalyzer. 4SALE is written in JAVA and therefore platform independent. The software is freely available and distributed from the website at http://4sale.bioapps.biozentrum.uni-wuerzburg.de

  9. Nuclear Receptor HNF4α Binding Sequences are Widespread in Alu Repeats

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    Bolotin Eugene

    2011-11-01

    Full Text Available Abstract Background Alu repeats, which account for ~10% of the human genome, were originally considered to be junk DNA. Recent studies, however, suggest that they may contain transcription factor binding sites and hence possibly play a role in regulating gene expression. Results Here, we show that binding sites for a highly conserved member of the nuclear receptor superfamily of ligand-dependent transcription factors, hepatocyte nuclear factor 4alpha (HNF4α, NR2A1, are highly prevalent in Alu repeats. We employ high throughput protein binding microarrays (PBMs to show that HNF4α binds > 66 unique sequences in Alu repeats that are present in ~1.2 million locations in the human genome. We use chromatin immunoprecipitation (ChIP to demonstrate that HNF4α binds Alu elements in the promoters of target genes (ABCC3, APOA4, APOM, ATPIF1, CANX, FEMT1A, GSTM4, IL32, IP6K2, PRLR, PRODH2, SOCS2, TTR and luciferase assays to show that at least some of those Alu elements can modulate HNF4α-mediated transactivation in vivo (APOM, PRODH2, TTR, APOA4. HNF4α-Alu elements are enriched in promoters of genes involved in RNA processing and a sizeable fraction are in regions of accessible chromatin. Comparative genomics analysis suggests that there may have been a gain in HNF4α binding sites in Alu elements during evolution and that non Alu repeats, such as Tiggers, also contain HNF4α sites. Conclusions Our findings suggest that HNF4α, in addition to regulating gene expression via high affinity binding sites, may also modulate transcription via low affinity sites in Alu repeats.

  10. Regulation of Na+/K+ ATPase transport velocity by RNA editing.

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    Claudia Colina

    Full Text Available Because firing properties and metabolic rates vary widely, neurons require different transport rates from their Na(+/K(+ pumps in order to maintain ion homeostasis. In this study we show that Na(+/K(+ pump activity is tightly regulated by a novel process, RNA editing. Three codons within the squid Na(+/K(+ ATPase gene can be recoded at the RNA level, and the efficiency of conversion for each varies dramatically, and independently, between tissues. At one site, a highly conserved isoleucine in the seventh transmembrane span can be converted to a valine, a change that shifts the pump's intrinsic voltage dependence. Mechanistically, the removal of a single methyl group specifically targets the process of Na(+ release to the extracellular solution, causing a higher turnover rate at the resting membrane potential.

  11. [CRISPR/Cas: a novel way of RNA-guided genome editing].

    Science.gov (United States)

    Li, Jun; Zhang, Yi; Chen, Kun-Ling; Shan, Qi-Wei; Wang, Yan-Peng; Liang, Zhen; Gao, Cai-Xia

    2013-11-01

    Bacteria and archaea have evolved an adaptive immune system, known as type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system, which uses short RNA to direct the degradation of target sequences present in invading viral and plasmid DNAs. Recent advances in CRISPR/Cas system provide an improved method for genome editing, showing robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci. It is the latest technology to modify genome DNA specifically and effectively following zinc finger nucleases (ZFNs) and TALE nucleases (TALENs). Compared with ZFNs and TALENs, CRISPR/Cas is much simpler and easier to engineer. This review summarizes recent progress, and discusses the prospects of CRISPR/Cas system, with an emphasis on its structure, principle, applications and potential challenges.

  12. Is plant mitochondrial RNA editing a source of phylogenetic incongruence? An answer from in silico and in vivo data sets

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    Quagliariello Carla

    2008-03-01

    Full Text Available Abstract Background In plant mitochondria, the post-transcriptional RNA editing process converts C to U at a number of specific sites of the mRNA sequence and usually restores phylogenetically conserved codons and the encoded amino acid residues. Sites undergoing RNA editing evolve at a higher rate than sites not modified by the process. As a result, editing sites strongly affect the evolution of plant mitochondrial genomes, representing an important source of sequence variability and potentially informative characters. To date no clear and convincing evidence has established whether or not editing sites really affect the topology of reconstructed phylogenetic trees. For this reason, we investigated here the effect of RNA editing on the tree building process of twenty different plant mitochondrial gene sequences and by means of computer simulations. Results Based on our simulation study we suggest that the editing ‘noise’ in tree topology inference is mainly manifested at the cDNA level. In particular, editing sites tend to confuse tree topologies when artificial genomic and cDNA sequences are generated shorter than 500 bp and with an editing percentage higher than 5.0%. Similar results have been also obtained with genuine plant mitochondrial genes. In this latter instance, indeed, the topology incongruence increases when the editing percentage goes up from about 3.0 to 14.0%. However, when the average gene length is higher than 1,000 bp (rps3, matR and atp1 no differences in the comparison between inferred genomic and cDNA topologies could be detected. Conclusions Our findings by the here reported in silico and in vivo computer simulation system seem to strongly suggest that editing sites contribute in the generation of misleading phylogenetic trees if the analyzed mitochondrial gene sequence is highly edited (higher than 3.0% and reduced in length (shorter than 500 bp. In the current lack of direct experimental evidence the results

  13. RED: A Java-MySQL Software for Identifying and Visualizing RNA Editing Sites Using Rule-Based and Statistical Filters.

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    Yongmei Sun

    Full Text Available RNA editing is one of the post- or co-transcriptional processes that can lead to amino acid substitutions in protein sequences, alternative pre-mRNA splicing, and changes in gene expression levels. Although several methods have been suggested to identify RNA editing sites, there remains challenges to be addressed in distinguishing true RNA editing sites from its counterparts on genome and technical artifacts. In addition, there lacks a software framework to identify and visualize potential RNA editing sites. Here, we presented a software - 'RED' (RNA Editing sites Detector - for the identification of RNA editing sites by integrating multiple rule-based and statistical filters. The potential RNA editing sites can be visualized at the genome and the site levels by graphical user interface (GUI. To improve performance, we used MySQL database management system (DBMS for high-throughput data storage and query. We demonstrated the validity and utility of RED by identifying the presence and absence of C→U RNA-editing sites experimentally validated, in comparison with REDItools, a command line tool to perform high-throughput investigation of RNA editing. In an analysis of a sample data-set with 28 experimentally validated C→U RNA editing sites, RED had sensitivity and specificity of 0.64 and 0.5. In comparison, REDItools had a better sensitivity (0.75 but similar specificity (0.5. RED is an easy-to-use, platform-independent Java-based software, and can be applied to RNA-seq data without or with DNA sequencing data. The package is freely available under the GPLv3 license at http://github.com/REDetector/RED or https://sourceforge.net/projects/redetector.

  14. Adenosine-to-inosine RNA editing affects trafficking of the gamma-aminobutyric acid type A (GABA(A)) receptor.

    Science.gov (United States)

    Daniel, Chammiran; Wahlstedt, Helene; Ohlson, Johan; Björk, Petra; Ohman, Marie

    2011-01-21

    Recoding by adenosine-to-inosine RNA editing plays an important role in diversifying proteins involved in neurotransmission. We have previously shown that the Gabra-3 transcript, coding for the α3 subunit of the GABA(A) receptor is edited in mouse, causing an isoleucine to methionine (I/M) change. Here we show that this editing event is evolutionarily conserved from human to chicken. Analyzing recombinant GABA(A) receptor subunits expressed in HEK293 cells, our results suggest that editing at the I/M site in α3 has functional consequences on receptor expression. We demonstrate that I/M editing reduces the cell surface and the total number of α3 subunits. The reduction in cell surface levels is independent of the subunit combination as it is observed for α3 in combination with either the β2 or the β3 subunit. Further, an amino acid substitution at the corresponding I/M site in the α1 subunit has a similar effect on cell surface presentation, indicating the importance of this site for receptor trafficking. We show that the I/M editing during brain development is inversely related to the α3 protein abundance. Our results suggest that editing controls trafficking of α3-containing receptors and may therefore facilitate the switch of subunit compositions during development as well as the subcellular distribution of α subunits in the adult brain. PMID:21030585

  15. Adenosine-to-Inosine RNA Editing Affects Trafficking of the γ-Aminobutyric Acid Type A (GABAA) Receptor*

    Science.gov (United States)

    Daniel, Chammiran; Wahlstedt, Helene; Ohlson, Johan; Björk, Petra; Öhman, Marie

    2011-01-01

    Recoding by adenosine-to-inosine RNA editing plays an important role in diversifying proteins involved in neurotransmission. We have previously shown that the Gabra-3 transcript, coding for the α3 subunit of the GABAA receptor is edited in mouse, causing an isoleucine to methionine (I/M) change. Here we show that this editing event is evolutionarily conserved from human to chicken. Analyzing recombinant GABAA receptor subunits expressed in HEK293 cells, our results suggest that editing at the I/M site in α3 has functional consequences on receptor expression. We demonstrate that I/M editing reduces the cell surface and the total number of α3 subunits. The reduction in cell surface levels is independent of the subunit combination as it is observed for α3 in combination with either the β2 or the β3 subunit. Further, an amino acid substitution at the corresponding I/M site in the α1 subunit has a similar effect on cell surface presentation, indicating the importance of this site for receptor trafficking. We show that the I/M editing during brain development is inversely related to the α3 protein abundance. Our results suggest that editing controls trafficking of α3-containing receptors and may therefore facilitate the switch of subunit compositions during development as well as the subcellular distribution of α subunits in the adult brain. PMID:21030585

  16. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA

    Science.gov (United States)

    Zhang, Yi; Liang, Zhen; Zong, Yuan; Wang, Yanpeng; Liu, Jinxing; Chen, Kunling; Qiu, Jin-Long; Gao, Caixia

    2016-01-01

    Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research. PMID:27558837

  17. Alu retrotransposons promote differentiation of human carcinoma cells through the aryl hydrocarbon receptor

    Science.gov (United States)

    Morales-Hernández, Antonio; González-Rico, Francisco J.; Román, Angel C.; Rico-Leo, Eva; Alvarez-Barrientos, Alberto; Sánchez, Laura; Macia, Ángela; Heras, Sara R.; García-Pérez, José L.; Merino, Jaime M.; Fernández-Salguero, Pedro M.

    2016-01-01

    Cell differentiation is a central process in development and in cancer growth and dissemination. OCT4 (POU5F1) and NANOG are essential for cell stemness and pluripotency; yet, the mechanisms that regulate their expression remain largely unknown. Repetitive elements account for almost half of the Human Genome; still, their role in gene regulation is poorly understood. Here, we show that the dioxin receptor (AHR) leads to differentiation of human carcinoma cells through the transcriptional upregulation of Alu retrotransposons, whose RNA transcripts can repress pluripotency genes. Despite the genome-wide presence of Alu elements, we provide evidences that those located at the NANOG and OCT4 promoters bind AHR, are transcribed by RNA polymerase-III and repress NANOG and OCT4 in differentiated cells. OCT4 and NANOG repression likely involves processing of Alu-derived transcripts through the miRNA machinery involving the Microprocessor and RISC. Consistently, stable AHR knockdown led to basal undifferentiation, impaired Alus transcription and blockade of OCT4 and NANOG repression. We suggest that transcripts produced from AHR-regulated Alu retrotransposons may control the expression of stemness genes OCT4 and NANOG during differentiation of carcinoma cells. The control of discrete Alu elements by specific transcription factors may have a dynamic role in genome regulation under physiological and diseased conditions. PMID:26883630

  18. Alu retrotransposons promote differentiation of human carcinoma cells through the aryl hydrocarbon receptor.

    Science.gov (United States)

    Morales-Hernández, Antonio; González-Rico, Francisco J; Román, Angel C; Rico-Leo, Eva; Alvarez-Barrientos, Alberto; Sánchez, Laura; Macia, Ángela; Heras, Sara R; García-Pérez, José L; Merino, Jaime M; Fernández-Salguero, Pedro M

    2016-06-01

    Cell differentiation is a central process in development and in cancer growth and dissemination. OCT4 (POU5F1) and NANOG are essential for cell stemness and pluripotency; yet, the mechanisms that regulate their expression remain largely unknown. Repetitive elements account for almost half of the Human Genome; still, their role in gene regulation is poorly understood. Here, we show that the dioxin receptor (AHR) leads to differentiation of human carcinoma cells through the transcriptional upregulation of Alu retrotransposons, whose RNA transcripts can repress pluripotency genes. Despite the genome-wide presence of Alu elements, we provide evidences that those located at the NANOG and OCT4 promoters bind AHR, are transcribed by RNA polymerase-III and repress NANOG and OCT4 in differentiated cells. OCT4 and NANOG repression likely involves processing of Alu-derived transcripts through the miRNA machinery involving the Microprocessor and RISC. Consistently, stable AHR knockdown led to basal undifferentiation, impaired Alus transcription and blockade of OCT4 and NANOG repression. We suggest that transcripts produced from AHR-regulated Alu retrotransposons may control the expression of stemness genes OCT4 and NANOG during differentiation of carcinoma cells. The control of discrete Alu elements by specific transcription factors may have a dynamic role in genome regulation under physiological and diseased conditions. PMID:26883630

  19. A survey of PPR proteins identifies DYW domains like those of land plant RNA editing factors in diverse eukaryotes

    OpenAIRE

    Schallenberg-Rüdinger, Mareike; Lenz, Henning; Polsakiewicz, Monika; Gott, Jonatha M.; Knoop, Volker

    2013-01-01

    The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among th...

  20. The essential role of AMPA receptor GluR2 subunit RNA editing in the normal and diseased brain

    Directory of Open Access Journals (Sweden)

    Amanda Lorraine Wright

    2012-04-01

    Full Text Available AMPA receptors are comprised of different combinations of GluR1-GluR4 (also known as GluA1-GluA4 and GluR-A to GluR-D subunits. The GluR2 subunit is subject to Q/R site RNA editing by the ADAR2 enzyme, which converts a codon for glutamine (Q, present in the GluR2 gene, to a codon for arginine (R found in the mRNA. AMPA receptors are calcium (Ca2+-permeable if they contain the unedited GluR2(Q subunit or if they lack the GluR2 subunit. While most AMPA receptors in the brain contain the edited GluR2(R subunit and are therefore Ca2+-impermeable, recent evidence suggests that Ca2+-permeable GluR2-lacking AMPA receptors are important in synaptic plasticity and learning. However, the presence of Ca2+-permeable AMPA receptors containing unedited GluR2 leads to excitotoxic cell loss. Recent studies have indicated that RNA editing of GluR2 is deregulated in diseases, such as amyotrophic lateral sclerosis (ALS, as well in acute neurodegenerative conditions, such as ischemia. More recently, studies have investigated the regulation of RNA editing and possible causes for its deregulation during disease. In this review, we will explore the role of GluR2 RNA editing in the healthy and diseased brain and outline new insights into the mechanisms that control this process.

  1. A survey of PPR proteins identifies DYW domains like those of land plant RNA editing factors in diverse eukaryotes.

    Science.gov (United States)

    Schallenberg-Rüdinger, Mareike; Lenz, Henning; Polsakiewicz, Monika; Gott, Jonatha M; Knoop, Volker

    2013-01-01

    The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among those carboxyterminal domain additions to plant PPR proteins, the so-called DYW domain is particularly interesting given its similarity to cytidine deaminases. The frequency of organelle C-to-U RNA editing and the diversity of DYW-type PPR proteins correlate well in plants and both were recently identified outside of land plants, in the protist Naegleria gruberi. Here we present a systematic survey of PPR protein genes and report on the identification of additional DYW-type PPR proteins in the protists Acanthamoeba castellanii, Malawimonas jakobiformis, and Physarum polycephalum. Moreover, DYW domains were also found in basal branches of multi-cellular lineages outside of land plants, including the alga Nitella flexilis and the rotifers Adineta ricciae and Philodina roseola. Intriguingly, the well-characterized and curious patterns of mitochondrial RNA editing in the slime mold Physarum also include examples of C-to-U changes. Finally, we identify candidate sites for mitochondrial RNA editing in Malawimonas, further supporting a link between DYW-type PPR proteins and C-to-U editing, which may have remained hitherto unnoticed in additional eukaryote lineages. PMID:23899506

  2. Region-specific alterations of A-to-I RNA editing of serotonin 2c receptor in the cortex of suicides with major depression.

    Science.gov (United States)

    Weissmann, D; van der Laan, S; Underwood, M D; Salvetat, N; Cavarec, L; Vincent, L; Molina, F; Mann, J J; Arango, V; Pujol, J F

    2016-01-01

    Brain region-specific abnormalities in serotonergic transmission appear to underlie suicidal behavior. Alterations of RNA editing on the serotonin receptor 2C (HTR2C) pre-mRNA in the brain of suicides produce transcripts that attenuate 5-HT2CR signaling by impairing intracellular G-protein coupling and subsequent intracellular signal transduction. In brain, the distribution of RNA-editing enzymes catalyzing deamination (A-to-I modification) shows regional variation, including within the cerebral cortex. We tested the hypothesis that altered pre-mRNA 5-HT2CR receptor editing in suicide is region-specific. To this end, we investigated the complete 5-HT2CR mRNA-editing profile in two architectonically distinct cortical areas involved in mood regulation and decision-making in a clinically well-characterized cohort of age- and sex-matched non-psychiatric drug-free controls and depressed suicides. By using an original biochemical detection method, that is, capillary electrophoresis single-stranded conformational polymorphism (CE-SSCP), we corroborated the 5-HT2CR mRNA-editing profile previously described in the dorsolateral prefrontal cortex (Brodmann area 9 (BA9)). Editing of 5-HT2CR mRNA displayed clear regional difference when comparing dorsolateral prefrontal cortex (BA9) and anterior cingulate cortex (BA24). Compared with non-psychiatric control individuals, alterations of editing levels of 5-HT2CR mRNA were detected in both cortical areas of depressed suicides. A marked increase in editing on 5-HT2CR was especially observed in the anterior cingulate cortex in suicides, implicating this cortical area in suicide risk. The results suggest that region-specific changes in RNA editing of 5-HT2CR mRNA and deficient receptor function likely contribute to the etiology of major depressive disorder or suicide. PMID:27576167

  3. The association of Alu repeats with the generation of potential AU-rich elements (ARE at 3' untranslated regions.

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    Bhak Jonghwa

    2004-12-01

    Full Text Available Abstract Background A significant portion (about 8% in the human genome of mammalian mRNA sequences contains AU (Adenine and Uracil rich elements or AREs at their 3' untranslated regions (UTR. These mRNA sequences are usually stable. However, an increasing number of observations have been made of unstable species, possibly depending on certain elements such as Alu repeats. ARE motifs are repeats of the tetramer AUUU and a monomer A at the end of the repeats ((AUUUnA. The importance of AREs in biology is that they make certain mRNA unstable. Proto-oncogene, such as c-fos, c-myc, and c-jun in humans, are associated with AREs. Although it has been known that the increased number of ARE motifs caused the decrease of the half-life of mRNA containing ARE repeats, the exact mechanism is as of yet unknown. We analyzed the occurrences of AREs and Alu and propose a possible mechanism for how human mRNA could acquire and keep AREs at its 3' UTR originating from Alu repeats. Results Interspersed in the human genome, Alu repeats occupy 5% of the 3' UTR of mRNA sequences. Alu has poly-adenine (poly-A regions at its end, which lead to poly-thymine (poly-T regions at the end of its complementary Alu. It has been found that AREs are present at the poly-T regions. From the 3' UTR of the NCBI's reference mRNA sequence database, we found nearly 40% (38.5% of ARE (Class I were associated with Alu sequences (Table 1 within one mismatch allowance in ARE sequences. Other ARE classes had statistically significant associations as well. This is far from a random occurrence given their limited quantity. At each ARE class, random distribution was simulated 1,000 times, and it was shown that there is a special relationship between ARE patterns and the Alu repeats. Table 1 Defined ARE classes. (Symbol marks are used in this study instead of full sequences. Symbol ARE sequence Class I (AUUU5A AUUUAUUUAUUUAUUUAUUUA Class II (AUUU4A AUUUAUUUAUUUAUUUA Class III U(AUUU3AU

  4. Striking differences in RNA editing requirements to express the rps4 gene in magnolia and sunflower mitochondria.

    Science.gov (United States)

    Regina, Teresa M R; Lopez, Loredana; Picardi, Ernesto; Quagliariello, Carla

    2002-03-01

    The ribosomal protein S4 gene (rps4) has been identified as a single copy sequence in the mitochondrial genomes of two distant higher plants, Magnolia and Helianthus. Sequence analysis revealed that the rps4 genes present in the magnolia and sunflower mitochondrial genomes encode S4 polypeptides of 352 and 331 amino acids, respectively, longer than their counterparts in liverwort and bacteria. Expression of the rps4 genes in the investigated higher plant mitochondria was confirmed by Western blot analysis. In Helianthus, one of two short nucleotide insertions at the 3'-end introduces in the coding region a premature termination codon. Northern hybridizations and reverse transcription-polymerase chain reaction analysis demonstrated that the monocistronic RNA transcripts generated from the rps4 locus in Magnolia and Helianthus mitochondria are modified by RNA editing at 28 and 13 positions, respectively. Although evolutionarily conserved, RNA editing requirements of the rps4 appear more extensive in Magnolia than in Helianthus and in the other higher plants so far investigated. Furthermore, our analysis also suggests that selection of editing sites is RNA sequence-specific in a duplicated sequence context. PMID:11943458

  5. Germline Chromothripsis Driven by L1-Mediated Retrotransposition and Alu/Alu Homologous Recombination

    DEFF Research Database (Denmark)

    Nazaryan-Petersen, Lusine; Bertelsen, Birgitte; Bak, Mads;

    2016-01-01

    L1-endonuclease potential target sites in other breakpoints. In addition, we found four Alu elements flanking the 110-kb deletion and associated with an inversion. We suggest that chromatin looping mediated by homologous Alu elements may have brought distal DNA regions into close proximity...

  6. Alterations of RNA Editing for the Mitochondrial ATP9 Gene in a New orf220-type Cytoplasmic Male-sterile Line of Stem Mustard (Brassica juncea var. tumida)

    Institute of Scientific and Technical Information of China (English)

    Jing-Hua Yang; Ming-Fang Zhang; Jing-Quan Yu

    2007-01-01

    RNA editing for the mitochondrial ATP9 gene of encoding regions has been observed in both cytoplasmic malesterile and maintainer lines of stem mustard, where its editing capacity varied spatially and temporally in the cytoplasmic male sterility (CMS) line. There were four RNA editing sites for the mitochondrial ATP9 gene according to its normal editing sites in mustard, of which three sites occurred as C-to-U changes and one as a U-to-C change.As a result, the hydrophobicity of deduced ATP9 protein was reduced due to the conversions at its 17th, 45th and 64th positions. Meanwhile, the conservation of deduced ATP9 protein was enhanced by changes at the 56th position.Loss of a specific editing site for ATP9 was observed in juvenile roots, senile roots, senile leaves and floret buds of the CMS line. Comparatively, complete RNA editing for ATP9 gene was retained in juvenile roots, juvenile leaves and floret buds of its maintainer line; however, the loss of a specific editing site for ATP9 gene occurred at senile roots and senile leaves in its maintainer line. These observations allow us to produce a hypothesis that the dysfunction of a specific mitochondrisl gene arising from RNA editing could probably be a factor triggering CMS and organ senescence through unknown cross-talk pathways during development.

  7. Alu Elements as Novel Regulators of Gene Expression in Type 1 Diabetes Susceptibility Genes?

    DEFF Research Database (Denmark)

    Kaur, Simranjeet; Pociot, Flemming

    2015-01-01

    Despite numerous studies implicating Alu repeat elements in various diseases, there is sparse information available with respect to the potential functional and biological roles of the repeat elements in Type 1 diabetes (T1D). Therefore, we performed a genome-wide sequence analysis of T1D candidate...... genes to identify embedded Alu elements within these genes. We observed significant enrichment of Alu elements within the T1D genes (p-value Alus revealed significant enrichment for immune......-mediated processes (p-value Alus (IRAlus) within their 3' untranslated regions (UTRs) that are known to regulate the expression of host mRNAs by generating double stranded RNA duplexes. Our in silico analysis predicted the formation of duplex structures...

  8. A distant cis acting intronic element induces site-selective RNA editing

    DEFF Research Database (Denmark)

    Daniel, Chammiran; Venø, Morten Trillingsgaard; Ekdahl, Ylva;

    2012-01-01

    Transcripts have been found to be site selectively edited from adenosine-to-inosine (A-to-I) in the mammalian brain, mostly in genes involved in neurotransmission. While A-to-I editing occurs at double-stranded structures, other structural requirements are largely unknown. We have investigated th...

  9. Altered mRNA editing and expression of ionotropic glutamate receptors after kainic acid exposure in cyclooxygenase-2 deficient mice.

    Directory of Open Access Journals (Sweden)

    Luca Caracciolo

    Full Text Available Kainic acid (KA binds to the AMPA/KA receptors and induces seizures that result in inflammation, oxidative damage and neuronal death. We previously showed that cyclooxygenase-2 deficient (COX-2(-/- mice are more vulnerable to KA-induced excitotoxicity. Here, we investigated whether the increased susceptibility of COX-2(-/- mice to KA is associated with altered mRNA expression and editing of glutamate receptors. The expression of AMPA GluR2, GluR3 and KA GluR6 was increased in vehicle-injected COX-2(-/- mice compared to wild type (WT mice in hippocampus and cortex, whereas gene expression of NMDA receptors was decreased. KA treatment decreased the expression of AMPA, KA and NMDA receptors in the hippocampus, with a significant effect in COX-2(-/- mice. Furthermore, we analyzed RNA editing levels and found that the level of GluR3 R/G editing site was selectively increased in the hippocampus and decreased in the cortex in COX-2(-/- compared with WT mice. After KA, GluR4 R/G editing site, flip form, was increased in the hippocampus of COX-2(-/- mice. Treatment of WT mice with the COX-2 inhibitor celecoxib for two weeks decreased the expression of AMPA/KA and NMDAR subunits after KA, as observed in COX-2(-/- mice. After KA exposure, COX-2(-/- mice showed increased mRNA expression of markers of inflammation and oxidative stress, such as cytokines (TNF-α, IL-1β and IL-6, inducible nitric oxide synthase (iNOS, microglia (CD11b and astrocyte (GFAP. Thus, COX-2 gene deletion can exacerbate the inflammatory response to KA. We suggest that COX-2 plays a role in attenuating glutamate excitotoxicity by modulating RNA editing of AMPA/KA and mRNA expression of all ionotropic glutamate receptor subunits and, in turn, neuronal excitability. These changes may contribute to the increased vulnerability of COX-2(-/- mice to KA. The overstimulation of glutamate receptors as a consequence of COX-2 gene deletion suggests a functional coupling between COX-2 and the

  10. Crystallization and X-ray diffraction analysis of the Trp/amber editing site of hepatitis delta virus (+)RNA: a case of rational design

    International Nuclear Information System (INIS)

    Well diffracting decamer crystals of the hepatitis delta virus RNA-editing site were prepared, but exhibited merohedral twinning and base averaging owing to duplex symmetry. A longer asymmetric construct that includes additional flanking RNA sequences has been crystallized that does not appear to exhibit these defects. RNA editing by mammalian ADAR1 (Adenosine Deaminase Acting on RNA) is required for the life cycle of the hepatitis delta virus (HDV). Editing extends the single viral open reading frame to yield two protein products of alternate length. ADARs are believed to recognize double-stranded RNA substrates via a ‘structure-based’ readout mechanism. Crystals of 10-mer duplexes representing the HDV RNA-editing site diffracted to 1.35 Å resolution, but suffered from merohedral twinning and averaging of the base registry. Expansion of the construct to include two flanking 3 × 1 internal loops yielded crystals in the primitive tetragonal space group P41212 or P43212. X-ray diffraction data were collected to 2.8 Å resolution, revealing a unit cell with parameters a = 62.5, c = 63.5 Å. The crystallization and X-ray analysis of multiple forms of the HDV RNA-editing substrate, encounters with common RNA crystal-growth defects and a strategy to overcome these problems are reported

  11. Crystallization and X-ray diffraction analysis of the Trp/amber editing site of hepatitis delta virus (+)RNA: a case of rational design

    Energy Technology Data Exchange (ETDEWEB)

    MacElrevey, Celeste; Wedekind, Joseph E., E-mail: joseph-wedekind@urmc.rochester.edu [Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 (United States)

    2005-12-01

    Well diffracting decamer crystals of the hepatitis delta virus RNA-editing site were prepared, but exhibited merohedral twinning and base averaging owing to duplex symmetry. A longer asymmetric construct that includes additional flanking RNA sequences has been crystallized that does not appear to exhibit these defects. RNA editing by mammalian ADAR1 (Adenosine Deaminase Acting on RNA) is required for the life cycle of the hepatitis delta virus (HDV). Editing extends the single viral open reading frame to yield two protein products of alternate length. ADARs are believed to recognize double-stranded RNA substrates via a ‘structure-based’ readout mechanism. Crystals of 10-mer duplexes representing the HDV RNA-editing site diffracted to 1.35 Å resolution, but suffered from merohedral twinning and averaging of the base registry. Expansion of the construct to include two flanking 3 × 1 internal loops yielded crystals in the primitive tetragonal space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2. X-ray diffraction data were collected to 2.8 Å resolution, revealing a unit cell with parameters a = 62.5, c = 63.5 Å. The crystallization and X-ray analysis of multiple forms of the HDV RNA-editing substrate, encounters with common RNA crystal-growth defects and a strategy to overcome these problems are reported.

  12. The Basques according to polymorphic Alu insertions.

    Science.gov (United States)

    de Pancorbo, M M; López-Martínez, M; Martínez-Bouzas, C; Castro, A; Fernández-Fernández, I; de Mayolo, G A; de Mayolo, A A; de Mayolo, P A; Rowold, D J; Herrera, R J

    2001-08-01

    Polymorphic Alu insertions provide a set of DNA markers of interest in human population genetics. Approximately 1000-2000 of these insertions have not reached fixation within the human genome. Each one of these polymorphic loci most probably resulted from a unique insertional event, and therefore all individuals possessing the insertion are related by descent not just state. In addition, the direction of mutational change is toward the gain of the Alu element at a particular locus. Therefore, the improved knowledge of both the ancestral state and the direction of mutational change greatly facilitates the analysis of population relationships. As a result, Alu insertion polymorphisms represent a significant tool for population genetic studies. In this study, polymorphic Alu insertions have been employed to ascertain phylogenetic relationships among Basque groups and worldwide populations. The Basques are considered to be a geographic isolate with a unique language and customs. They may be direct descendants of Cro-Magnon enclaves from the upper Paleolithic (38,000 to 10,000 years). The Basques are distributed among narrow valleys in northeastern Spain with little migration between them until recently. This characteristic may have had an effect on allelic frequency distributions. With the aim of studying this possible effect, we have analyzed six autosomal polymorphic Alu loci from four different sites within the Spanish Basque region in order to ascertain any genetic heterogeneity among the Basques. The results are consistent with a lack of homogeneity among these four autochthonous Basque groups. PMID:11511929

  13. Transcriptome, genetic editing, and microRNA divergence substantiate sympatric speciation of blind mole rat, Spalax.

    Science.gov (United States)

    Li, Kexin; Wang, Liuyang; Knisbacher, Binyamin A; Xu, Qinqin; Levanon, Erez Y; Wang, Huihua; Frenkel-Morgenstern, Milana; Tagore, Satabdi; Fang, Xiaodong; Bazak, Lily; Buchumenski, Ilana; Zhao, Yang; Lövy, Matěj; Li, Xiangfeng; Han, Lijuan; Frenkel, Zeev; Beiles, Avigdor; Cao, Yi Bin; Wang, Zhen Long; Nevo, Eviatar

    2016-07-01

    Incipient sympatric speciation in blind mole rat, Spalax galili, in Israel, caused by sharp ecological divergence of abutting chalk-basalt ecologies, has been proposed previously based on mitochondrial and whole-genome nuclear DNA. Here, we present new evidence, including transcriptome, DNA editing, microRNA, and codon usage, substantiating earlier evidence for adaptive divergence in the abutting chalk and basalt populations. Genetic divergence, based on the previous and new evidence, is ongoing despite restricted gene flow between the two populations. The principal component analysis, neighbor-joining tree, and genetic structure analysis of the transcriptome clearly show the clustered divergent two mole rat populations. Gene-expression level analysis indicates that the population transcriptome divergence is displayed not only by soil divergence but also by sex. Gene ontology enrichment of the differentially expressed genes from the two abutting soil populations highlights reproductive isolation. Alternative splicing variation of the two abutting soil populations displays two distinct splicing patterns. L-shaped FST distribution indicates that the two populations have undergone divergence with gene flow. Transcriptome divergent genes highlight neurogenetics and nutrition characterizing the chalk population, and energetics, metabolism, musculature, and sensory perception characterizing the abutting basalt population. Remarkably, microRNAs also display divergence between the two populations. The GC content is significantly higher in chalk than in basalt, and stress-response genes mostly prefer nonoptimal codons. The multiple lines of evidence of ecological-genomic and genetic divergence highlight that natural selection overrules the gene flow between the two abutting populations, substantiating the sharp ecological chalk-basalt divergence driving sympatric speciation.

  14. The mitochondrial PPR protein LOVASTATIN INSENSITIVE 1 plays regulatory roles in cytosolic and plastidial isoprenoid biosynthesis through RNA editing.

    Science.gov (United States)

    Tang, Jianwei; Kobayashi, Keiko; Suzuki, Masashi; Matsumoto, Shogo; Muranaka, Toshiya

    2010-02-01

    Unlike animals, plants synthesize isoprenoids via two pathways, the cytosolic mevalonate (MVA) pathway and the plastidial 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway. Little information is known about the mechanisms that regulate these complex biosynthetic networks over multiple organelles. To understand such regulatory mechanisms of the biosynthesis of isoprenoids in plants, we previously characterized the Arabidopsis mutant, lovastatin insensitive 1 (loi1), which is resistant to lovastatin and clomazone, specific inhibitors of the MVA and MEP pathways, respectively. LOI1 encodes a pentatricopeptide repeat (PPR) protein localized in mitochondria that is thought to have RNA binding ability and function in post-transcriptional regulation of mitochondrial gene expression. LOI1 belongs to the DYW subclass of PPR proteins, which is hypothesized to be correlated with RNA editing. As a result of analysis of RNA editing of mitochondrial genes in loi1, a defect in RNA editing of three genes, nad4, ccb203 and cox3, was identified in loi1. These genes are related to the respiratory chain. Wild type (WT) treated with some respiration inhibitors mimicked the loi1 phenotype. Interestingly, HMG-CoA reductase activity of WT treated with lovastatin combined with antimycin A, an inhibitor of complex III in the respiratory chain, was higher than that of WT treated with only lovastatin, despite the lack of alteration of transcript or protein levels of HMGR. These results suggest that HMGR enzyme activity is regulated through the respiratory cytochrome pathway. Although various mechanisms exist for isoprenoid biosynthesis, our studies demonstrate the novel possibility that mitochondrial respiration plays potentially regulatory roles in isoprenoid biosynthesis.

  15. EdiPy: a resource to simulate the evolution of plant mitochondrial genes under the RNA editing.

    Science.gov (United States)

    Picardi, Ernesto; Quagliariello, Carla

    2006-02-01

    EdiPy is an online resource appropriately designed to simulate the evolution of plant mitochondrial genes in a biologically realistic fashion. EdiPy takes into account the presence of sites subjected to RNA editing and provides multiple artificial alignments corresponding to both genomic and cDNA sequences. Each artificial data set can successively be submitted to main and widespread evolutionary and phylogenetic software packages such as PAUP, Phyml, PAML and Phylip. As an online bioinformatic resource, EdiPy is available at the following web page: http://biologia.unical.it/py_script/index.html.

  16. The DYW Subgroup PPR Protein MEF35 Targets RNA Editing Sites in the Mitochondrial rpl16, nad4 and cob mRNAs in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Nadja Brehme

    Full Text Available RNA editing in plant mitochondria and plastids alters specific nucleotides from cytidine (C to uridine (U mostly in mRNAs. A number of PLS-class PPR proteins have been characterized as RNA recognition factors for specific RNA editing sites, all containing a C-terminal extension, the E domain, and some an additional DYW domain, named after the characteristic C-terminal amino acid triplet of this domain. Presently the recognition factors for more than 300 mitochondrial editing sites are still unidentified. In order to characterize these missing factors, the recently proposed computational prediction tool could be of use to assign target RNA editing sites to PPR proteins of yet unknown function. Using this target prediction approach we identified the nuclear gene MEF35 (Mitochondrial Editing Factor 35 to be required for RNA editing at three sites in mitochondria of Arabidopsis thaliana. The MEF35 protein contains eleven PPR repeats and E and DYW extensions at the C-terminus. Two T-DNA insertion mutants, one inserted just upstream and the other inside the reading frame encoding the DYW domain, show loss of editing at a site in each of the mRNAs for protein 16 in the large ribosomal subunit (site rpl16-209, for cytochrome b (cob-286 and for subunit 4 of complex I (nad4-1373, respectively. Editing is restored upon introduction of the wild type MEF35 gene in the reading frame mutant. The MEF35 protein interacts in Y2H assays with the mitochondrial MORF1 and MORF8 proteins, mutation of the latter also influences editing at two of the three MEF35 target sites. Homozygous mutant plants develop indistinguishably from wild type plants, although the RPL16 and COB/CYTB proteins are essential and the amino acids encoded after the editing events are conserved in most plant species. These results demonstrate the feasibility of the computational target prediction to screen for target RNA editing sites of E domain containing PLS-class PPR proteins.

  17. Research Progress on Relationship of PPR Proteins with RNA Editing%PPR蛋白参与RNA编辑机制的研究进展

    Institute of Scientific and Technical Information of China (English)

    何鹏; 陈海燕; 俞嘉宁

    2013-01-01

    RNA editing is a post-transcription phenomenon of gene processing and modification at specific nucleotide sites,which including nucleotide insertion/deletion or nucleotide conversion.RNA editing mainly occurs at genomic of mitochondria and chloroplast in higher plants.It has many important biology functions,but the mechanism of this phenomenon is still unclear.During recently years,the PPR proteins,might be as trans-acting factor of RNA editing,has been put forward and become a research focus in molecular biology.In this review,we introduced PPR proteins,RNA editing and possibility interaction pattern between PPR proteins and RNA editing.%RNA编辑是一种发生在转录后核苷酸特异位点的加工修饰现象,包括核苷酸的插入、删除和改变.高等植物中RNA编辑主要发生在线粒体与叶绿体中,具有重要的生物学功能,其机制仍在探索中.而PPR蛋白作为RNA编辑的反式作用因子,成为近几年来分子生物学的研究热点.该文就PPR蛋白、RNA编辑及PPR蛋白参与RNA编辑的机制等进行了综述.

  18. Elucidation of the RNA Recognition Code for Pentatricopeptide Repeat Proteins Involved in Organelle RNA Editing in Plants

    OpenAIRE

    Yagi, Yusuke; Hayashi, Shimpei; Kobayashi, Keiko; Hirayama, Takashi; Nakamura, Takahiro

    2013-01-01

    Pentatricopeptide repeat (PPR) proteins are eukaryotic RNA-binding proteins that are commonly found in plants. Organelle transcript processing and stability are mediated by PPR proteins in a gene-specific manner through recognition by tandem arrays of degenerate 35-amino-acid repeating units, the PPR motifs. However, the sequence-specific RNA recognition mechanism of the PPR protein remains largely unknown. Here, we show the principle underlying RNA recognition for PPR proteins involved in RN...

  19. Multifunctional G-rich and RRM-containing domains of TbRGG2 perform separate yet essential functions in trypanosome RNA editing.

    Science.gov (United States)

    Foda, Bardees M; Downey, Kurtis M; Fisk, John C; Read, Laurie K

    2012-09-01

    Efficient editing of Trypanosoma brucei mitochondrial RNAs involves the actions of multiple accessory factors. T. brucei RGG2 (TbRGG2) is an essential protein crucial for initiation and 3'-to-5' progression of editing. TbRGG2 comprises an N-terminal G-rich region containing GWG and RG repeats and a C-terminal RNA recognition motif (RRM)-containing domain. Here, we perform in vitro and in vivo separation-of-function studies to interrogate the mechanism of TbRGG2 action in RNA editing. TbRGG2 preferentially binds preedited mRNA in vitro with high affinity attributable to its G-rich region. RNA-annealing and -melting activities are separable, carried out primarily by the G-rich and RRM domains, respectively. In vivo, the G-rich domain partially complements TbRGG2 knockdown, but the RRM domain is also required. Notably, TbRGG2's RNA-melting activity is dispensable for RNA editing in vivo. Interactions between TbRGG2 and MRB1 complex proteins are mediated by both G-rich and RRM-containing domains, depending on the binding partner. Overall, our results are consistent with a model in which the high-affinity RNA binding and RNA-annealing activities of the G-rich domain are essential for RNA editing in vivo. The RRM domain may have key functions involving interactions with the MRB1 complex and/or regulation of the activities of the G-rich domain. PMID:22798390

  20. The mRNA expression of apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G in peripheral blood mononuclear cells in patients with chronic hepatitis C and its regulation by interferon-α

    Institute of Scientific and Technical Information of China (English)

    蔡卫平

    2013-01-01

    Objective To study the mRNA expression of apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G(APOBEC3G) in the peripheral blood mononuclear cells(PBMC) in patients with chronic hepatitis C(CHC) and its regulation by exogenous interferon-α

  1. Functional and structural insights revealed by molecular dynamics simulations of an essential RNA editing ligase in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Rommie E Amaro

    Full Text Available RNA editing ligase 1 (TbREL1 is required for the survival of both the insect and bloodstream forms of Trypanosoma brucei, the parasite responsible for the devastating tropical disease African sleeping sickness. The type of RNA editing that TbREL1 is involved in is unique to the trypanosomes, and no close human homolog is known to exist. In addition, the high-resolution crystal structure revealed several unique features of the active site, making this enzyme a promising target for structure-based drug design. In this work, two 20 ns atomistic molecular dynamics (MD simulations are employed to investigate the dynamics of TbREL1, both with and without the ATP substrate present. The flexibility of the active site, dynamics of conserved residues and crystallized water molecules, and the interactions between TbREL1 and the ATP substrate are investigated and discussed in the context of TbREL1's function. Differences in local and global motion upon ATP binding suggest that two peripheral loops, unique to the trypanosomes, may be involved in interdomain signaling events. Notably, a significant structural rearrangement of the enzyme's active site occurs during the apo simulations, opening an additional cavity adjacent to the ATP binding site that could be exploited in the development of effective inhibitors directed against this protozoan parasite. Finally, ensemble averaged electrostatics calculations over the MD simulations reveal a novel putative RNA binding site, a discovery that has previously eluded scientists. Ultimately, we use the insights gained through the MD simulations to make several predictions and recommendations, which we anticipate will help direct future experimental studies and structure-based drug discovery efforts against this vital enzyme.

  2. Behavioral Analysis of Different ALU Architectures

    Directory of Open Access Journals (Sweden)

    G.V.V.S.R.Krishna

    2012-06-01

    Full Text Available Digital design is an amazing and very broad field. The applications of digital design are present in our daily life, including Computers, calculators, video cameras etc. In fact, there will be always need for high speed and low power digital products which makes digital design a future growing business. Low power and high speed is challenging work in processor design. Implementing power optimization on all components of the processor is a choice. One of the most basicoperational units in theprocessor is an (Arithmetic logic unit ALU. ALU is a critical component of a microprocessor and is the core component of central processing unit. Furthermore, it is the heart of the instruction execution portion of every computer. It has the capability of performing a number of Arithmetic and logical operations such as addition, subtraction, complement, bit shifts and magnitude comparisons. Hence the speed, circuit delay, layout density, and power consumption trade-off is important for the portable digital system designers. This paper proposed to design and compare different parameters like low power, number of transistors and high speed of ALU’s .It compares the conventional CMOS ALU with Pseudo NMOS logic and MOS Logics. These different circuit parameters are compared with TANNER 13.1 using IBM SCN CMOS 65nm technology at room temperature. Results of comparison are shows that the Pseudo NMOS logic requires a lower number of transistors and performs well in terms of delay other than remaining architectures.

  3. Genome-wide tracking of unmethylated DNA Alu repeats in normal and cancer cells

    DEFF Research Database (Denmark)

    Rodriguez, Jairo; Vives, Laura; Jordà, Mireia;

    2008-01-01

    the highest prevalence of the SmaI site (AluY: 42%; AluS: 18%, AluJ: 5%) but the lower rates of unmethylation (AluY: 1.65%; AluS: 3.1%, AluJ: 12%). Data are consistent with a stronger silencing pressure on the youngest repetitive elements, which are closer to genes. Further insights into the functional...

  4. Effective Alu repeat based RT-Qpcr normalization in cancer cell perturbation experiments.

    Directory of Open Access Journals (Sweden)

    Ali Rihani

    Full Text Available BACKGROUND: Measuring messenger RNA (mRNA levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations. These manipulations may result in dramatic alterations in gene expression levels, even of assumed reference genes. In this study, we evaluated the expression levels of 11 commonly used reference genes as internal controls for normalization of 19 experiments that include neuroblastoma, T-ALL, melanoma, breast cancer, non small cell lung cancer (NSCL, acute myeloid leukemia (AML, prostate cancer, colorectal cancer, and cervical cancer cell lines subjected to various perturbations. RESULTS: The geNorm algorithm in the software package qbase+ was used to rank the candidate reference genes according to their expression stability. We observed that the stability of most of the candidate reference genes varies greatly in perturbation experiments. Expressed Alu repeats show relatively stable expression regardless of experimental condition. These Alu repeats are ranked among the best reference assays in all perturbation experiments and display acceptable average expression stability values (M<0.5. CONCLUSIONS: We propose the use of Alu repeats as a reference assay when performing cancer cell perturbation experiments.

  5. Alu repeats as markers for forensic DNA analyses

    Energy Technology Data Exchange (ETDEWEB)

    Batzer, M.A.; Alegria-Hartman, M. [Lawrence Livermore National Lab., CA (United States); Kass, D.H. [Louisiana State Univ., New Orleans, LA (United States)] [and others

    1994-01-01

    The Human-Specific (HS) subfamily of Alu sequences is comprised of a group of 500 nearly identical members which are almost exclusively restricted to the human genome. Individual subfamily members share an average of 98.9% nucleotide identity with the HS subfamily consensus sequence, and have an average age of 2.8 million years. We have developed a Polymerase Chain Reaction (PCR) based assay using primers complementary to the 5 inch and 3 inch unique flanking DNA sequences from each HS Alu that allow the locus to be assayed for the presence or absence of the Alu repeat. The dimorphic HS Alu sequences probably inserted in the human genome after the radiation of modem humans (within the last 200,000-one million years) and represent a unique source of information for human population genetics and forensic DNA analyses. These sites can be developed into Dimorphic Alu Sequence Tagged Sites (DASTS) for the Human Genome Project. HS Alu family member insertions differ from other types of polymorphism (e.g. Variable Number of Tandem Repeat [VNTR] or Restriction Fragment Length Polymorphism [RFLP]) in that polymorphisms due to Alu insertions arise as a result of a unique event which has occurred only one time in the human population and spread through the population from that point. Therefore, individuals that share HS Alu repeats inherited these elements from a common ancestor. Most VNTR and RFLP polymorphisms may arise multiple times in parallel within a population.

  6. Oligophrenin-1 (OPHN1, a gene involved in X-linked intellectual disability, undergoes RNA editing and alternative splicing during human brain development.

    Directory of Open Access Journals (Sweden)

    Sabina Barresi

    Full Text Available Oligophrenin-1 (OPHN1 encodes for a Rho-GTPase-activating protein, important for dendritic morphogenesis and synaptic function. Mutations in this gene have been identified in patients with X-linked intellectual disability associated with cerebellar hypoplasia. ADAR enzymes are responsible for A-to-I RNA editing, an essential post-transcriptional RNA modification contributing to transcriptome and proteome diversification. Specifically, ADAR2 activity is essential for brain development and function. Herein, we show that the OPHN1 transcript undergoes post-transcriptional modifications such as A-to-I RNA editing and alternative splicing in human brain and other tissues. We found that OPHN1 editing is detectable already at the 18th week of gestation in human brain with a boost of editing at weeks 20 to 33, concomitantly with OPHN1 expression increase and the appearance of a novel OPHN1 splicing isoform. Our results demonstrate that multiple post-transcriptional events occur on OPHN1, a gene playing an important role in brain function and development.

  7. The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing.

    Science.gov (United States)

    de Solis, Christopher A; Ho, Anthony; Holehonnur, Roopashri; Ploski, Jonathan E

    2016-01-01

    The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox) and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV). Specifically, we developed an inducible gRNA (gRNAi) AAV vector that is designed to express the gRNA from a H1/TO promoter. This AAV vector is also designed to express the Tet repressor (TetR) to regulate the expression of the gRNAi in a Dox dependent manner. We show that H1/TO promoters of varying length and a U6/TO promoter can edit DNA with similar efficiency in vitro, in a Dox dependent manner. We also demonstrate that our inducible gRNAi vector can be used to edit the genomes of neurons in vivo within the mouse brain in a Dox dependent manner. Genome editing can be induced in vivo with this system by supplying animals Dox containing food for as little as 1 day. This system might be cross compatible with many existing S. pyogenes Cas9 systems (i.e., Cas9 mouse, CRISPRi, etc.), and therefore it likely can be used to render these systems inducible as well. PMID:27587996

  8. The development of a viral mediated CRISPR/Cas9 system with doxycycline dependent gRNA expression for inducible in vitro and in vivo genome editing.

    Directory of Open Access Journals (Sweden)

    Christopher A. de Solis

    2016-08-01

    Full Text Available The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV. Specifically, we developed an inducible gRNA (gRNAi AAV vector that is designed to express the gRNA from a H1/TO promoter. This AAV vector is also designed to express the Tet repressor (TetR to regulate the expression of the gRNAi in a Dox dependent manner. We show that H1/TO promoters of varying length and a U6/TO promoter can edit DNA with similar efficiency in vitro, in a Dox dependent manner. We also demonstrate that our inducible gRNAi vector can be used to edit the genomes of neurons in vivo within the mouse brain in a Dox dependent manner. Genome editing can be induced in vivo with this system by supplying animals Dox containing food for as little as one day. This system might be cross compatible with many existing S. pyogenes Cas9 systems (i.e. Cas9 mouse, CRISPRi, etc., and therefore it likely can be used to render these systems inducible as well.

  9. Alu repeats as markers for human population genetics

    Energy Technology Data Exchange (ETDEWEB)

    Batzer, M.A.; Alegria-Hartman, M. [Lawrence Livermore National Lab., CA (United States); Bazan, H. [Louisiana State Univ., New Orleans, LA (United States). Medical Center] [and others

    1993-09-01

    The Human-Specific (HS) subfamily of Alu sequences is comprised of a group of 500 nearly identical members which are almost exclusively restricted to the human genome. Individual subfamily members share an average of 97.9% nucleotide identity with each other and an average of 98.9% nucleotide identity with the HS subfamily consensus sequence. HS Alu family members are thought to be derived from a single source ``master`` gene, and have an average age of 2.8 million years. We have developed a Polymerase Chain Reaction (PCR) based assay using primers complementary to the 5 in. and 3 in. unique flanking DNA sequences from each HS Alu that allows the locus to be assayed for the presence or absence of an Alu repeat. Individual HS Alu sequences were found to be either monomorphic or dimorphic for the presence or absence of each repeat. The monomorphic HS Alu family members inserted in the human genome after the human/great ape divergence (which is thought to have occurred 4--6 million years ago), but before the radiation of modem man. The dimorphic HS Alu sequences inserted in the human genome after the radiation of modem man (within the last 200,000-one million years) and represent a unique source of information for human population genetics and forensic DNA analyses. These sites can be developed into Dimorphic Alu Sequence Tagged Sites (DASTS) for the Human Genome Project as well. HS Alu family member insertion dimorphism differs from other types of polymorphism (e.g. Variable Number of Tandem Repeat [VNTR] or Restriction Fragment Length Polymorphism [RFLP]) because individuals share HS Alu family member insertions based upon identity by descent from a common ancestor as a result of a single event which occurred one time within the human population. The VNTR and RFLP polymorphisms may arise multiple times within a population and are identical by state only.

  10. News Editing. Second Edition.

    Science.gov (United States)

    Westley, Bruce H.

    A revision of the first edition of "News Editing," this is a textbook for the newspaper editor. The duties of the editor are detailed, as are those of other newspaper employees. Among the basic editing skills the author includes suggestions for sentence structure, word usage, and vocabulary. Examples are given of editing for objectivity, handling…

  11. Genetic and epigenetic variations contributed by Alu retrotransposition

    Directory of Open Access Journals (Sweden)

    de Andrade Alexandre

    2011-12-01

    Full Text Available Abstract Background De novo retrotransposition of Alu elements has been recognized as a major driver for insertion polymorphisms in human populations. In this study, we exploited Alu-anchored bisulfite PCR libraries to identify evolutionarily recent Alu element insertions, and to investigate their genetic and epigenetic variation. Results A total of 327 putatively recent Alu insertions were identified, altogether represented by 1,762 sequence reads. Nearly all such de novo retrotransposition events (316/327 were novel. Forty-seven out of forty-nine randomly selected events, corresponding to nineteen genomic loci, were sequence-verified. Alu element insertions remained hemizygous in one or more individuals in sixteen of the nineteen genomic loci. The Alu elements were found to be enriched for young Alu families with characteristic sequence features, such as the presence of a longer poly(A tail. In addition, we documented the occurrence of a duplication of the AT-rich target site in their immediate flanking sequences, a hallmark of retrotransposition. Furthermore, we found the sequence motif (TT/AAAA that is recognized by the ORF2P protein encoded by LINE-1 in their 5'-flanking regions, consistent with the fact that Alu retrotransposition is facilitated by LINE-1 elements. While most of these Alu elements were heavily methylated, we identified an Alu localized 1.5 kb downstream of TOMM5 that exhibited a completely unmethylated left arm. Interestingly, we observed differential methylation of its immediate 5' and 3' flanking CpG dinucleotides, in concordance with the unmethylated and methylated statuses of its internal 5' and 3' sequences, respectively. Importantly, TOMM5's CpG island and the 3 Alu repeats and 1 MIR element localized upstream of this newly inserted Alu were also found to be unmethylated. Methylation analyses of two additional genomic loci revealed no methylation differences in CpG dinucleotides flanking the Alu insertion sites in

  12. Alu-Alu Recombination Underlying the First Large Genomic Deletion in GlcNAc-Phosphotransferase Alpha/Beta (GNPTAB) Gene in a MLII Alpha/Beta Patient

    DEFF Research Database (Denmark)

    Coutinho, F; da Silva Santos, L; Lacerda, L;

    2012-01-01

    to the identification of a 21 bp repetitive motif in introns 18 and 19. Further analysis revealed that both the 5' and 3' breakpoints were located within highly homologous Alu elements (Alu-Sz in intron 18 and Alu-Sq2, in intron 19), suggesting that this deletion has probably resulted from Alu-Alu unequal homologous......), and a third in which exon 19 was substituted by a pseudoexon inclusion consisting of a 62 bp fragment from intron 18 (p.Arg1145Serfs*16). Interestingly, this 62 bp fragment corresponds to the Alu-Sz element integrated in intron 18.This represents the first description of a large deletion identified...

  13. High conservation of a 5' element required for RNA editing of a C target in chloroplast psbE transcripts.

    Science.gov (United States)

    Hayes, Michael L; Hanson, Maureen R

    2008-09-01

    C-to-U editing modifies 30-40 distinct nucleotides within higher-plant chloroplast transcripts. Many C targets are located at the same position in homologous genes from different plants; these either could have emerged independently or could share a common origin. The 5' sequence GCCGUU, required for editing of C214 in tobacco psbE in vitro, is one of the few identified editing cis-elements. We investigated psbE sequences from many plant species to determine in what lineage(s) editing of psbE C214 emerged and whether the cis-element identified in tobacco is conserved in plants with a C214. The GCCGUU sequence is present at a high frequency in plants that carry a C214 in psbE. However, Sciadopitys verticillata (Pinophyta) edits C214 despite the presence of nucleotide differences compared to the conserved cis-element. The C214 site in psbE genes is represented in members of four branches of spermatophytes but not in gnetophytes, resulting in the parsimonious prediction that editing of psbE C214 was present in the ancestor of spermatophytes. Extracts from chloroplasts from a species that has a difference in the motif and lacks the C target are incapable of editing tobacco psbE C214 substrates, implying that the critical trans-acting protein factors were not retained without a C target. Because noncoding sequences are less constrained than coding regions, we analyzed sequences 5' to two C editing targets located within coding regions to search for possible editing-related conserved elements. Putative editing cis-elements were uncovered in the 5' UTRs near editing sites psbL C2 and ndhD C2.

  14. The use of dimorphic Alu insertions in human DNA fingerprinting

    Energy Technology Data Exchange (ETDEWEB)

    Novick, G.E.; Gonzalez, T.; Garrison, J.; Novick, C.C.; Herrera, R.J. [Florida International Univ., Miami, FL (United States). Dept. of Biological Sciences; Batzer, M.A. [Lawrence Livermore National Lab., CA (United States); Deininger, P.L. [Louisiana State Univ., New Orleans, LA (United States). Medical Center

    1992-12-04

    We have characterized certain Human Specific Alu Insertions as either dimorphic (TPA25, PV92, APO), sightly dimorphic (C2N4 and C4N4) or monomorphic (C3N1, C4N6, C4N2, C4N5, C4N8), based on studies of Caucasian, Asian, American Black and African Black populations. Our approach is based upon: (1) PCR amplification using primers directed to the sequences that flank the site of insertion of the different Alu elements studied; (2) gel electrophoresis and scoring according to the presence or absence of an Alu insertion in one or both homologous chromosomes; (3) allelic frequencies calculated and compared according to Hardy-Weinberg equilibrium. Our DNA fingerprinting procedure using PCR amplification of dimorphic Human Specific Alu insertions, is stable enough to be used not only as a tool for genetic mapping but also to characterize populations, study migrational patterns and track the inheritance of human genetic disorders.

  15. Origin and diversification of minisatellites derived from human Alu sequences.

    Science.gov (United States)

    Jurka, Jerzy; Gentles, Andrew J

    2006-01-01

    We analyze minisatellites derived from Alu fragments corresponding approximately to the first 44 bases of human Alu consensus sequences from different subfamilies. The origin of Alu-derived minisatellites appears to have been mediated by short flanking repeats, as first proposed by Haber and Louis [Haber, J.E., Louis, E.J., 1998. Minisatellite origins in yeast and humans. Genomics 48, 132-135.]. We also present evidence for base substitutions and deletions introduced to minisatellites by gene conversion with partially similar but unrelated flanking regions. Segments flanked by short direct repeats are relatively common in different regions of Alu and other repetitive sequences. Our analysis shows that they can be effectively used in comparative studies of the overall sequence context which may contribute to instability of DNA segments flanked by short direct repeats.

  16. Transcriptional Slippage and RNA Editing Increase the Diversity of Transcripts in Chloroplasts: Insight from Deep Sequencing of Vigna radiata Genome and Transcriptome.

    Directory of Open Access Journals (Sweden)

    Ching-Ping Lin

    Full Text Available We performed deep sequencing of the nuclear and organellar genomes of three mungbean genotypes: Vigna radiata ssp. sublobata TC1966, V. radiata var. radiata NM92 and the recombinant inbred line RIL59 derived from a cross between TC1966 and NM92. Moreover, we performed deep sequencing of the RIL59 transcriptome to investigate transcript variability. The mungbean chloroplast genome has a quadripartite structure including a pair of inverted repeats separated by two single copy regions. A total of 213 simple sequence repeats were identified in the chloroplast genomes of NM92 and RIL59; 78 single nucleotide variants and nine indels were discovered in comparing the chloroplast genomes of TC1966 and NM92. Analysis of the mungbean chloroplast transcriptome revealed mRNAs that were affected by transcriptional slippage and RNA editing. Transcriptional slippage frequency was positively correlated with the length of simple sequence repeats of the mungbean chloroplast genome (R2=0.9911. In total, 41 C-to-U editing sites were found in 23 chloroplast genes and in one intergenic spacer. No editing site that swapped U to C was found. A combination of bioinformatics and experimental methods revealed that the plastid-encoded RNA polymerase-transcribed genes psbF and ndhA are affected by transcriptional slippage in mungbean and in main lineages of land plants, including three dicots (Glycine max, Brassica rapa, and Nicotiana tabacum, two monocots (Oryza sativa and Zea mays, two gymnosperms (Pinus taeda and Ginkgo biloba and one moss (Physcomitrella patens. Transcript analysis of the rps2 gene showed that transcriptional slippage could affect transcripts at single sequence repeat regions with poly-A runs. It showed that transcriptional slippage together with incomplete RNA editing may cause sequence diversity of transcripts in chloroplasts of land plants.

  17. Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery.

    Science.gov (United States)

    Wang, Jianbin; DeClercq, Joshua J; Hayward, Samuel B; Li, Patrick Wai-Lun; Shivak, David A; Gregory, Philip D; Lee, Gary; Holmes, Michael C

    2016-02-18

    The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise in recent clinical trials. Nuclease-driven genome editing provides a method in which to precisely target genetic changes to further enhance T cell function in vivo. We describe the development of a highly efficient method to genome edit both primary human CD8 and CD4 T cells by homology-directed repair at a pre-defined site of the genome. Two different homology donor templates were evaluated, representing both minor gene editing events (restriction site insertion) to mimic gene correction, or the more significant insertion of a larger gene cassette. By combining zinc finger nuclease mRNA delivery with AAV6 delivery of a homologous donor we could gene correct 41% of CCR5 or 55% of PPP1R12C (AAVS1) alleles in CD8(+) T cells and gene targeting of a GFP transgene cassette in >40% of CD8(+) and CD4(+) T cells at both the CCR5 and AAVS1 safe harbor locus, potentially providing a robust genome editing tool for T cell-based immunotherapy. PMID:26527725

  18. The Arabidopsis thaliana RNA Editing FactorSLO2, which Affects the Mitochondrial ElectronTransport Chain, Participates in Multiple Stressand Hormone Resoonses

    Institute of Scientific and Technical Information of China (English)

    2014-01-01

    Recently, we reported that the novel mitochondrial RNA editing factor SLO2 is essential for mitochondrialelectron transport, and vital for plant growth through regulation of carbon and energy metabolism. Here, we show thatmutation in SL02 causes hypersensitivity to ABA and insensitivity to ethylene, suggesting a link with stress responses.Indeed, slo2 mutants are hypersensitive to salt and osmotic stress during the germination stage, while adult plantsshow increased drought and salt tolerance. Moreover, slo2 mutants are more susceptible to Botrytis cinerea infection.An increased expression of nuclear-encoded stress-responsive genes, as well as mitochondrial-encoded NAD genes ofcomplex I and genes of the alternative respiratory pathway, was observed in slo2 mutants, further enhanced by ABAtreatment. In addition, H202 accumulation and altered amino acid levels were recorded in slo2 mutants. We conclude thatSLO2 is required for plant sensitivity to ABA, ethylene, biotic, and abiotic stress. Although two stress-related RNA editingfactors were reported very recently, this study demonstrates a unique role of SLO2, and further supports a link betweenmitochondrial RNA editing events and stress response.

  19. A knowledgebase of the human Alu repetitive elements.

    Science.gov (United States)

    Mallona, Izaskun; Jordà, Mireia; Peinado, Miguel A

    2016-04-01

    Alu elements are the most abundant retrotransposons in the human genome with more than one million copies. Alu repeats have been reported to participate in multiple processes related with genome regulation and compartmentalization. Moreover, they have been involved in the facilitation of pathological mutations in many diseases, including cancer. The contribution of Alus and other repeats in genomic regulation is often overlooked because their study poses technical and analytical challenges hardly attainable with conventional strategies. Here we propose the integration of ontology-based semantic methods to query a knowledgebase for the human Alus. The knowledgebase for the human Alus leverages Sequence (SO) and Gene Ontologies (GO) and is devoted to address functional and genetic information in the genomic context of the Alus. For each Alu element, the closest gene and transcript are stored, as well their functional annotation according to GO, the state of the chromatin and the transcription factors binding sites inside the Alu. The model uses Web Ontology Language (OWL) and Semantic Web Rule Language (SWRL). As a case of use and to illustrate the utility of the tool, we have evaluated the epigenetic states of Alu repeats associated with gene promoters according to their transcriptional activity. The ontology is easily extendable, offering a scaffold for the inclusion of new experimental data. The RDF/XML formalization is freely available at http://aluontology.sourceforge.net/. PMID:26827622

  20. A knowledgebase of the human Alu repetitive elements.

    Science.gov (United States)

    Mallona, Izaskun; Jordà, Mireia; Peinado, Miguel A

    2016-04-01

    Alu elements are the most abundant retrotransposons in the human genome with more than one million copies. Alu repeats have been reported to participate in multiple processes related with genome regulation and compartmentalization. Moreover, they have been involved in the facilitation of pathological mutations in many diseases, including cancer. The contribution of Alus and other repeats in genomic regulation is often overlooked because their study poses technical and analytical challenges hardly attainable with conventional strategies. Here we propose the integration of ontology-based semantic methods to query a knowledgebase for the human Alus. The knowledgebase for the human Alus leverages Sequence (SO) and Gene Ontologies (GO) and is devoted to address functional and genetic information in the genomic context of the Alus. For each Alu element, the closest gene and transcript are stored, as well their functional annotation according to GO, the state of the chromatin and the transcription factors binding sites inside the Alu. The model uses Web Ontology Language (OWL) and Semantic Web Rule Language (SWRL). As a case of use and to illustrate the utility of the tool, we have evaluated the epigenetic states of Alu repeats associated with gene promoters according to their transcriptional activity. The ontology is easily extendable, offering a scaffold for the inclusion of new experimental data. The RDF/XML formalization is freely available at http://aluontology.sourceforge.net/.

  1. Inhibition of LINE-1 and Alu retrotransposition by exosomes encapsidating APOBEC3G and APOBEC3F.

    Science.gov (United States)

    Khatua, Atanu K; Taylor, Harry E; Hildreth, James E K; Popik, Waldemar

    2010-04-25

    Human cytidine deaminases, including APOBEC3G (A3G) and A3F, are part of a cellular defense system against retroviruses and retroelements including non-LTR retrotransposons LINE-1 (L1) and Alu. Expression of cellular A3 proteins is sufficient for inhibition of L1 and Alu retrotransposition, but the effect of A3 proteins transferred in exosomes on retroelement mobilization is unknown. Here, we demonstrate for the first time that exosomes secreted by CD4(+)H9 T cells and mature monocyte-derived dendritic cells encapsidate A3G and A3F and inhibit L1 and Alu retrotransposition. A3G is the major contributor to the inhibitory activity of exosomes, however, the contribution of A3F in H9 exosomes cannot be excluded. Additionally, we show that exosomes encapsidate mRNAs coding for A3 proteins. A3G mRNA, and less so A3F, was enriched in exosomes secreted by H9 cells. Exosomal A3G mRNA was functional in vitro. Whether exosomes inhibit retrotransposons in vivo requires further investigation.

  2. Structure of a putative trans-editing enzyme for prolyl-tRNA synthetase from Aeropyrum pernix K1 at 1.7 Å resolution

    Energy Technology Data Exchange (ETDEWEB)

    Murayama, Kazutaka; Kato-Murayama, Miyuki; Katsura, Kazushige; Uchikubo-Kamo, Tomomi; Yamaguchi-Hirafuji, Machiko; Kawazoe, Masahito; Akasaka, Ryogo; Hanawa-Suetsugu, Kyoko; Hori-Takemoto, Chie [RIKEN Genomic Sciences Center, Yokohama (Japan); Terada, Takaho [RIKEN Genomic Sciences Center, Yokohama (Japan); RIKEN Harima Institute at SPring-8, Hyogo (Japan); Shirouzu, Mikako [RIKEN Harima Institute at SPring-8, Hyogo (Japan); Yokoyama, Shigeyuki, E-mail: yokoyama@biochem.s.u-tokyo.ac.jp [RIKEN Genomic Sciences Center, Yokohama (Japan); RIKEN Harima Institute at SPring-8, Hyogo (Japan); Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo (Japan)

    2005-01-01

    The three-dimensional structure of the APE2540 protein from A. pernix K1 has been determined by the multiple anomalous dispersion method at 1.7 Å resolution. The structure includes two monomers in the asymmetric unit and shares structural similarity with the YbaK protein or cysteinyl-tRNA{sup Pro} deacylase from H. influenzae. The crystal structure of APE2540, the putative trans-editing enzyme ProX from Aeropyrum pernix K1, was determined in a high-throughput manner. The crystal belongs to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 47.4, b = 58.9, c = 53.6 Å, β = 106.8°. The structure was solved by the multiwavelength anomalous dispersion method at 1.7 Å and refined to an R factor of 16.8% (R{sub free} = 20.5%). The crystal structure includes two protein molecules in the asymmetric unit. Each monomer consists of eight β-strands and seven α-helices. A structure-homology search revealed similarity between the trans-editing enzyme YbaK (or cysteinyl-tRNA{sup Pro} deacylase) from Haemophilus influenzae (HI1434; 22% sequence identity) and putative ProX proteins from Caulobacter crescentus (16%) and Agrobacterium tumefaciens (21%)

  3. Recognition of tRNALeu by Aquifex aeolicus leucyl-tRNA synthetase during the aminoacylation and editing steps

    OpenAIRE

    Yao, Peng; Zhu, Bin; Jaeger, Sophie; Eriani, Gilbert; Wang, En-Duo

    2008-01-01

    Recognition of tRNA by the cognate aminoacyl-tRNA synthetase during translation is crucial to ensure the correct expression of the genetic code. To understand tRNALeu recognition sets and their evolution, the recognition of tRNALeu by the leucyl-tRNA synthetase (LeuRS) from the primitive hyperthermophilic bacterium Aquifex aeolicus was studied by RNA probing and mutagenesis. The results show that the base A73; the core structure of tRNA formed by the tertiary interactions U8–A14, G18–U55 and ...

  4. International distribution and age estimation of the Portuguese BRCA2 c.156_157insAlu founder mutation

    DEFF Research Database (Denmark)

    Peixoto, Ana; Santos, Catarina; Pinheiro, Manuela;

    2011-01-01

    The c.156_157insAlu BRCA2 mutation has so far only been reported in hereditary breast/ovarian cancer (HBOC) families of Portuguese origin. Since this mutation is not detectable using the commonly used screening methodologies and must be specifically sought, we screened for this rearrangement...... in a total of 5,443 suspected HBOC families from several countries. Whereas the c.156_157insAlu BRCA2 mutation was detected in 11 of 149 suspected HBOC families from Portugal, representing 37.9% of all deleterious mutations, in other countries it was detected only in one proband living in France and in four...... regarding the production of the BRCA2 full length RNA and the transcript lacking exon 3 in c.156_157insAlu BRCA2 mutation carriers and in controls. The cumulative incidence of breast cancer in carriers did not differ from that of other BRCA2 and BRCA1 pathogenic mutations. We recommend that all suspected...

  5. Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analyses of threonyl-tRNA synthetase editing domain from Aeropyrum pernix

    International Nuclear Information System (INIS)

    The editing domain of threonyl-tRNA synthetase from the archaeon Aeropyrum pernix has been overexpressed, purified and crystallized. The crystal diffracted to a resolution of 1.66 Å. The proofreading function of aminoacyl-tRNA synthetases is crucial in maintaining the fidelity of protein synthesis. Most archaeal threonyl-tRNA synthetases (ThrRSs) possess a unique proofreading domain unrelated to their eukaryotic/bacterial counterpart. The crystal structure of this domain from the archaeon Pyrococcus abysii in complex with its cognate and noncognate substrate analogues had given insights into its catalytic and discriminatory mechanisms. To probe further into the mechanistic and evolutionary aspects of this domain, work has been extended to another archaeon Aeropyrum pernix. The organism possesses two proteins corresponding to threonyl-tRNA synthetase, i.e. ThrRS1 and ThrRS2, encoded by two different genes, thrS1 and thrS2, respectively. ThrRS1 is responsible for aminoacylation and ThrRS2 for proofreading activity. Here the purification, crystallization and preliminary X-ray crystallographic investigation of the N-terminal proofreading domain of ThrRS2 from A. pernix is reported. The crystals belong to either the P41212 or P43212 space group and consist of one monomer per asymmetric unit

  6. Co-occurrence of TDP-43 mislocalization with reduced activity of an RNA editing enzyme, ADAR2, in aged mouse motor neurons.

    Science.gov (United States)

    Hideyama, Takuto; Teramoto, Sayaka; Hachiga, Kosuke; Yamashita, Takenari; Kwak, Shin

    2012-01-01

    TDP-43 pathology in spinal motor neurons is a neuropathological hallmark of sporadic amyotrophic lateral sclerosis (ALS) and has recently been shown to be closely associated with the downregulation of an RNA editing enzyme called adenosine deaminase acting on RNA 2 (ADAR2) in the motor neurons of sporadic ALS patients. Because TDP-43 pathology is found more frequently in the brains of elderly patients, we investigated the age-related changes in the TDP-43 localization and ADAR2 activity in mouse motor neurons. We found that ADAR2 was developmentally upregulated, and its mRNA expression level was progressively decreased in the spinal cords of aged mice. Motor neurons normally exhibit nuclear ADAR2 and TDP-43 immunoreactivity, whereas fast fatigable motor neurons in aged mice demonstrated a loss of ADAR2 and abnormal TDP-43 localization. Importantly, these motor neurons expressed significant amounts of the Q/R site-unedited AMPA receptor subunit 2 (GluA2) mRNA. Because expression of unedited GluA2 has been demonstrated as a lethality-causing molecular abnormality observed in the motor neurons, these results suggest that age-related decreases in ADAR2 activity play a mechanistic role in aging and serve as one of risk factors for ALS.

  7. Gene editing by co-transformation of TALEN and chimeric RNA/DNA oligonucleotides on the rice OsEPSPS gene and the inheritance of mutations.

    Directory of Open Access Journals (Sweden)

    Mugui Wang

    Full Text Available Although several site-specific nucleases (SSNs, such as zinc-finger nucleases (ZFNs, transcription activator-like effector nucleases (TALENs, and the clustered regularly interspaced short palindromic repeat (CRISPR/Cas, have emerged as powerful tools for targeted gene editing in many organisms, to date, gene targeting (GT in plants remains a formidable challenge. In the present study, we attempted to substitute a single base in situ on the rice OsEPSPS gene by co-transformation of TALEN with chimeric RNA/DNA oligonucleotides (COs, including different strand composition such as RNA/DNA (C1 or DNA/RNA (C2 but contained the same target base to be substituted. In contrast to zero GT event obtained by the co-transformation of TALEN with homologous recombination plasmid (HRP, we obtained one mutant showing target base substitution although accompanied by undesired deletion of 12 bases downstream the target site from the co-transformation of TALEN and C1. In addition to this typical event, we also obtained 16 mutants with different length of base deletions around the target site among 105 calli lines derived from transformation of TALEN alone (4/19 as well as co-transformation of TELAN with either HRP (5/30 or C1 (2/25 or C2 (5/31. Further analysis demonstrated that the homozygous gene-edited mutants without foreign gene insertion could be obtained in one generation. The induced mutations in transgenic generation were also capable to pass to the next generation stably. However, the genotypes of mutants did not segregate normally in T1 population, probably due to lethal mutations. Phenotypic assessments in T1 generation showed that the heterozygous plants with either one or three bases deletion on target sequence, called d1 and d3, were more sensitive to glyphosate and the heterozygous d1 plants had significantly lower seed-setting rate than wild-type.

  8. 基于转录组测序数据识别黑猩猩RNA编辑位点%Identification of RNA Editing Sites in Chimpanzee by Transcriptome-wide Sequencing Data

    Institute of Scientific and Technical Information of China (English)

    王端青; 何涛; 汪莉; 王玉民; 邵卫东

    2012-01-01

    RNA editing is a widespread post-transcriptional modification mechanism that alters genetic information at the RNA level by nucleotide insertions, deletions or substitutions, which can contribute to the diversification of the transcriptome and proteome. Although tens of thousands of A-to-I RNA editing events have been found in humans, there is limited knowledge of RNA editing in other nonhuman primates. For exploring the mechanism as well as potential functions of the RNA editing events in chimpanzee, we identified RNA editing sites based on chimpanzee RNA-Seq data here. By aligning between RNA-Seq data and chimpanzee genome sequences with TopHat software, all RNA-DNA mismatch sites were regarded as a candidate set. Low quality sites were filtered out by using both genome and transcriptome sequencing quality scores. The other filters containing uncertainty of sequencing at 3'-terminial positions, read coverage, SNP sites and estimated editing level were also applied on the candidate set. Statistical tests based on the Binomial distribution and Bonferroni multiple testing correction were performed on each candidate site to remove random errors between genome and transcriptome. Then, we detected tissue- and sex-specific RNA editing sites using bioinformatics approaches based on the Fisher's exact test and the Bonferroni multiple testing correction. The Two Sample Logo software was used to analyze the feature of the sequences surrounding the RNA editing site. A total of 8 334 RNA editing sites were identified in chimpanzee transcriptome and all 12 possible categories of discordances were observed. The top four distributions were A-to-G, U-to-C, G-to-A and C-to-U editing sites, which contained 1 995, 1 452, 1 293 and 1 101 sites, respectively. Forty-one editing sites alter amino acid residues, one of them creates a new stop codon which may shorten the KRT31 protein and affect its activity. Three editing sites damage the binding of microRNA potentially. Six hundred and

  9. Identification of human-specific AluS elements through comparative genomics.

    Science.gov (United States)

    Lee, Jae; Kim, Yun-Ji; Mun, Seyoung; Kim, Heui-Soo; Han, Kyudong

    2015-01-25

    Mobile elements are responsible for ~45% of the human genome. Among them is the Alu element, accounting for 10% of the human genome (>1.1million copies). Several studies of Alu elements have reported that they are frequently involved in human genetic diseases and genomic rearrangements. In this study, we investigated the AluS subfamily, which is a relatively old Alu subfamily and has the highest copy number in primate genomes. Previously, a set of 263 human-specific AluS insertions was identified in the human genome. To validate these, we compared each of the human-specific AluS loci with its pre-insertion site in other primate genomes, including chimpanzee, gorilla, and orangutan. We obtained 24 putative human-specific AluS candidates via the in silico analysis and manual inspection, and then tried to verify them using PCR amplification and DNA sequencing. Through the PCR product sequencing, we were able to detect two instances of near-parallel Alu insertions in nearby sites that led to computational false negatives. Finally, we computationally and experimentally verified 23 human-specific AluS elements. We reported three alternative Alu insertion events, which are accompanied by filler DNA and/or Alu retrotransposition mediated-deletion. Bisulfite sequencing was carried out to examine DNA methylation levels of human-specific AluS elements. The results showed that fixed AluS elements are hypermethylated compared with polymorphic elements, indicating a possible relation between DNA methylation and Alu fixation in the human genome. PMID:25447892

  10. Methylation status of individual CpG sites within Alu elements in the human genome and Alu hypomethylation in gastric carcinomas

    International Nuclear Information System (INIS)

    Alu methylation is correlated with the overall level of DNA methylation and recombination activity of the genome. However, the maintenance and methylation status of each CpG site within Alu elements (Alu) and its methylation status have not well characterized. This information is useful for understanding natural status of Alu in the genome and helpful for developing an optimal assay to quantify Alu hypomethylation. Bisulfite clone sequencing was carried out in 14 human gastric samples initially. A Cac8I COBRA-DHPLC assay was developed to detect methylated-Alu proportion in cell lines and 48 paired gastric carcinomas and 55 gastritis samples. DHPLC data were statistically interpreted using SPSS version 16.0. From the results of 427 Alu bisulfite clone sequences, we found that only 27.2% of CpG sites within Alu elements were preserved (4.6 of 17 analyzed CpGs, A ~ Q) and that 86.6% of remaining-CpGs were methylated. Deamination was the main reason for low preservation of methylation targets. A high correlation coefficient of methylation was observed between Alu clones and CpG site J (0.963), A (0.950), H (0.946), D (0.945). Comethylation of the sites H and J were used as an indicator of the proportion of methylated-Alu in a Cac8I COBRA-DHPLC assay. Validation studies showed that hypermethylation or hypomethylation of Alu elements in human cell lines could be detected sensitively by the assay after treatment with 5-aza-dC and M.SssI, respectively. The proportion of methylated-Alu copies in gastric carcinomas (3.01%) was significantly lower than that in the corresponding normal samples (3.19%) and gastritis biopsies (3.23%). Most Alu CpG sites are deaminated in the genome. 27% of Alu CpG sites represented in our amplification products. 87% of the remaining CpG sites are methylated. Alu hypomethylation in primary gastric carcinomas could be detected with the Cac8I COBRA-DHPLC assay quantitatively

  11. CRISPR/CAS9-Mediated Genome Editing of miRNA-155 Inhibits Proinflammatory Cytokine Production by RAW264.7 Cells

    Directory of Open Access Journals (Sweden)

    Weixia Jing

    2015-01-01

    Full Text Available MicroRNA 155 (miR-155 is a key proinflammatory regulator in clinical and experimental rheumatoid arthritis (RA. Here we generated a miR-155 genome knockout (GKO RAW264.7 macrophage cell line using the clustered regulatory interspaced short palindromic repeat (CRISPR/CRISPR-associated protein 9 (CAS9 technology. While upregulating the Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1, the miR-155 GKO line is severely impaired in producing proinflammatory cytokines but slightly increased in osteoclastogenesis upon treatment with receptor activator of nuclear factor-κB ligand (RANKL. Taken together, our results suggest that genome editing of miR-155 holds the potential as a therapeutic strategy in RA.

  12. 肿瘤中GLI1RNA编辑的下调及其潜在功能分析%Down-regulation of RNA editing of GLI1 in tumors and analysis of its potential function

    Institute of Scientific and Technical Information of China (English)

    张颖; 胡亚欧; 何涛; 侯小强; 汪莉; 张部昌; 王玉民

    2012-01-01

    Objective To detect the A-to-[ RNA editing in the coding region of cancer associated gene GUI, compare the difference in the editing level between tumor and normal cell lines , and to analyze the potential effect of this editing event in the hope of discovering the role of the RNA editing in the occurrence and progression of cancer . Methods The identification of the A-to-[ RNA editing event and the comparison of the editing level were carried out by comparing the cDNA sequences with their corresponding genomic templates based on PCR , RT-PCR and sequencing methods. The potential functional implications of RNA editing were analyzed by multiple bioinformatics tools . Results An A-to-I RNA editing site mapped to chr 12: 56150891 changed the arginine residue at position 701 of the GUI protein to a glutamine residue, which was detected in the human brain , breast, small intestine tissues and multiple cell lines. Compared with normal controls, the editing level of this site decreased in the hepatoma and breast cancer cell lines . By bioinformatics analysis, this editing event altered amino acid residue and was predicted to destroy putative exonic splicing enhancers ( ESE) and affect putative protein tertiary structure. Conclusion This A-to-I RNA editing event in the coding region of GUI occurs in many diverse human tissues and cell lines. Down^-egulation of RNA editing of GUI in hepatoma and breast cancer cell lines may be associated with the occurrence and progression of cancer .%目的 检测癌相关基因GLI1编码区发生的A-to-I RNA编辑,比较编辑水平在肿瘤与正常细胞系中的差异,并分析编辑事件的潜在影响,以期揭示其与癌症发生发展的关联.方法 通过PCR、RT-PCR及测序的方法检测GLI1编码区DNA和RNA序列上的差异,以此来识别该区域上的A-to-I RNA编辑事件和比较肿瘤与正常细胞系中编辑位点的编辑水平差异.使用多种生物信息学工具分析编辑事

  13. Marketingový mix firmy ALU KOLA CB

    OpenAIRE

    Urban, Karel

    2011-01-01

    This bachelor thesis is focused on a marketing mix practical application in my own company ALU KOLA CB. My company sells alloy wheels and tyres for personal cars. In a literary review are introduced and explained terms marketing, marketing mix and its parts - product, price, place and promotion. In a practical part of this thesis are these terms applied on my company. The end of this part contains results and improvement suggestions.

  14. Design, Analysis, Implementation and Synthesis of 16 bit Reversible ALU by using Xilinx 12.2

    Directory of Open Access Journals (Sweden)

    S.Anusha

    2014-04-01

    Full Text Available In the modern world, Arithmetic Logic Unit (ALU is one of the most crucial components of any system and is used in many appliances like calculators, cell phones, and computers and so on. An arithmetic logic unit is a multi-functional circuit that conditionally performs one of several possible functions on two operands A and B depending on control inputs. This paper proposes the design of programmable reversible logic gate structures, targeted for the ALU implementation and their use in the realization of an efficient reversible ALU. Reversible or information-lossless circuits have applications in digital signal processing, communication, computer graphics and cryptography. This ALU consists of thirteen operations, 5 arithmetic, 4 logical operations and 4 shifting operations. All the modules are being designed using the basic reversible gates. Using reversible logic gates instead of traditional logic AND/OR gates, a reversible ALU whose function is the same as traditional ALU is constructed. Comparing with the number of input bits and the discarded bits of the traditional ALU, the reversible ALU significantly reduce the use and loss of information bits. The proposed reversible 16-bit ALU reduces the information bits use and loss by reusing the logic information bits logically and realizes the goal of lowering power consumption of logic circuits. Programmable reversible logic gates are realized in Verilog by using XILINX 12.2. Key words:

  15. A comparison of 100 human genes using an alu element-based instability model.

    Directory of Open Access Journals (Sweden)

    George W Cook

    Full Text Available The human retrotransposon with the highest copy number is the Alu element. The human genome contains over one million Alu elements that collectively account for over ten percent of our DNA. Full-length Alu elements are randomly distributed throughout the genome in both forward and reverse orientations. However, full-length widely spaced Alu pairs having two Alus in the same (direct orientation are statistically more prevalent than Alu pairs having two Alus in the opposite (inverted orientation. The cause of this phenomenon is unknown. It has been hypothesized that this imbalance is the consequence of anomalous inverted Alu pair interactions. One proposed mechanism suggests that inverted Alu pairs can ectopically interact, exposing both ends of each Alu element making up the pair to a potential double-strand break, or "hit". This hypothesized "two-hit" (two double-strand breaks potential per Alu element was used to develop a model for comparing the relative instabilities of human genes. The model incorporates both 1 the two-hit double-strand break potential of Alu elements and 2 the probability of exon-damaging deletions extending from these double-strand breaks. This model was used to compare the relative instabilities of 50 deletion-prone cancer genes and 50 randomly selected genes from the human genome. The output of the Alu element-based genomic instability model developed here is shown to coincide with the observed instability of deletion-prone cancer genes. The 50 cancer genes are collectively estimated to be 58% more unstable than the randomly chosen genes using this model. Seven of the deletion-prone cancer genes, ATM, BRCA1, FANCA, FANCD2, MSH2, NCOR1 and PBRM1, were among the most unstable 10% of the 100 genes analyzed. This algorithm may lay the foundation for comparing genetic risks posed by structural variations that are unique to specific individuals, families and people groups.

  16. Analysis of the features and source gene composition of the AluYg6 subfamily of human retrotransposons

    Directory of Open Access Journals (Sweden)

    Brookfield John FY

    2007-07-01

    Full Text Available Abstract Background Alu elements are a family of SINE retrotransposons in primates. They are classified into subfamilies according to specific diagnostic mutations from the general Alu consensus. It is now believed that there may be several retrotranspositionally-competent source genes within an Alu subfamily. To investigate the evolution of young Alu elements it is critical to have access to complete subfamilies, which, following the release of the final human genome assembly, can now be obtained using in silico methods. Results 380 elements belonging to the young AluYg6 subfamily were identified in the human genome, a number significantly exceeding prior expectations. An AluYg6 element was also identified in the chimpanzee genome, indicating that the subfamily is older than previously estimated, and appears to have undergone a period of dormancy before its expansion. The relative contributions of back mutation and gene conversion to variation at the six diagnostic positions are examined, and cases of complete forward gene conversion events are reported. Two small subfamilies derived from AluYg6 have been identified, named AluYg6a2 and AluYg5b3, which contain 40 and 27 members, respectively. These small subfamilies are used to illustrate the ambiguity regarding Alu subfamily definition, and to assess the contribution of secondary source genes to the AluYg6 subfamily. Conclusion The number of elements in the AluYg6 subfamily greatly exceeds prior expectations, indicating that the current knowledge of young Alu subfamilies is incomplete, and that prior analyses that have been carried out using these data may have generated inaccurate results. A definition of primary and secondary source genes has been provided, and it has been shown that several source genes have contributed to the proliferation of the AluYg6 subfamily. Access to the sequence data for the complete AluYg6 subfamily will be invaluable in future computational analyses investigating

  17. To edit or not to edit: regulation of ADAR editing specificity and efficiency.

    Science.gov (United States)

    Deffit, Sarah N; Hundley, Heather A

    2016-01-01

    Hundreds to millions of adenosine (A)-to-inosine (I) modifications are present in eukaryotic transcriptomes and play an essential role in the creation of proteomic and phenotypic diversity. As adenosine and inosine have different base-pairing properties, the functional consequences of these modifications or 'edits' include altering coding potential, splicing, and miRNA-mediated gene silencing of transcripts. However, rather than serving as a static control of gene expression, A-to-I editing provides a means to dynamically rewire the genetic code during development and in a cell-type specific manner. Interestingly, during normal development, in specific cells, and in both neuropathological diseases and cancers, the extent of RNA editing does not directly correlate with levels of the substrate mRNA or the adenosine deaminase that act on RNA (ADAR) editing enzymes, implying that cellular factors are required for spatiotemporal regulation of A-to-I editing. The factors that affect the specificity and extent of ADAR activity have been thoroughly dissected in vitro. Yet, we still lack a complete understanding of how specific ADAR family members can selectively deaminate certain adenosines while others cannot. Additionally, in the cellular environment, ADAR specificity and editing efficiency is likely to be influenced by cellular factors, which is currently an area of intense investigation. Data from many groups have suggested two main mechanisms for controlling A-to-I editing in the cell: (1) regulating ADAR accessibility to target RNAs and (2) protein-protein interactions that directly alter ADAR enzymatic activity. Recent studies suggest cis- and trans-acting RNA elements, heterodimerization and RNA-binding proteins play important roles in regulating RNA editing levels in vivo. WIREs RNA 2016, 7:113-127. doi: 10.1002/wrna.1319. PMID:26612708

  18. Orangutan Alu quiescence reveals possible source element: support for ancient backseat drivers

    Directory of Open Access Journals (Sweden)

    Walker Jerilyn A

    2012-04-01

    Full Text Available Abstract Background Sequence analysis of the orangutan genome revealed that recent proliferative activity of Alu elements has been uncharacteristically quiescent in the Pongo (orangutan lineage, compared with all previously studied primate genomes. With relatively few young polymorphic insertions, the genomic landscape of the orangutan seemed like the ideal place to search for a driver, or source element, of Alu retrotransposition. Results Here we report the identification of a nearly pristine insertion possessing all the known putative hallmarks of a retrotranspositionally competent Alu element. It is located in an intronic sequence of the DGKB gene on chromosome 7 and is highly conserved in Hominidae (the great apes, but absent from Hylobatidae (gibbon and siamang. We provide evidence for the evolution of a lineage-specific subfamily of this shared Alu insertion in orangutans and possibly the lineage leading to humans. In the orangutan genome, this insertion contains three orangutan-specific diagnostic mutations which are characteristic of the youngest polymorphic Alu subfamily, AluYe5b5_Pongo. In the Homininae lineage (human, chimpanzee and gorilla, this insertion has acquired three different mutations which are also found in a single human-specific Alu insertion. Conclusions This seemingly stealth-like amplification, ongoing at a very low rate over millions of years of evolution, suggests that this shared insertion may represent an ancient backseat driver of Alu element expansion.

  19. Alu Insertions and Genetic Diversity: A Preliminary Investigation by an Undergraduate Bioinformatics Class

    Science.gov (United States)

    Elwess, Nancy L.; Duprey, Stephen L.; Harney, Lindesay A.; Langman, Jessie E.; Marino, Tara C.; Martinez, Carolina; McKeon, Lauren L.; Moss, Chantel I. E.; Myrie, Sasha S.; Taylor, Luke Ryan

    2008-01-01

    "Alu"-insertion polymorphisms were used by an undergraduate Bioinformatics class to study how these insertion sites could be the basis for an investigation in human population genetics. Based on the students' investigation, both allele and genotype "Alu" frequencies were determined for African-American and Japanese populations as well as a…

  20. Super-resolution imaging of fluorescently labeled, endogenous RNA Polymerase II in living cells with CRISPR/Cas9-mediated gene editing

    Science.gov (United States)

    Cho, Won-Ki; Jayanth, Namrata; Mullen, Susan; Tan, Tzer Han; Jung, Yoon J.; Cissé, Ibrahim I.

    2016-01-01

    Live cell imaging of mammalian RNA polymerase II (Pol II) has previously relied on random insertions of exogenous, mutant Pol II coupled with the degradation of endogenous Pol II using a toxin, α-amanitin. Therefore, it has been unclear whether over-expression of labeled Pol II under an exogenous promoter may have played a role in reported Pol II dynamics in vivo. Here we label the endogenous Pol II in mouse embryonic fibroblast (MEF) cells using the CRISPR/Cas9 gene editing system. Using single-molecule based super-resolution imaging in the living cells, we captured endogenous Pol II clusters. Consistent with previous studies, we observed that Pol II clusters were short-lived (cluster lifetime ~8 s) in living cells. Moreover, dynamic responses to serum-stimulation, and drug-mediated transcription inhibition were all in agreement with previous observations in the exogenous Pol II MEF cell line. Our findings suggest that previous exogenously tagged Pol II faithfully recapitulated the endogenous polymerase clustering dynamics in living cells, and our approach may in principle be used to directly label transcription factors for live cell imaging. PMID:27782203

  1. Design Approach for Fault Recoverable ALU with Improved Fault Tolerance

    Directory of Open Access Journals (Sweden)

    Ankit K V

    2015-08-01

    Full Text Available A new design for fault tolerant and fault recoverable ALU System has been proposed in this paper. Reliability is one of the most critical factors that have to be considered during the designing phase of any IC. In critical applications like Medical equipment & Military applications this reliability factor plays a very critical role in determining the acceptance of product. Insertion of special modules in the main design for reliability enhancement will give considerable amount of area & power penalty. So, a novel approach to this problem is to find ways for reusing the already available components in digital system in efficient way to implement recoverable methodologies. Triple Modular Redundancy (TMR has traditionally used for protecting digital logic from the SEUs (single event upset by triplicating the critical components of the system to give fault tolerance to system. ScTMR- Scan chain-based error recovery TMR technique provides recovery for all internal faults. ScTMR uses a roll-forward approach and employs the scan chain implemented in the circuits for testability purposes to recover the system to fault-free state. The proposed design will incorporate a ScTMR controller over TMR system of ALU and will make the system fault tolerant and fault recoverable. Hence, proposed design will be more efficient & reliable to use in critical applications, than any other design present till today.

  2. Effects of Alu elements on global nucleosome positioning in the human genome

    Directory of Open Access Journals (Sweden)

    Yamashita Riu

    2010-05-01

    Full Text Available Abstract Background Understanding the genome sequence-specific positioning of nucleosomes is essential to understand various cellular processes, such as transcriptional regulation and replication. As a typical example, the 10-bp periodicity of AA/TT and GC dinucleotides has been reported in several species, but it is still unclear whether this feature can be observed in the whole genomes of all eukaryotes. Results With Fourier analysis, we found that this is not the case: 84-bp and 167-bp periodicities are prevalent in primates. The 167-bp periodicity is intriguing because it is almost equal to the sum of the lengths of a nucleosomal unit and its linker region. After masking Alu elements, these periodicities were greatly diminished. Next, using two independent large-scale sets of nucleosome mapping data, we analyzed the distribution of nucleosomes in the vicinity of Alu elements and showed that (1 there are one or two fixed slot(s for nucleosome positioning within the Alu element and (2 the positioning of neighboring nucleosomes seems to be in phase, more or less, with the presence of Alu elements. Furthermore, (3 these effects of Alu elements on nucleosome positioning are consistent with inactivation of promoter activity in Alu elements. Conclusions Our discoveries suggest that the principle governing nucleosome positioning differs greatly across species and that the Alu family is an important factor in primate genomes.

  3. The genome editing revolution

    DEFF Research Database (Denmark)

    Stella, Stefano; Montoya, Guillermo

    2016-01-01

    the previously available DNA binding templates, zinc fingers and meganucleases. Recently, the area experimented a quantum leap because of the introduction of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (clustered regularly interspaced short palindromic...... sequence). This ribonucleoprotein complex protects bacteria from invading DNAs, and it was adapted to be used in genome editing. The CRISPR ribonucleic acid (RNA) molecule guides to the specific DNA site the Cas9 nuclease to cleave the DNA target. Two years and more than 1000 publications later, the CRISPR......-Cas system has become the main tool for genome editing in many laboratories. Currently the targeted genome editing technology has been used in many fields and may be a possible approach for human gene therapy. Furthermore, it can also be used to modifying the genomes of model organisms for studying human...

  4. Alu and LINE-1 hypomethylation is associated with HER2 enriched subtype of breast cancer.

    Science.gov (United States)

    Park, So Yeon; Seo, An Na; Jung, Hae Yoen; Gwak, Jae Moon; Jung, Namhee; Cho, Nam-Yun; Kang, Gyeong Hoon

    2014-01-01

    The changes in DNA methylation status in cancer cells are characterized by hypermethylation of promoter CpG islands and diffuse genomic hypomethylation. Alu and long interspersed nucleotide element-1 (LINE-1) are non-coding genomic repetitive sequences and methylation of these elements can be used as a surrogate marker for genome-wide methylation status. This study was designed to evaluate the changes of Alu and LINE-1 hypomethylation during breast cancer progression from normal to pre-invasive lesions and invasive breast cancer (IBC), and their relationship with characteristics of IBC. We analyzed the methylation status of Alu and LINE-1 in 145 cases of breast samples including normal breast tissue, atypical ductal hyperplasia/flat epithelial atypia (ADH/FEA), ductal carcinoma in situ (DCIS) and IBC, and another set of 129 cases of IBC by pyrosequencing. Alu methylation showed no significant changes during multistep progression of breast cancer, although it tended to decrease during the transition from DCIS to IBC. In contrast, LINE-1 methylation significantly decreased from normal to ADH/FEA, while it was similar in ADH/FEA, DCIS and IBC. In IBC, Alu hypomethylation correlated with negative estrogen receptor (ER) status, and LINE-1 hypomethylation was associated with negative ER status, ERBB2 (HER2) amplification and p53 overexpression. Alu and LINE-1 methylation status was significantly different between breast cancer subtypes, and the HER2 enriched subtype had lowest methylation levels. In survival analyses, low Alu methylation status tended to be associated with poor disease-free survival of the patients. Our findings suggest that LINE-1 hypomethylation is an early event and Alu hypomethylation is probably a late event during breast cancer progression, and prominent hypomethylation of Alu and LINE-1 in HER2 enriched subtype may be related to chromosomal instability of this specific subtype.

  5. Design of an Efficient ALU Using Low-Power Dual Mode Logic

    Directory of Open Access Journals (Sweden)

    K. Vinay Kumar

    2014-05-01

    Full Text Available The dual mode logic is an efficient model, which is starts working in between the static and dynamic mode of operations. Since both of the static and dynamic modes having some disadvantages like speed and power dissipations. In this paper we are going to implement a faster and efficient ALU using the DML mode of logic. A performance valuation of designed DML ALU is done with respect to the ordinary normal ALU. And we are implementing this on CADENCE Platform in 180 nm technology. And for a variation of length and width ratio’s (W/L how the design will work is going to be done. Key words -

  6. Learning Stochastic Tree Edit Distance

    OpenAIRE

    Bernard, Marc; Habrard, Amaury; Sebban, Marc

    2006-01-01

    Trees provide a suited structural representation to deal with complex tasks such as web information extraction, RNA secondary structure prediction, or conversion of tree structured documents. In this context, many applications require the calculation of similarities between tree pairs. The most studied distance is likely the tree edit distance for which improvements in terms of complexity have been achieved during the last decade. However, this classic edit distance usually uses a priori fixe...

  7. Comparative Analysis of ALU Implementation with RCA and Sklansky Adders In ASIC Design Flow

    Directory of Open Access Journals (Sweden)

    Abdul Rehman Buzdar

    2016-07-01

    Full Text Available An Arithmetic Logic Unit (ALU is the heart of every central processing unit (CPU which performs basic operations like addition, subtraction, multiplication, division and bitwise logic operations on binary numbers. This paper deals with implementation of a basic ALU unit using two different types of adder circuits, a ripple carry adder and a sklansky type adder. The ALU is designed using application specific integrated circuit (ASIC platform where VHDL hardware description language and standard cells are used. The target process technology is 130nm CMOS from the foundry ST Microelectronics. The Cadence EDA tools are used for the ASIC implementation. A comparative analysis is provided for the two ALU circuits designed in terms of area, power and timing requirements.

  8. Higher Alu methylation levels in catch-up growth in twenty-year-old offsprings.

    Directory of Open Access Journals (Sweden)

    Kittipan Rerkasem

    Full Text Available Alu elements and long interspersed element-1 (LINE-1 or L1 are two major human intersperse repetitive sequences. Lower Alu methylation, but not LINE-1, has been observed in blood cells of people in old age, and in menopausal women having lower bone mass and osteoporosis. Nevertheless, Alu methylation levels also vary among young individuals. Here, we explored phenotypes at birth that are associated with Alu methylation levels in young people. In 2010, 249 twenty-years-old volunteers whose mothers had participated in a study association between birth weight (BW and nutrition during pregnancy in 1990, were invited to take part in our present study. In this study, the LINE-1 and Alu methylation levels and patterns were measured in peripheral mononuclear cells and correlated with various nutritional parameters during intrauterine and postnatal period of offspring. This included the amount of maternal intake during pregnancy, the mother's weight gain during pregnancy, birth weight, birth length, and the rate of weight gain in the first year of life. Catch-up growth (CUG was defined when weight during the first year was >0.67 of the standard score, according to WHO data. No association with LINE-1 methylation was identified. The mean level of Alu methylation in the CUG group was significantly higher than those non-CUG (39.61% and 33.66 % respectively, P < 0.0001. The positive correlation between the history of CUG in the first year and higher Alu methylation indicates the role of Alu methylation, not only in aging cells, but also in the human growth process. Moreover, here is the first study that demonstrated the association between a phenotype during the newborn period and intersperse repetitive sequences methylation during young adulthood.

  9. Non-GMO genetically edited crop plants.

    Science.gov (United States)

    Kanchiswamy, Chidananda Nagamangala; Malnoy, Mickael; Velasco, Riccardo; Kim, Jin-Soo; Viola, Roberto

    2015-09-01

    Direct delivery of purified Cas9 protein with guide RNA into plant cells, as opposed to plasmid-mediated delivery, displays high efficiency and reduced off-target effects. Following regeneration from edited cells, the ensuing plant is also likely to bypass genetically modified organism (GMO) legislation as the genome editing complex is degraded in the recipient cells.

  10. Non-GMO genetically edited crop plants.

    Science.gov (United States)

    Kanchiswamy, Chidananda Nagamangala; Malnoy, Mickael; Velasco, Riccardo; Kim, Jin-Soo; Viola, Roberto

    2015-09-01

    Direct delivery of purified Cas9 protein with guide RNA into plant cells, as opposed to plasmid-mediated delivery, displays high efficiency and reduced off-target effects. Following regeneration from edited cells, the ensuing plant is also likely to bypass genetically modified organism (GMO) legislation as the genome editing complex is degraded in the recipient cells. PMID:25978870

  11. Cluster editing

    DEFF Research Database (Denmark)

    Böcker, S.; Baumbach, Jan

    2013-01-01

    The Cluster Editing problem asks to transform a graph into a disjoint union of cliques using a minimum number of edge modifications. Although the problem has been proven NP-complete several times, it has nevertheless attracted much research both from the theoretical and the applied side. The...... algorithms for biological problems. © 2013 Springer-Verlag....... problem has been the inspiration for numerous algorithms in bioinformatics, aiming at clustering entities such as genes, proteins, phenotypes, or patients. In this paper, we review exact and heuristic methods that have been proposed for the Cluster Editing problem, and also applications of these...

  12. Gated Clock Implementation of Arithmetic Logic Unit (ALU

    Directory of Open Access Journals (Sweden)

    Dr. Neelam R. Prakash

    2013-05-01

    Full Text Available Low power design has emerged as one of the challenging area in today’s ASIC (Application specific integrated circuit design. With continuous decrease in transistor size, power density is increasing and there is an urgent need for reduction in total power consumption. Clock gating is one most effective technique for low power synchronous circuit design. Clock gating technique in low power design is used to reduce the dynamic power consumption. Clock signal in a synchronous circuit is used for synchronization only and hence does not carry any important information. Since clock is applied to each block of a synchronous circuit, and clock switches for every cycle, clock power is the major part of dynamic power consumption in synchronous circuits. Clock gating is a well known technique to reduce clock power. In clock gating clock to an idle block is disabled. Thus significant amount of power consumption is reduced by employing clock gating. In this paper an ALU design is proposed employing Gated clock for its operation. Design simulation has been performed on Xilinx ISE design suite, and power calculation is done by Xilinx Xpower analyzer. Results show that approximately 17% of total clock power consumption is reduced by gated clock implementation.

  13. 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. PMID:27112566

  14. Multilinguals and Wikipedia Editing

    CERN Document Server

    Hale, Scott A

    2013-01-01

    This article analyzes one month of edits to Wikipedia in order to examine the role of users editing multiple language editions (referred to as multilingual users). Such multilingual users may serve an important function in diffusing information across different language editions of the project, and prior work has suggested this could reduce the level of self-focus bias in each edition. This study finds multilingual users are much more active than their single-edition (monolingual) counterparts. They are found in all language editions, but smaller-sized editions with fewer users have a higher percentage of multilingual users than larger-sized editions. About a quarter of multilingual users always edit the same articles in multiple languages, while just over 40% of multilingual users edit different articles in different languages. When non-English users do edit a second language edition, that edition is most frequently English. Nonetheless, several regional and linguistic cross-editing patterns are also present...

  15. A SINE in the genome of the cephalochordate amphioxus is an Alu element

    Directory of Open Access Journals (Sweden)

    2006-04-01

    Full Text Available Transposable elements of about 300 bp, termed “short interspersed nucleotide elements or SINEs are common in eukaryotes. However, Alu elements, SINEs containing restriction sites for the AluI enzyme, have been known only from primates. Here I report the first SINE found in the genome of the cephalochordate, amphioxus. It is an Alu element of 375 bp that does not share substantial identity with any genomic sequences in vertebrates. It was identified because it was located in the FoxD regulatory region in a cosmid derived from one individual, but absent from the two FoxD alleles of BACs from a second individual. However, searches of sequences of BACs and genomic traces from this second individual gave an estimate of 50-100 copies in the amphioxus genome. The finding of an Alu element in amphioxus raises the question of whether Alu elements in amphioxus and primates arose by convergent evolution or by inheritance from a common ancestor. Genome-wide analyses of transposable elements in amphioxus and other chordates such as tunicates, agnathans and cartilaginous fishes could well provide the answer.

  16. Chromosomal aberrations induced by the restriction endonucleases Alu I and Bam HI: comparison with X-rays

    International Nuclear Information System (INIS)

    Dose-effect relationships for the frequencies of polycentric chromosomes induced by the restriction endonucleases Alu I and Bam HI and by X-rays in Chinese hamster ovary (CHO) cells were analyzed and compared. 1 Gy of X-rays produce the same frequency of polycentric chromosomes as 2 units Alu I and 7.9 units Bam HI. (author)

  17. SSTL Based Low Power Thermal Efficient WLAN Specific 32bit ALU Design on 28nm FPGA

    DEFF Research Database (Denmark)

    Kalia, Kartik; Pandey, Bishwajeet; Das, Teerath;

    2016-01-01

    In this paper we have designed a Thermal energy efficient 32Bit ALU for network processor, the main objective of this design is to provide better thermal efficiency with respect to existing designs. For that purpose we have used six different members of SSTL I/Os standards on 28nm technology along...... with consideration of airflow toward hit sink and different frequency on which ALU operate in network processor or any WLAN devices. We have done total power analysis of WLAN operating on different frequencies. We have considered a set of frequencies, which are based on IEEE 802.11 standards. First we did...... at minimum and maximum temperature as compared to all other considered I/O standards. This design has application where 32bit ALU design is considered for designing an electronic device such as WLAN. The design can be implemented on different nano chips for better efficiency depending upon the design...

  18. APOBEC3G oligomerization is associated with the inhibition of both Alu and LINE-1 retrotransposition.

    Directory of Open Access Journals (Sweden)

    Takayoshi Koyama

    Full Text Available Alu and LINE-1 (L1, which constitute ~11% and ~17% of the human genome, respectively, are transposable non-LTR retroelements. They transpose not only in germ cells but also in somatic cells, occasionally causing cancer. We have previously demonstrated that antiretroviral restriction factors, human APOBEC3 (hA3 proteins (A-H, differentially inhibit L1 retrotransposition. In this present study, we found that hA3 members also restrict Alu retrotransposition at differential levels that correlate with those observed previously for L1 inhibition. Through deletion analyses based on the best-characterized hA3 member human APOBEC3G (hA3G, its N-terminal 30 amino acids were required for its inhibitory activity against Alu retrotransposition. The inhibitory effect of hA3G on Alu retrotransposition was associated with its oligomerization that was affected by the deletion of its N-terminal 30 amino acids. Through structural modeling, the amino acids 24 to 28 of hA3G were predicted to be located at the interface of the dimer. The mutation of these residues resulted in abrogated hA3G oligomerization, and consistently abolished the inhibitory activity of hA3G against Alu retrotransposition. Importantly, the anti-L1 activity of hA3G was also associated with hA3G oligomerization. These results suggest that the inhibitory activities of hA3G against Alu and L1 retrotransposition might involve a common mechanism.

  19. A young Alu subfamily amplified independently in human and African great apes lineages.

    OpenAIRE

    Zietkiewicz, E; Richer, C.; Makalowski, W; Jurka, J; Labuda, D

    1994-01-01

    A variety of Alu subfamilies amplified in primate genomes at different evolutionary time periods. Alu Sb2 belongs to a group of young subfamilies with a characteristic two-nucleotide deletion at positions 65/66. It consists of repeats having a 7-nucleotide duplication of a sequence segment involving positions 246 through 252. The presence of Sb2 inserts was examined in five genomic loci in 120 human DNA samples as well as in DNAs of higher primates. The lack of the insertional polymorphism se...

  20. Alu-mediated diverse and complex pathogenic copy-number variants within human chromosome 17 at p13.3.

    Science.gov (United States)

    Gu, Shen; Yuan, Bo; Campbell, Ian M; Beck, Christine R; Carvalho, Claudia M B; Nagamani, Sandesh C S; Erez, Ayelet; Patel, Ankita; Bacino, Carlos A; Shaw, Chad A; Stankiewicz, Paweł; Cheung, Sau Wai; Bi, Weimin; Lupski, James R

    2015-07-15

    Alu repetitive elements are known to be major contributors to genome instability by generating Alu-mediated copy-number variants (CNVs). Most of the reported Alu-mediated CNVs are simple deletions and duplications, and the mechanism underlying Alu-Alu-mediated rearrangement has been attributed to non-allelic homologous recombination (NAHR). Chromosome 17 at the p13.3 genomic region lacks extensive low-copy repeat architecture; however, it is highly enriched for Alu repetitive elements, with a fraction of 30% of total sequence annotated in the human reference genome, compared with the 10% genome-wide and 18% on chromosome 17. We conducted mechanistic studies of the 17p13.3 CNVs by performing high-density oligonucleotide array comparative genomic hybridization, specifically interrogating the 17p13.3 region with ∼150 bp per probe density; CNV breakpoint junctions were mapped to nucleotide resolution by polymerase chain reaction and Sanger sequencing. Studied rearrangements include 5 interstitial deletions, 14 tandem duplications, 7 terminal deletions and 13 complex genomic rearrangements (CGRs). Within the 17p13.3 region, Alu-Alu-mediated rearrangements were identified in 80% of the interstitial deletions, 46% of the tandem duplications and 50% of the CGRs, indicating that this mechanism was a major contributor for formation of breakpoint junctions. Our studies suggest that Alu repetitive elements facilitate formation of non-recurrent CNVs, CGRs and other structural aberrations of chromosome 17 at p13.3. The common observation of Alu-mediated rearrangement in CGRs and breakpoint junction sequences analysis further demonstrates that this type of mechanism is unlikely attributed to NAHR, but rather may be due to a recombination-coupled DNA replicative repair process.

  1. Circular RNAs are abundant, conserved, and associated with ALU repeats

    OpenAIRE

    Jeck, William R.; Sorrentino, Jessica A.; Wang, Kai; Slevin, Michael K.; Christin E Burd; Liu, Jinze; Marzluff, William F.; Sharpless, Norman E.

    2013-01-01

    Using a novel approach to identify exonic circular RNAs (ecircRNAs), the authors show widespread production of circular RNAs from a significant fraction of expressed genes in murine and human cells. They demonstrate that several of these noncoding transcripts are stable and abundant and can be targeted by siRNA. Bioinformatic analysis of the entire ecircRNA complement revealed gene features associated with RNA circularization.

  2. Genome editing comes of age.

    Science.gov (United States)

    Kim, Jin-Soo

    2016-09-01

    Genome editing harnesses programmable nucleases to cut and paste genetic information in a targeted manner in living cells and organisms. Here, I review the development of programmable nucleases, including zinc finger nucleases (ZFNs), TAL (transcription-activator-like) effector nucleases (TALENs) and CRISPR (cluster of regularly interspaced palindromic repeats)-Cas9 (CRISPR-associated protein 9) RNA-guided endonucleases (RGENs). I specifically highlight the key advances that set the foundation for the rapid and widespread implementation of CRISPR-Cas9 genome editing approaches that has revolutionized the field. PMID:27490630

  3. Convergent Evolution of Fern-Specific Mitochondrial Group II Intron atp1i361g2 and Its Ancient Source Paralogue rps3i249g2 and Independent Losses of Intron and RNA Editing among Pteridaceae

    Science.gov (United States)

    Zumkeller, Simon Maria; Knoop, Volker; Knie, Nils

    2016-01-01

    Mitochondrial intron patterns are highly divergent between the major land plant clades. An intron in the atp1 gene, atp1i361g2, is an example for a group II intron specific to monilophytes (ferns). Here, we report that atp1i361g2 is lost independently at least 4 times in the fern family Pteridaceae. Such plant organelle intron losses have previously been found to be accompanied by loss of RNA editing sites in the flanking exon regions as a consequence of genomic recombination of mature cDNA. Instead, we now observe that RNA editing events in both directions of pyrimidine exchange (C-to-U and U-to-C) are retained in atp1 exons after loss of the intron in Pteris argyraea/biaurita and in Actiniopteris and Onychium. We find that atp1i361g2 has significant similarity with intron rps3i249g2 present in lycophytes and gymnosperms, which we now also find highly conserved in ferns. We conclude that atp1i361g2 may have originated from the more ancestral rps3i249g2 paralogue by a reverse splicing copy event early in the evolution of monilophytes. Secondary structure elements of the two introns, most characteristically their domains III, show strikingly convergent evolution in the monilophytes. Moreover, the intron paralogue rps3i249g2 reveals relaxed evolution in taxa where the atp1i361g2 paralogue is lost. Our findings may reflect convergent evolution of the two related mitochondrial introns exerted by co-evolution with an intron-binding protein simultaneously acting on the two paralogues. PMID:27492234

  4. Structure of a putative trans-editing enzyme for prolyl-tRNA synthetase from Aeropyrum pernix K1 at 1.7 Å resolution

    International Nuclear Information System (INIS)

    The three-dimensional structure of the APE2540 protein from A. pernix K1 has been determined by the multiple anomalous dispersion method at 1.7 Å resolution. The structure includes two monomers in the asymmetric unit and shares structural similarity with the YbaK protein or cysteinyl-tRNAPro deacylase from H. influenzae. The crystal structure of APE2540, the putative trans-editing enzyme ProX from Aeropyrum pernix K1, was determined in a high-throughput manner. The crystal belongs to the monoclinic space group P21, with unit-cell parameters a = 47.4, b = 58.9, c = 53.6 Å, β = 106.8°. The structure was solved by the multiwavelength anomalous dispersion method at 1.7 Å and refined to an R factor of 16.8% (Rfree = 20.5%). The crystal structure includes two protein molecules in the asymmetric unit. Each monomer consists of eight β-strands and seven α-helices. A structure-homology search revealed similarity between the trans-editing enzyme YbaK (or cysteinyl-tRNAPro deacylase) from Haemophilus influenzae (HI1434; 22% sequence identity) and putative ProX proteins from Caulobacter crescentus (16%) and Agrobacterium tumefaciens (21%)

  5. Mapping a mathematical expression onto a Montium ALU using GNU Bison

    NARCIS (Netherlands)

    Rosien, M.A.J.; Smit, G.J.M.

    2004-01-01

    The Montium processing tile [1], [4] contains a number of complex ALUs which can perform many different operations in many different ways. In the Chameleon tool flow [2], it is necessary to automatically determine whether a certain mathematical expression can be mapped onto an ALU and to automatical

  6. Widespread Alu repeat-driven expansion of consensus DR2 retinoic acid response elements during primate evolution

    Directory of Open Access Journals (Sweden)

    Wang Tian-Tian

    2007-01-01

    Full Text Available Abstract Background Nuclear receptors are hormone-regulated transcription factors whose signaling controls numerous aspects of development and physiology. Many receptors recognize DNA hormone response elements formed by direct repeats of RGKTCA motifs separated by 1 to 5 bp (DR1-DR5. Although many known such response elements are conserved in the mouse and human genomes, it is unclear to which extent transcriptional regulation by nuclear receptors has evolved specifically in primates. Results We have mapped the positions of all consensus DR-type hormone response elements in the human genome, and found that DR2 motifs, recognized by retinoic acid receptors (RARs, are heavily overrepresented (108,582 elements. 90% of these are present in Alu repeats, which also contain lesser numbers of other consensus DRs, including 50% of consensus DR4 motifs. Few DR2s are in potentially mobile AluY elements and the vast majority are also present in chimp and macaque. 95.5% of Alu-DR2s are distributed throughout subclasses of AluS repeats, and arose largely through deamination of a methylated CpG dinucleotide in a non-consensus motif present in AluS sequences. We find that Alu-DR2 motifs are located adjacent to numerous known retinoic acid target genes, and show by chromatin immunoprecipitation assays in squamous carcinoma cells that several of these elements recruit RARs in vivo. These findings are supported by ChIP-on-chip data from retinoic acid-treated HL60 cells revealing RAR binding to several Alu-DR2 motifs. Conclusion These data provide strong support for the notion that Alu-mediated expansion of DR elements contributed to the evolution of gene regulation by RARs and other nuclear receptors in primates and humans.

  7. Selection of sgRNA for Efficient Editing of TCR Gene by CRISPR-Cas9 System%CRISPR-Cas9系统定向编辑TCR基因的sgRNA筛选

    Institute of Scientific and Technical Information of China (English)

    邵红伟; 陈辉; 彭鑫; 徐畅; 张广献; 黄树林

    2015-01-01

    为构建靶向T细胞抗原受体( T Cell Receptor, TCR)基因的CRISPR-Cas9基因组编辑系统,基于pX458质粒构建靶向TCR基因β链C区的CRISPR-Cas-sgRNA质粒,将其转染HepG2细胞系,用流式细胞术检测转染效率;转染48 h后提取HepG2细胞基因组DNA,扩增含有编辑位点的片段,测序分析该片段的峰图改变;对出现双峰的扩增片段做T-A克隆后测序分析,确定基因编辑发生的位置,并结合转染效率计算基因编辑效率.结果表明,成功构建含有3种sgRNA序列( N1、 N2、 S1)的pX458-sgRNA质粒,其转染效率分别为38.5%(N1)、39.7%(N2)和24.2%(S1);基因组PCR产物测序分析发现, S1组扩增片段在打靶位置出现杂峰; T-A克隆测序发现,20克隆有4个发生了基因编辑(20%),结合转染效率(24.2%)可知,编辑效率约为83%.可见,本文成功构建靶向 TCR 基因的 CRISPR -Cas -sgRNA质粒,并鉴定出基因编辑效率较高的一种sgRNA序列.%To establish a TCR-targeted CRISPR-Cas9 system for study of TCR functions, the CRISPR-Cas-sgRNA constructs targeting TRBC were made based on pX458 vector and transferred into HepG2 . The transfection efficiencies were detected by flow cytometry ( FCM) after 48 h. Subsequently, the genomic DNA of HepG2 was extracted and a fragment covering target sequence was amplified by PCR and analyzed by se-quencing. The fragment exhibiting overlapping peaks in target sequence was subject to T-A cloning. The se-quencing results were then analyzed to confirm the occurrence of indel and calculate the editing efficiency. The results showed that three pX458-sgRNA constructs (N1、 N2、 S1) were made. The transfection efficien-cies were 38. 5% (N1), 39. 7% (N2) and 24. 2% (S1), respectively. Sequencing results of S1 fragment exhibited overlapping peaks. After cloning and sequencing, 4 of 20 S1 clones showed sequence alteration. Considering the 24. 2% transfection efficiency, the indel efficiency induced by the sgRNA-S1 is

  8. Development of two highly sensitive forensic sex determination assays based on human DYZ1 and Alu repetitive DNA elements.

    Science.gov (United States)

    Fazi, Amanda; Gobeski, Brianne; Foran, David

    2014-11-01

    Sex determination is a critical component of forensic identification, the standard genetic method for which is detection of the single copy amelogenin gene that has differing homologues on the X and Y chromosomes. However, this assay may not be sensitive enough when DNA samples are minute or highly compromised, thus other strategies for sex determination are needed. In the current research, two ultrasensitive sexing assays, based on real-time PCR and pyrosequencing, were developed targeting the highly repetitive elements DYZ1 on the Y chromosome and Alu on the autosomes. The DYZ1/Alu strategy was compared to amelogenin for overall sensitivity based on high molecular weight and degraded DNA, followed by assaying the sex of 34 touch DNA samples and DNA from 30 hair shafts. The real-time DYZ1/Alu assay proved to be approximately 1500 times more sensitive than its amelogenin counterpart based on high molecular weight DNA, and even more sensitive when sexing degraded DNA. The pyrosequencing DYZ1/Alu assay correctly sexed 26 of the touch DNAs, compared to six using amelogenin. Hair shaft DNAs showed equally improved sexing results using the DYZ1/Alu assays. Overall, both DYZ1/Alu assays were far more sensitive and accurate than was the amelogenin assay, and thus show great utility for sexing poor quality and low quantity DNA evidence. PMID:25168471

  9. The agents of natural genome editing

    Institute of Scientific and Technical Information of China (English)

    Guenther Witzany

    2011-01-01

    The DNA serves as a stable information storage medium and every protein which is needed by the cell is produced from this blueprint via an RNA intermediate code. More recently it was found that an abundance of various RNA elements cooperate in a variety of steps and substeps as regulatory and catalytic units with multiple competencies to act on RNA transcripts. Natural genome editing on one side is the competent agent*driven generation and integration of meaningful DNA nucleotide sequences into pre-existing genomic content arrangements, and the ability to (re-)combine and (re-)regulate them according to context-dependent (i.e. adaptational) purposes of the host organism. Natural genome editing on the other side designates the integration of all RNA activities acting on RNA transcripts without altering DNA-encoded genes. If we take the genetic code seriously as a natural code, there must be agents that are competent to act on this code because no natural code codes itself as no natural language speaks itself. As code editing agents,viral and subviral agents have been suggested because there are several indicators that demonstrate viruses competent in both RNA and DNA natural genome editing.

  10. The coding region of the human c-mos pseudogene contains Alu repeat insertions.

    Science.gov (United States)

    Zabarovsky, E R; Chumakov, I M; Prassolov, V S; Kisselev, L L

    1984-10-01

    We have determined the nucleotide sequence of an 841-bp fragment derived from a segment of the human genome previously cloned by Chumakov et al. [Gene 17 (1982) 19-26] and Zabarovsky et al. [Gene 23 (1983) 379-384] and containing regions homologous to the viral mos gene probe. This sequence displays homology with part of the coding region of the human and murine c-mos genes, contains several termination codons, and is interrupted by two Alu-family elements flanked by short direct repeats. Probably, the progenitor of the human c-mos gene was duplicated approximately at the time of mammalian divergence, was converted to a pseudogene, and acquired insertions of two Alu elements.

  11. Identification of homogeneously staining regions by G-banding and chromosome microdissection, and FISH marker selection using human Alu sequence primers in a scleractinian coral Coelastrea aspera Verrill, 1866 (Cnidaria).

    Science.gov (United States)

    Taguchi, Takahiro; Kubota, Satoshi; Mezaki, Takuma; Tagami, Erika; Sekida, Satoko; Nakachi, Shu; Okuda, Kazuo; Tominaga, Akira

    2016-01-01

    Karyotype analysis was performed on the scleractinian coral Coelastrea aspera Verrill, 1866, commonly found along temperate coasts in Japan (30-35°N) and in coastal waters in the Indian and Pacific oceans. G-banding of Coelastrea aspera was successfully performed, although the banding pattern was not as clear as that in mammals. The karyogram clearly revealed that this coral had a homogeneously staining region (hsr) in chromosome 11. This hsr consisted of ribosomal RNA (rRNA) related genes, which was demonstrated by fluorescence in situ hybridization (FISH) with probes generated using 28S ribosomal DNA (rDNA) primers and those generated through chromosome microdissection. In addition, we conducted silver-stained nucleolus organizer region (Ag-NOR) analysis and found Ag depositions in the interphase nuclei but not on rRNA gene loci and hsr(s) in the mitotic stage. The hsr of this coral was observed in approximately 50% of the metaphase spreads analyzed. This may explain the diversity of coral rDNA based on the molecular study of sequence analysis. Furthermore, it was discovered that human telomere and Alu repeated sequences were present in this Coelastrea aspera. Probes derived from human Alu sequences are expected to play an important role in the classification of corals. Overall, our data can be of great value in discriminating among scleractinian coral species and understanding their genetics, including chromosomal evolution. PMID:27186338

  12. Development and irradiation testing of Al-U3Si2 at Chalk River Laboratories

    International Nuclear Information System (INIS)

    Mini-elements containing Al-64 wt% U3Si2 (3.15 gU/cm3), with three discrete U3Si2 particle-size distributions, have been irradiated up to 93 at% burnup in the NRU reactor. The uranium silicide (U-7.0Si) was used in the as-cast condition, and contained up to 4 wt% free uranium in the U3Si2 matrix. Post-irradiation examinations (PIE) of the high-burnup elements have been recently completed. PIE included underwater and hot-cell examinations, immersion density measurements, neutron radiography, optical and scanning electron microscopy (SEM) with wavelength dispersion X-ray (WDX) analysis, and computerized image analysis of the fission-gas bubble-size distributions. The results show that the Al-U3Si2 swelled less than Al-U3Si fuel previously irradiated under similar conditions in NRU, and no significant swelling dependence on particle-size distribution was observed. Al-U3Si2 core volume increases ranged from 4.2 to 4.7 vol%, compared to 5.8 to 6.8 vol% for Al-U3Si fuel with identical uranium loadings. SEM examinations revealed that the U3Si2 (U-7.0Si) particles contained regions with relatively ordered, very dense populations of sub-micron fission-gas bubbles. In contrast, the gas bubbles are randomly distributed within U3Si (U-3.96Si) particles, vary widely in size, and small bubbles coalesce to form larger bubbles. The capability of U3Si2 to retain fission gas in small bubbles accounts for the lower swelling. (author)

  13. The levels of edit, second edition

    Science.gov (United States)

    Vanburen, R.; Buehler, M. F.

    1980-01-01

    The editorial process is analyzed, and five levels of edit are identified. These levels represent cumulative combinations of nine types of edit: Coordination, Policy, Integrity, Screening, Copy Clarification, Format, Mechanical Style, Language, and Substantive. The levels and types of edit, although developed for specific use with external reports at the Jet Propulsion Laboratory, cover the general range of technical editing, especially as it applies to an in-house technical publications organization. Each type of edit is set forth in terms of groups of actions to be performed by editor. The edit-level concept has enhanced understanding and communication among editors, authors, and publications managers concerning the specific editorial work to be done on each manuscript. It has also proved useful as a management tool for estimating and monitoring cost.

  14. In situ hybridization of bat chromosomes with human (TTAGGGn probe, after previous digestion with Alu I

    Directory of Open Access Journals (Sweden)

    Karina de Cassia Faria

    2002-01-01

    Full Text Available The purpose of this work was to verify the ability of the enzyme Alu I to cleave and/or remove satellite DNA sequences from heterochromatic regions in chromosomes of bats, by identifying the occurrence of modifications in the pattern of fluorescence in situ hybridization with telomeric DNA. The localization and fluorescence intensity of the telomeric DNA sites of the Alu-digested and undigested chromosomes of species Eumops glaucinus, Carollia perspicillata, and Platyrrhinus lineatus were analyzed. Telomeric sequences were detected at the termini of chromosomes of all three species, although, in C. perspicillata, the signals were very faint or absent in most chromosomes. This finding was interpreted as being due to a reduced number of copies of the telomeric repeat, resulting from extensive telomeric association and/or rearrangements undergone by the chromosomes of Carollia. Fluorescent signals were also observed in centromeric and pericentromeric regions in several two-arm chromosomes of E. glaucinus and C. perspicillata. In E. glaucinus and P. lineatus, some interstitial and terminal telomeric sites were observed to be in association with regions of constitutive heterochromatin and ribosomal DNA (NORs. After digestion, these telomeric sites showed a significant decrease in signal intensity, indicating that enzyme Alu I cleaves and/or removes part of the satellite DNA present in these regions. These results suggest that the telomeric sequence is a component of the heterochromatin, and that the C-band- positive regions of bat chromosomes have a different DNA composition.

  15. Genetic variation among world populations: inferences from 100 Alu insertion polymorphisms.

    Science.gov (United States)

    Watkins, W Scott; Rogers, Alan R; Ostler, Christopher T; Wooding, Steve; Bamshad, Michael J; Brassington, Anna-Marie E; Carroll, Marion L; Nguyen, Son V; Walker, Jerilyn A; Prasad, B V Ravi; Reddy, P Govinda; Das, Pradipta K; Batzer, Mark A; Jorde, Lynn B

    2003-07-01

    We examine the distribution and structure of human genetic diversity for 710 individuals representing 31 populations from Africa, East Asia, Europe, and India using 100 Alu insertion polymorphisms from all 22 autosomes. Alu diversity is highest in Africans (0.349) and lowest in Europeans (0.297). Alu insertion frequency is lowest in Africans (0.463) and higher in Indians (0.544), E. Asians (0.557), and Europeans (0.559). Large genetic distances are observed among African populations and between African and non-African populations. The root of a neighbor-joining network is located closest to the African populations. These findings are consistent with an African origin of modern humans and with a bottleneck effect in the human populations that left Africa to colonize the rest of the world. Genetic distances among all pairs of populations show a significant product-moment correlation with geographic distances (r = 0.69, P distance estimates. These analyses also demonstrate that markers with higher F(ST) values have greater resolving power and produce more consistent genetic distance estimates.

  16. Wikipedia editing dynamics

    Science.gov (United States)

    Gandica, Y.; Carvalho, J.; Sampaio dos Aidos, F.

    2015-01-01

    A model for the probabilistic function followed in editing Wikipedia is presented and compared with simulations and real data. It is argued that the probability of editing is proportional to the editor's number of previous edits (preferential attachment), to the editor's fitness, and to an aging factor. Using these simple ingredients, it is possible to reproduce the results obtained for Wikipedia editing dynamics for a collection of single pages as well as the averaged results. Using a stochastic process framework, a recursive equation was obtained for the average of the number of edits per editor that seems to describe the editing behavior in Wikipedia.

  17. Wikipedia edition dynamics

    CERN Document Server

    Gandica, Y; Carvalho, J

    2014-01-01

    A model for the probabilistic function followed in Wikipedia edition is presented and compared with simulations and real data. It is argued that the probability to edit is proportional to the editor's number of previous editions (preferential attachment), to the editor's fitness and to an ageing factor. Using these simple ingredients, it is possible to reproduce the results obtained for Wikipedia edition dynamics for a collection of single pages as well as the averaged results. Using a stochastic process framework, a recursive equation was obtained for the average of the number of editions per editor that seems to describe the editing behaviour in Wikipedia.

  18. Human nucleosomes: special role of CG dinucleotides and Alu-nucleosomes

    Directory of Open Access Journals (Sweden)

    Trifonov Edward N

    2011-05-01

    Full Text Available Abstract Background The periodical occurrence of dinucleotides with a period of 10.4 bases now is undeniably a hallmark of nucleosome positioning. Whereas many eukaryotic genomes contain visible and even strong signals for periodic distribution of dinucleotides, the human genome is rather featureless in this respect. The exact sequence features in the human genome that govern the nucleosome positioning remain largely unknown. Results When analyzing the human genome sequence with the positional autocorrelation method, we found that only the dinucleotide CG shows the 10.4 base periodicity, which is indicative of the presence of nucleosomes. There is a high occurrence of CG dinucleotides that are either 31 (10.4 × 3 or 62 (10.4 × 6 base pairs apart from one another - a sequence bias known to be characteristic of Alu-sequences. In a similar analysis with repetitive sequences removed, peaks of repeating CG motifs can be seen at positions 10, 21 and 31, the nearest integers of multiples of 10.4. Conclusions Although the CG dinucleotides are dominant, other elements of the standard nucleosome positioning pattern are present in the human genome as well. The positional autocorrelation analysis of the human genome demonstrates that the CG dinucleotide is, indeed, one visible element of the human nucleosome positioning pattern, which appears both in Alu sequences and in sequences without repeats. The dominant role that CG dinucleotides play in organizing human chromatin is to indicate the involvement of human nucleosomes in tuning the regulation of gene expression and chromatin structure, which is very likely due to cytosine-methylation/-demethylation in CG dinucleotides contained in the human nucleosomes. This is further confirmed by the positions of CG-periodical nucleosomes on Alu sequences. Alu repeats appear as monomers, dimers and trimers, harboring two to six nucleosomes in a run. Considering the exceptional role CG dinucleotides play in the

  19. Edit wars in Wikipedia

    CERN Document Server

    Sumi, Róbert; Rung, András; Kornai, András; Kertész, János

    2011-01-01

    We present a new, efficient method for automatically detecting severe conflicts `edit wars' in Wikipedia and evaluate this method on six different language WPs. We discuss how the number of edits, reverts, the length of discussions, the burstiness of edits and reverts deviate in such pages from those following the general workflow, and argue that earlier work has significantly over-estimated the contentiousness of the Wikipedia editing process.

  20. DNA-free genome editing methods for targeted crop improvement.

    Science.gov (United States)

    Kanchiswamy, Chidananda Nagamangala

    2016-07-01

    Evolution of the next-generation clustered, regularly interspaced, short palindromic repeat/Cas9 (CRISPR/Cas9) genome editing tools, ribonucleoprotein (RNA)-guided endonuclease (RGEN) RNPs, is paving the way for developing DNA-free genetically edited crop plants. In this review, I discuss the various methods of RGEN RNPs tool delivery into plant cells and their limitations to adopt this technology to numerous crop plants. Furthermore, focus is given on the importance of developing DNA-free genome edited crop plants, including perennial crop plants. The possible regulation on the DNA-free, next-generation genome-edited crop plants is also highlighted. PMID:27100964

  1. Microlunatus cavernae sp. nov., a novel actinobacterium isolated from Alu ancient cave, Yunnan, South-West China.

    Science.gov (United States)

    Cheng, Juan; Chen, Wei; Huo-Zhang, Bing; Nimaichand, Salam; Zhou, En-Min; Lu, Xin-Hua; Klenk, Hans-Peter; Li, Wen-Jun

    2013-07-01

    A Gram-positive, coccoid, non-endospore-forming actinobacterium, designated YIM C01117(T), was isolated from a soil sample collected from Alu ancient cave, Yunnan province, south-west China. Based on the 16S rRNA gene sequence analysis, strain YIM C01117(T) was shown to belong to the genus Microlunatus, with highest sequence similarity of 97.4 % to Microlunatus soli DSM 21800(T). The whole genomic DNA relatedness as shown by the DNA-DNA hybridization study between YIM C01117(T) and M. soli DSM 21800(T) had a low value (47 ± 2 %). Strain YIM C01117(T) was determined to contain LL-diaminopimelic acid with Gly, Glu and Ala amino acids (A3γ' type) in the cell wall. Whole-cell hydrolysates were found to contain glucose, galactose, mannose and ribose. The major polar lipids were determined to be phosphatidylglycerol and diphosphatidylglycerol. The predominant menaquinone system present is MK-9(H4), while the major fatty acids were identified to be anteiso-C15:0 (24.1 %), iso-C16:0 (22.3 %) and iso-C15:0 (11.4 %). The G+C content of the genomic DNA was determined to be 65.9 mol%. The chemotaxonomic and genotypic data support the affiliation of the strain YIM C01117(T) to the genus Microlunatus. The results of physiological and biochemical tests allow strain YIM C01117(T) to be differentiated phenotypically from recognized Microlunatus species. Strain YIM C01117(T) is therefore considered to represent a novel species of the genus Microlunatus, for which the name Microlunatus cavernae sp. nov. is proposed. The type strain is YIM C01117(T) (= DSM 26248(T) = JCM 18536(T)).

  2. A genomewide screen for suppressors of Alu-mediated rearrangements reveals a role for PIF1.

    Directory of Open Access Journals (Sweden)

    Karen M Chisholm

    Full Text Available Alu-mediated rearrangement of tumor suppressor genes occurs frequently during carcinogenesis. In breast cancer, this mechanism contributes to loss of the wild-type BRCA1 allele in inherited disease and to loss of heterozygosity in sporadic cancer. To identify genes required for suppression of Alu-mediated recombination we performed a genomewide screen of a collection of 4672 yeast gene deletion mutants using a direct repeat recombination assay. The primary screen and subsequent analysis identified 12 candidate genes including TSA, ELG1, and RRM3, which are known to play a significant role in maintaining genomic stability. Genetic analysis of the corresponding human homologs was performed in sporadic breast tumors and in inherited BRCA1-associated carcinomas. Sequencing of these genes in high risk breast cancer families revealed a potential role for the helicase PIF1 in cancer predisposition. PIF1 variant L319P was identified in three breast cancer families; importantly, this variant, which is predicted to be functionally damaging, was not identified in a large series of controls nor has it been reported in either dbSNP or the 1000 Genomes Project. In Schizosaccharomyces pombe, Pfh1 is required to maintain both mitochondrial and nuclear genomic integrity. Functional studies in yeast of human PIF1 L319P revealed that this variant cannot complement the essential functions of Pfh1 in either the nucleus or mitochondria. Our results provide a global view of nonessential genes involved in suppressing Alu-mediated recombination and implicate variation in PIF1 in breast cancer predisposition.

  3. Aberrant methylation and associated transcriptional mobilization of Alu elements contributes to genomic instability in hypoxia.

    Science.gov (United States)

    Pal, Arnab; Srivastava, Tapasya; Sharma, Manish K; Mehndiratta, Mohit; Das, Prerna; Sinha, Subrata; Chattopadhyay, Parthaprasad

    2010-11-01

    Hypoxia is an integral part of tumorigenesis and contributes extensively to the neoplastic phenotype including drug resistance and genomic instability. It has also been reported that hypoxia results in global demethylation. Because a majority of the cytosine-phosphate-guanine (CpG) islands are found within the repeat elements of DNA, and are usually methylated under normoxic conditions, we suggested that retrotransposable Alu or short interspersed nuclear elements (SINEs) which show altered methylation and associated changes of gene expression during hypoxia, could be associated with genomic instability. U87MG glioblastoma cells were cultured in 0.1% O₂ for 6 weeks and compared with cells cultured in 21% O₂ for the same duration. Real-time PCR analysis showed a significant increase in SINE and reverse transcriptase coding long interspersed nuclear element (LINE) transcripts during hypoxia. Sequencing of bisulphite treated DNA as well as the Combined Bisulfite Restriction Analysis (COBRA) assay showed that the SINE loci studied underwent significant hypomethylation though there was patchy hypermethylation at a few sites. The inter-alu PCR profile of DNA from cells cultured under 6-week hypoxia, its 4-week revert back to normoxia and 6-week normoxia showed several changes in the band pattern indicating increased alu mediated genomic alteration. Our results show that aberrant methylation leading to increased transcription of SINE and reverse transcriptase associated LINE elements could lead to increased genomic instability in hypoxia. This might be a cause of genetic heterogeneity in tumours especially in variegated hypoxic environment and lead to a development of foci of more aggressive tumour cells.

  4. POMEN IN MERJENJE ORGANIZACIJSKE KULTURE. PRIMER PODJETJA ALPOS ALU D.O.O.

    OpenAIRE

    Senica, Damjana

    2013-01-01

    Diplomsko delo obravnava organizacijsko kulturo (OK), ki je splet vrednot, prepričanj, stališč in spoznanj o tem, kako organizacija deluje ali naj bi delovala. Ob teoretični obravnavi OK je bila v nalogi opravljena tudi analiza OK v podjetju Alpos Alu d.o.o., ki je zaradi splošne gospodarske krize v zapletenem položaju, zato je še posebej zanimivo za obravnavo. Z metodo OCAI avtorjev Camerona in Quinna sem prišla do ugotovitve, da v podjetju prevladuje kultura trga, zaposleni pa si žel...

  5. Novel Reversible TSG Gate and Its Application for Designing Components of Primitive Reversible/Quantum ALU

    OpenAIRE

    Thapliyal, Himanshu; Srinivas, M. B

    2006-01-01

    In recent years, reversible logic has emerged as a promising computing paradigm having application in low power CMOS, quantum computing, nanotechnology, and optical computing. The classical set of gates such as AND, OR, and EXOR are not reversible. This paper utilizes a new 4 * 4 reversible gate called TSG gate to build the components of a primitive reversible/quantum ALU. The most significant aspect of the TSG gate is that it can work singly as a reversible full adder, that is reversible ful...

  6. Editing efficiency of a Drosophila gene correlates with a distant splice site selection

    OpenAIRE

    AGRAWAL, RITESH; Stormo, Gary D.

    2005-01-01

    RNA editing and alternative splicing are two processes that increase protein diversity. The relationship between the two processes is not well understood. There are a few examples of correlations between editing and alternative splicing, but these are all nearby effects. A search for alternative splicing among 16 edited genes in Drosophila reveals two novel instances of alternative splicing. In one example where alternative splicing occurs downstream of editing, a strong correlation between e...

  7. International distribution and age estimation of the Portuguese BRCA2 c.156_157insAlu founder mutation

    DEFF Research Database (Denmark)

    Peixoto, Ana; Santos, Catarina; Pinheiro, Manuela;

    2011-01-01

    The c.156_157insAlu BRCA2 mutation has so far only been reported in hereditary breast/ovarian cancer (HBOC) families of Portuguese origin. Since this mutation is not detectable using the commonly used screening methodologies and must be specifically sought, we screened for this rearrangement...... in a total of 5,443 suspected HBOC families from several countries. Whereas the c.156_157insAlu BRCA2 mutation was detected in 11 of 149 suspected HBOC families from Portugal, representing 37.9% of all deleterious mutations, in other countries it was detected only in one proband living in France and in four...

  8. The potential role of Alu Y in the development of resistance to SN38 (Irinotecan) or oxaliplatin in colorectal cancer

    DEFF Research Database (Denmark)

    Lin, Xue; Stenvang, Jan; Rasmussen, Mads Heilskov;

    2015-01-01

    or oxaliplatin resistant colorectal cancer cell line models. Moreover, we extended the RRBS analysis to tumor tissue from 14 patients with colorectal cancer who either did or did not benefit from capecitabine + oxaliplatin treatment. For the clinical samples, we applied a concept of 'DNA methylation entropy......' to estimate the diversity of DNA methylation states of the identified resistance phenotype-associated methylation loci observed in the cell line models. We identified different loci being characteristic for the different resistant cell lines. Interestingly, 53% of the identified loci were Alu sequences...... by mobility of Alu elements and stresses the importance of personalized strategies, using a systematic and dynamic view, for effective cancer therapy....

  9. Identifikasi Keragaman Genetik Gen Reseptor Hormon Pertumbuhan (GHR|Alu I pada Sapi Bali

    Directory of Open Access Journals (Sweden)

    Zulkharnaim

    2010-08-01

    Full Text Available Growth hormone receptor (GHR is one factor affecting animal growth. GHR is required by growth hormone (GH to carry out its effects on target tissues. The objective of the study was to estimate genetic diversity of the GHR|AluI gene in bali, limousin, simmental and pesisir cattle. Genotyping was performed on 248 animals, including 162 bali, 21 limousin, 17 simmental and 48 pesisir. Single nucleotide polymorphisms (SNP had been found in exon 10, coding for the cytoplasmic domain of GHR, which was located at position 81 bp (A/G induced amino acid substitutions Ser/Gly. Genotype frequencies of bali cattle AA (0.988, GG (0.006 and AG (0.006 were evidenced for the GHR AluI monomorphism, but mostly different from limousin GG (0.667, AA (0.238 and AG (0.095, simmental AG (0.529, GG (0.471 and AA (0.000, pesisir AA (0.604, GG (0.375 and AG (0.021 were the evidence of polymorphism. Homozigosity (monomorphism in bali cattle might be affected by adaptability in extreme environmental conditions such as poor nutrition and improper management practices. It also could be affected by natural selection and phenotype plasticity phenomena.

  10. An Alu element-associated hypermethylation variant of the POMC gene is associated with childhood obesity.

    Directory of Open Access Journals (Sweden)

    Peter Kuehnen

    Full Text Available The individual risk for common diseases not only depends on genetic but also on epigenetic polymorphisms. To assess the role of epigenetic variations in the individual risk for obesity, we have determined the methylation status of two CpG islands at the POMC locus in obese and normal-weight children. We found a hypermethylation variant targeting individual CpGs at the intron 2-exon 3 boundary of the POMC gene by bisulphite sequencing that was significantly associated with obesity. POMC exon 3 hypermethylation interferes with binding of the transcription enhancer P300 and reduces expression of the POMC transcript. Since intron 2 contains Alu elements that are known to influence methylation in their genomic vicinity, the exon 3 methylation variant seems to result from an Alu element-triggered default state of methylation boundary definition. Exon 3 hypermethylation in the POMC locus represents the first identified DNA methylation variant that is associated with the individual risk for obesity.

  11. Transcripts from a novel human KRAB zinc finger gene contain spliced Alu and endogenous retroviral segments

    Energy Technology Data Exchange (ETDEWEB)

    Baban, S.; Freeman, J.D.; Mager, D.L. [Univ. of British Columbia, Vancouver, British Columbia (Canada)

    1996-05-01

    During the course of an investigation into the potential effects of endogenous retroviruses on adjacent gene expression, we isolated two cDNA clones containing a small sequence segment belonging to the human endogenous retrovirus family, HERV-H. Characterization of the clones revealed that they represent transcripts from a novel KRAB zinc finger gene termed ZNF177. The two cDNA clones differ at their 5{prime} termini and in the presence of a 559-bp internal exon. The clone containing this internal exon has six imperfect zinc finger motifs followed by seven perfect copies of the C{sub 2}H{sub 2} type but has a frame shift between the KRAB domain and the downstream zinc finger region. The smaller clone lacks the six imperfect motifs and has an intact ORF. The 5{prime} putative untranslated regions of both cDNAs contain an 86-bp HERV-H env segment and a segment of an Alu repeat, both in the antisense orientation, that have been incorporated by splicing. RT-PCR experiments show evidence of alternative splicing but the majority of transcripts appear to contain the Alu and env segments. Genomic PCR and hybridization experiments suggest that a partial HERV-H element is integrated within the ZNF177 locus, which Southern analysis has shown to be a single-copy gene. Northern and RT-PCR analyses suggest that ZNF177 is transcribed at a low level in a variety of cell types. 41 refs., 8 figs.

  12. Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells.

    Science.gov (United States)

    Klawitter, Sabine; Fuchs, Nina V; Upton, Kyle R; Muñoz-Lopez, Martin; Shukla, Ruchi; Wang, Jichang; Garcia-Cañadas, Marta; Lopez-Ruiz, Cesar; Gerhardt, Daniel J; Sebe, Attila; Grabundzija, Ivana; Merkert, Sylvia; Gerdes, Patricia; Pulgarin, J Andres; Bock, Anja; Held, Ulrike; Witthuhn, Anett; Haase, Alexandra; Sarkadi, Balázs; Löwer, Johannes; Wolvetang, Ernst J; Martin, Ulrich; Ivics, Zoltán; Izsvák, Zsuzsanna; Garcia-Perez, Jose L; Faulkner, Geoffrey J; Schumann, Gerald G

    2016-01-08

    Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate in vitro to generate derivatives of the three primary germ layers. Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs.

  13. Regulation of nucleolus assembly by non-coding RNA polymerase II transcripts.

    Science.gov (United States)

    Caudron-Herger, Maïwen; Pankert, Teresa; Rippe, Karsten

    2016-05-01

    The nucleolus is a nuclear subcompartment for tightly regulated rRNA production and ribosome subunit biogenesis. It also acts as a cellular stress sensor and can release enriched factors in response to cellular stimuli. Accordingly, the content and structure of the nucleolus change dynamically, which is particularly evident during cell cycle progression: the nucleolus completely disassembles during mitosis and reassembles in interphase. Although the mechanisms that drive nucleolar (re)organization have been the subject of a number of studies, they are only partly understood. Recently, we identified Alu element-containing RNA polymerase II transcripts (aluRNAs) as important for nucleolar structure and rRNA synthesis. Integrating these findings with studies on the liquid droplet-like nature of the nucleolus leads us to propose a model on how RNA polymerase II transcripts could regulate the assembly of the nucleolus in response to external stimuli and during cell cycle progression.

  14. Recent Advances in Genome Editing Using CRISPR/Cas9

    OpenAIRE

    Ding, Yuduan; Li, Hong; Chen, Ling-Ling; Xie, Kabin

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is a versatile tool for genome engineering that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This simple RNA-guided genome-editing technology has become a revolutionary tool in biology and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing method, summarize the recent advances in CRISPR/Cas9 ...

  15. Multiplex editing system

    DEFF Research Database (Denmark)

    2015-01-01

    The present invention relates to a multiplex editing system. The system allows multiple editing of nucleic acid sequences such as genomic sequences, such as knockins of genes of interest in a genome, knockouts of genomic sequences and/or allele replacement. Also provided herein are a method...

  16. Quality text editing

    Directory of Open Access Journals (Sweden)

    Gyöngyi Bujdosó

    2009-10-01

    Full Text Available Text editing is more than the knowledge of word processing techniques. Originally typographers, printers, text editors were the ones qualified to edit texts, which were well structured, legible, easily understandable, clear, and were able to emphasize the coreof the text. Time has changed, and nowadays everyone has access to computers as well as to text editing software and most users believe that having these tools is enough to edit texts. However, text editing requires more skills. Texts appearing either in printed or inelectronic form reveal that most of the users do not realize that they are not qualified to edit and publish their works. Analyzing the ‘text-products’ of the last decade a tendency can clearly be drawn. More and more documents appear, which instead of emphasizingthe subject matter, are lost in the maze of unstructured text slices. Without further thoughts different font types, colors, sizes, strange arrangements of objects, etc. are applied. We present examples with the most common typographic and text editing errors. Our aim is to call the attention to these mistakes and persuadeusers to spend time to educate themselves in text editing. They have to realize that a well-structured text is able to strengthen the effect on the reader, thus the original message will reach the target group.

  17. A plant mitochondrial sequence transcribed in transgenic tobacco chloroplasts is not edited

    Energy Technology Data Exchange (ETDEWEB)

    Sutton, C.A.; Hanson, M.R. [Cornell Univ., Ithaca, NY (United States); Zoubenko, O.V.; Maliga, P. [State Univ. of New Jersey, Piscataway, NJ (United States)

    1995-03-01

    RNA editing occurs in two higher-plant organelles, chloroplasts, and mitochondria. Because chloroplasts and mitochondria exhibit some similarity in editing site selection, we investigated whether mitochondrial RNA sequences could be edited in chloroplasts. We produced transgenic tobacco plants that contained chimeric genes in which the second exon of a Petunia hybrida mitochondrial coxII gene was under the control of chloroplast gene regulatory sequences. coxII transcripts accumulated to low or high levels in transgenic chloroplasts containing chimeric genes with the plastid ribosomal protein gene rps16 or the rRNA operon promoter, respectively. Exon 2 of coxII was chosen because it carries seven editing sites and is edited in petunia mitochondria even when located in an abnormal context in an aberrant recombined gene. When editing of the coxII transcripts in transgenic chloroplasts was examined, no RNA editing at any of the usual sites was detected, nor was there any novel editing at any other sites. These results indicate that the RNA editing mechanisms of chloroplasts and mitochondria are not identical but must have at least some organelle-specific components. 33 refs., 5 figs.

  18. Translational arrest by a prokaryotic signal recognition particle is mediated by RNA interactions.

    Science.gov (United States)

    Beckert, Bertrand; Kedrov, Alexej; Sohmen, Daniel; Kempf, Georg; Wild, Klemens; Sinning, Irmgard; Stahlberg, Henning; Wilson, Daniel N; Beckmann, Roland

    2015-10-01

    The signal recognition particle (SRP) recognizes signal sequences of nascent polypeptides and targets ribosome-nascent chain complexes to membrane translocation sites. In eukaryotes, translating ribosomes are slowed down by the Alu domain of SRP to allow efficient targeting. In prokaryotes, however, little is known about the structure and function of Alu domain-containing SRPs. Here, we report a complete molecular model of SRP from the Gram-positive bacterium Bacillus subtilis, based on cryo-EM. The SRP comprises two subunits, 6S RNA and SRP54 or Ffh, and it facilitates elongation slowdown similarly to its eukaryotic counterpart. However, protein contacts with the small ribosomal subunit observed for the mammalian Alu domain are substituted in bacteria by RNA-RNA interactions of 6S RNA with the α-sarcin-ricin loop and helices H43 and H44 of 23S rRNA. Our findings provide a structural basis for cotranslational targeting and RNA-driven elongation arrest in prokaryotes. PMID:26344568

  19. CRISPR Genome Editing

    Science.gov (United States)

    A research article about a technique for gene editing known as CRISPR-Cas9. The technique has made it much easier and faster for cancer researchers to study mutations and test new therapeutic targets.

  20. Design and Implementation of Submicron Level 10T Full Adder in ALU Using Cell Based and SOC Technology

    Directory of Open Access Journals (Sweden)

    K.Swathi

    2014-09-01

    Full Text Available As technology scales into the nanometer regime leakage current, active power, delay and area are becoming important metric for the analysis and design of complex circuits. The main concern in mobile and battery based systems are leakage current and power dissipation. A transistor resizing approach for 10 transistor single bit full adder cells is used to determine optimal sleep transistor size which reduces power dissipation and leakage current. A submicron level 10-transistor single bit full adder cell is considered to achieve low leakage current, reduced power dissipation and high speed. In this paper initially 10T full adder cell is designed with submicron technique and later this is employed to design an ALU adder unit. The modified ALU is simulated and synthesized successfully on cadence 180nm technology.

  1. Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system

    OpenAIRE

    Cheong, Taek-Chin; Compagno, Mara; Chiarle, Roberto

    2016-01-01

    Applications of the CRISPR-Cas9 system to edit the genome have widely expanded to include DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings. Here we show the application of CRISPR-Cas9 technology to edit the mouse and human immunoglobulin (Ig) genes. By delivering Cas9 and guide-RNA (gRNA) with retro- or lenti-virus to IgM+ mouse B cells and hybridomas, we induce class-switch recombination (CSR) of the IgH chain to the desired subclass. Similarl...

  2. Analysis of the action of the restriction endonuclease AluI using three different comet assay protocols

    International Nuclear Information System (INIS)

    Background and purpose: the comet assay offers the opportunity to measure the amount of DNA damage and the effectiveness of DNA repair in single cells. In a first part, experiments are presented comparing three different protocols of the comet assay technique with respect to the analysis of the induction of DNA damage after X-irradiation in isolated human lymphocytes and CHO cells. In a second part, the restriction enzyme AluI, an agent producing DNA double-strand breaks exclusively, was introduced into CHO cells by electroporation and the effects were analyzed using the different comet assay protocols. The experiments were carried out in order to test the assertion that comet assay techniques can measure different types of DNA damages at different pH conditions of lysis and electrophoresis. Material and methods: three different comet assay protocols were used for the analysis of DNA damage in lymphocytes and CHO cells. Results: the results clearly indicate that among the three protocols the modified comet assay technique used by the authors showed the highest sensitivity in the radiotherapy-relevant dose range between 0 and 2 Gy. All three protocols were capable of detecting an effect by AluI. This effect, however, was clearly different from radiation effects. Whereas after radiation exposure all cell nuclei show a dose-dependent increase in DNA content in the comet tail, most of the cell nuclei were unaffected by an AluI uptake. Nevertheless, there was an effect by AluI that could be detected in all three assay versions: between 5% and 15% of the nuclei showed clearly abnormal comet morphologies. Conclusion: neither the strictly alkaline nor the strictly neutral comet assay is applicable in the radiation dose range of about 2 Gy. The restriction enzyme results show that other factors than just DNA strand breaks contribute to DNA migration into the tail of the comets. (orig.)

  3. Alu Sb2 subfamily is present in all higher primates but was most succesfully amplified in humans

    Energy Technology Data Exchange (ETDEWEB)

    Richer, C.; Zietkiewicz, E.; Labuda, D. [Universite de Montreal, Que (Canada)

    1994-09-01

    Alu repeats can be classified into subfamilies which amplified in primate genomes at different evolutionary time periods. A young Alu subfamily, Sb2, with a characteristic 7-nucleotide duplication at position 256, has been described in seven human loci. An Sb2 insertion found near the HD gene was unique to two HD families, indicating that Sb2 was still retropositionally active. Here, we have shown that the Sb2 insertion in the CHOL locus was similarly rare, being absent in 120 individuals of Caucasian, Oriental and Black origin. In contrast, Sb2 inserts in five other loci were found fixed (non-polymorphic), based on measurements in the same population sample, but absent from orthologous positions in higher apes. This suggest that Sb2 repeats spread relatively early in the human lineage following divergence from other primates and that these elements may be human-specific. By quantitative PCR, we investigated the presence of Sb2 sequences in different primate DNA, using one PCR primer anchored at the 5{prime} Alu-end and the other complementary to the duplicated Sb2-specific segment. With an Sb2-containing plasmid as a standard, we estimated the number of Sb2 repeats at 1500-1800 copies per human haploid equivalent; corresponding numbers in chimpanzee and gorilla were almost two orders of magnitude lower, while the signal observed in orangutan and gibbon DNAs was consistent with the presence of a single copy. The analysis of 22 human, 11 chimpanzee and 10 gorilla sequences indicates that the Alu Sb2 dispersed independently in these three primate lineages; gorilla consensus differs from the human Sb2 sequence by one position, while all chimpanzee repeats have their linker expanded by up to eight A-residues. Should they be thus considered as separate subfamilies? It is possible that sequence modifications with respect to the human consensus are responsible for poor retroposition of Sb2 in apes.

  4. RNA-DNA Differences Are Generated in Human Cells within Seconds After RNA Exits Pol II

    OpenAIRE

    Isabel X. Wang; Leighton J. Core; Hojoong Kwak; Lauren Brady; Alan Bruzel; Lee McDaniel; Allison L. Richards; Ming Wu; Christopher Grunseich; John T. Lis; Vivian G. Cheung

    2014-01-01

    RNA sequences are expected to be identical to their corresponding DNA sequences. Here, we found all 12 types of RNA-DNA sequence differences (RDDs) in nascent RNA. Our results show that RDDs begin to occur in RNA chains about 55 nucleotides from the RNA polymerase II (Pol II) active site. These RDDs occur so soon after transcription that they are incompatible with known deaminase-mediated RNA editing mechanisms. Moreover, the 55-nucleotide delay in appearance indicates they do not arise durin...

  5. A novel PCR technique using Alu-specific primers to identify unknown flanking sequences from the human genome

    Energy Technology Data Exchange (ETDEWEB)

    Minami, M.; Poussin, K.; Brechot, C.; Paterlini, P. [INSERM, Paris (France)

    1995-09-20

    The rapid and reproducible identification of new cellular DNA sequences is difficult to achieve with the currently available procedures. Here we describe a novel approach based on the polymerase chain reaction (PCR) using a primer specific to the known sequence and another directed to a human Alu repeat. To avoid undesirable amplifications between Alu sequences, primers are constructed with dUTPs and destroyed by uracil DNA glycosylase treatment after 10 initial cycles of amplification. Only desirable fragments are then further amplified with specific primers to the known region and to a tag sequence introduced in the Alu-specific primer. Using this protocol, we have successfully indentified cellular sequences flanking integrated hepatitis B virus DNA from the human genome of three hepatoma tissues. The method enables a direct specific amplification without any ligation or nonspecific annealing steps as required by previous PCR-based protocols. This rapid and straightforward approach will be a powerful tool for the study of viral integration sites, but is also widely applicable to other studies of the human genome. 39 refs., 4 figs.

  6. The editing sites in transcripts of functional genes of rice mitochondria

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    RNA editing exists extensively in the higher plant mitochondria, and is a required step for forming functional proteins. There may be some relationship between RNA editing and cytoplasmic male sterility (CMS), a kind of phenomenon that is attributed to mitochondrial genome mutations. The research materials used are the gametophytic male sterility line (A), maintainer line (B) and F1 hybrid (F1) of HL-type CMS rice. cDNAs and DNAs of atp6 and coxII have been obtained from A, B and F1 by PCR and RT-PCR. Comparing sequences of cDNAs and DNAs, 18 and 15 editing sites were found respectively in the transcripts of atp6 and coxII. A, B and F1 shared the same editing sites. RNA editing improves hydrophobicity and conservation of the predicted protein as compared with other organisms.

  7. Genome Editing in Sugarcane: Challenges Ahead

    Science.gov (United States)

    Mohan, Chakravarthi

    2016-01-01

    Genome editing opens new and unique opportunities for researchers to enhance crop production. Until 2013, the zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) were the key tools used for genome editing applications. The advent of RNA-guided engineered nucleases - the type II clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 (CRISPR-associated) system from Streptococcus pyogenes holds great potential since it is simple, effective and more versatile than ZFNs and TALENs. CRISPR/Cas9 system has already been successfully employed in several crop plants. Use of these techniques is in its infant stage in sugarcane. Jung and Altpeter (2016) have reported TALEN mediated approach for the first time to reduce lignin content in sugarcane to make it amenable for biofuel production. This is so far the only report describing genome editing in sugarcane. Large genome size, polyploidy, low transformation efficiency, transgene silencing and lack of high throughput screening techniques are certainly great challenges for genome editing in sugarcane which would be discussed in detail in this review. PMID:27790238

  8. Sequences more than 500 base pairs upstream of the human U3 small nuclear RNA gene stimulate the synthesis of U3 RNA in frog oocytes

    International Nuclear Information System (INIS)

    Small nuclear RNA (snRNA) genes contain strong promoters capable of initiating transcription once every 4 s. Studies on the human U1 snRNA gene, carried out in other laboratories, showed that sequences within 400 bp of the 5' flanking region are sufficient for maximal levels of transcription both in vivo and in frog oocytes [reviewed in Dahlberg and Lund (1988)]. The authors studied the expression of a human U3 snRNA gene by injecting 5' deletion mutants into frog oocytes. The results show that sequences more than 500 bp upstream of the U3 snRNA gene have a 2-3-fold stimulatory effect on the U3 snRNA synthesis. These results indicate that the human U3 snRNA gene is different from human U1 snRNA gene in containing regulatory elements more than 500 bp upstream. The U3 snRNA gene upstream sequences contain an AluI homologous sequence in the -1,200 region; these AluI sequences were transcribed in vitro and in frog oocytes but were not detectable in Hela cells

  9. 不同ALU实现方法的功耗研究%The Research on Power Dissipation of Different ALU Implementation Schemes

    Institute of Scientific and Technical Information of China (English)

    孙军凯; 蒋安平

    2011-01-01

    Low power is a challenging work in micro - processor design. Implementing power optimization on all components of the processor is a choice. One of the most basic components in micro -processor is the Arithmetic and Logic Unit (ALU). The architecture of ALU has several implications on power consumption, delay and area. There are three common ALU architectures: complex architecture, adder independent architecture and chain architecture. To find out which ALU architecture provides the best power efficiency, an 8 - bit ALU of the three different architectures is designed. Compared with other architectures,the power savings of complex architecture are 19. 38% and 33. 87% .%低功耗是微处理器设计中一项具有挑战性的工作.对每一个组成单元进行功耗优化是进行低功耗微处理器设计必不可少的一种方法.算术逻辑单元(Arithmetic and Logic Unit,ALU)是微处理器中最基本的组成单元之一.ALU的结构与功耗、延迟和面积有着复杂的联系.常用的ALU结构有三种:复合结构、加法器独立结构和链武结构.基于这三种结构,实现了一个8比特ALU,通过对这个8 - bit ALU进行功耗分析来研究ALU的结构对功耗的影响.研究结果表明:复合结构ALU具有最小的功耗,与其它两种结构的ALU相比,能分别节省19.38%和33.87%的功耗.

  10. Questioning the "melting pot": analysis of Alu inserts in three population samples from Uruguay.

    Science.gov (United States)

    Hidalgo, Pedro C; Mut, Patricia; Ackermann, Elizabeth; Figueiro, Gonzalo; Sans, Monica

    2014-01-01

    The way that immigrants integrate into recipient societies has been discussed for decades, mainly from the perspective of the social sciences. Uruguay, as other American countries, received diffferent waves of European immigrants, although the details of the process of assimilation, when it did occur, are unclear. In this study we used genetic markers to understand the process experienced by the Basques, one of the major migration waves that populated Uruguay, and their relation to other immigrants, as well as to Native American and African descendants. For this purpose, we analyzed the allele frequencies of 10 ALU loci (A25, ACE, APOA1, B65, D1, F13B, PV92, TPA25, HS2.43, and HS4.65) in three samples from Uruguay (two of Basque descendants, one of non-Basque descendants) from two locations: Montevideo and Trinidad. No departure from Hardy-Weinberg expectations was observed, with the exceptions of the APOA1 and D1 loci in the non-Basque descendants' samples. Our data show that the major genetic contribution in the three samples comes from Europe (78-88%), with minor African (10-15%) and Native American (0-10%) contributions. Genetic distances reveal that Basque descendants from Trinidad cluster with Europeans, whereas both Montevideo samples cluster together and are separate from other populations, showing two diffferent types of integration, related to the general characteristics of each regional population.

  11. Questioning the "melting pot": analysis of Alu inserts in three population samples from Uruguay.

    Science.gov (United States)

    Hidalgo, Pedro C; Mut, Patricia; Ackermann, Elizabeth; Figueiro, Gonzalo; Sans, Monica

    2014-01-01

    The way that immigrants integrate into recipient societies has been discussed for decades, mainly from the perspective of the social sciences. Uruguay, as other American countries, received diffferent waves of European immigrants, although the details of the process of assimilation, when it did occur, are unclear. In this study we used genetic markers to understand the process experienced by the Basques, one of the major migration waves that populated Uruguay, and their relation to other immigrants, as well as to Native American and African descendants. For this purpose, we analyzed the allele frequencies of 10 ALU loci (A25, ACE, APOA1, B65, D1, F13B, PV92, TPA25, HS2.43, and HS4.65) in three samples from Uruguay (two of Basque descendants, one of non-Basque descendants) from two locations: Montevideo and Trinidad. No departure from Hardy-Weinberg expectations was observed, with the exceptions of the APOA1 and D1 loci in the non-Basque descendants' samples. Our data show that the major genetic contribution in the three samples comes from Europe (78-88%), with minor African (10-15%) and Native American (0-10%) contributions. Genetic distances reveal that Basque descendants from Trinidad cluster with Europeans, whereas both Montevideo samples cluster together and are separate from other populations, showing two diffferent types of integration, related to the general characteristics of each regional population. PMID:25397699

  12. Precision genome editing

    DEFF Research Database (Denmark)

    Steentoft, Catharina; Bennett, Eric P; Schjoldager, Katrine Ter-Borch Gram;

    2014-01-01

    of glycobiology, primarily due to their low efficiencies, with resultant failure to impose substantial phenotypic consequences upon the final glycosylation products. Here, we review novel nuclease-based precision genome editing techniques enabling efficient and stable gene editing, including gene disruption...... by introducing single or double-stranded breaks at a defined genomic sequence. We here compare and contrast the different techniques and summarize their current applications, highlighting cases from the field of glycobiology as well as pointing to future opportunities. The emerging potential of precision gene...

  13. Edit Distance for Timed Automata

    OpenAIRE

    Chatterjee, Krishnendu; Ibsen-Jensen, Rasmus; Majumdar, Rupak

    2013-01-01

    The edit distance between two (untimed) traces is the minimum cost of a sequence of edit operations (insertion, deletion, or substitution) needed to transform one trace to the other. Edit distances have been extensively studied in the untimed setting, and form the basis for approximate matching of sequences in different domains such as coding theory, parsing, and speech recognition. In this paper, we lift the study of edit distances from untimed languages to the timed setting. We define ...

  14. CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani

    OpenAIRE

    Zhang, Wen-Wei; Matlashewski, Greg

    2015-01-01

    ABSTRACT The prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promo...

  15. Editing graphs for maximum effect

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, P.W.; Rhiner, R.W.

    1991-01-08

    The paper contains over eighty rules for editing graphs, arranged under nine major headings in a logical sequence for editing all the graphs in a manuscript. It is excerpted from a monograph used at the Lawrence Livermore National Laboratory to train beginning technical editors in editing graphs; a corresponding Hypercard stack is also used in this training. 6 refs., 4 figs.

  16. Beginning to edit physics

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, P.W.

    1995-02-01

    A physicist-turned-editor shows you the basics required for copyediting physics papers (physical quantities, symbols, units, scientific notation, the structure of mathematical expressions, the nature of graphs), and points the way to learning enough ``editorial physics`` to begin substantive editing.

  17. The Craft of Editing

    DEFF Research Database (Denmark)

    Moeran, Brian

    To edit is to make a choice, or series of choices. Will I write a rough draft of this essay in longhand, or hammer it out on my computer? If the latter, what font shall I use? Times New Roman, Book Antiqua, or Garamond? Once I get started, what style shall I adopt: realistic, confessional or impr...

  18. Simple Genome Editing of Rodent Intact Embryos by Electroporation.

    Directory of Open Access Journals (Sweden)

    Takehito Kaneko

    Full Text Available The clustered regularly interspaced short palindromic repeat (CRISPR/CRISPR-associated (Cas system is a powerful tool for genome editing in animals. Recently, new technology has been developed to genetically modify animals without using highly skilled techniques, such as pronuclear microinjection of endonucleases. Technique for animal knockout system by electroporation (TAKE method is a simple and effective technology that produces knockout rats by introducing endonuclease mRNAs into intact embryos using electroporation. Using TAKE method and CRISPR/Cas system, the present study successfully produced knockout and knock-in mice and rats. The mice and rats derived from embryos electroporated with Cas9 mRNA, gRNA and single-stranded oligodeoxynucleotide (ssODN comprised the edited targeted gene as a knockout (67% of mice and 88% of rats or knock-in (both 33%. The TAKE method could be widely used as a powerful tool to produce genetically modified animals by genome editing.

  19. Selection of tRNA charging quality control mechanisms that increase mistranslation of the genetic code

    DEFF Research Database (Denmark)

    Yadavalli, Srujana S; Ibba, Michael

    2013-01-01

    Mistranslation can follow two events during protein synthesis: production of non-cognate amino acid:transfer RNA (tRNA) pairs by aminoacyl-tRNA synthetases (aaRSs) and inaccurate selection of aminoacyl-tRNAs by the ribosome. Many aaRSs actively edit non-cognate amino acids, but editing mechanisms...

  20. Edit Distance for Pushdown Automata

    OpenAIRE

    Chatterjee, Krishnendu; Henzinger, Thomas A.; Ibsen-Jensen, Rasmus; Otop, Jan

    2015-01-01

    The edit distance between two words $w_1, w_2$ is the minimal number of word operations (letter insertions, deletions, and substitutions) necessary to transform $w_1$ to $w_2$. The edit distance generalizes to languages ${\\cal L}_1, {\\cal L}_2$, where the edit distance is the minimal number $k$ such that for every word from ${\\cal L}_1$ there exists a word in ${\\cal L}_2$ with edit distance at most $k$. We study the edit distance computation problem between pushdown automata and their subclas...

  1. A New Implementation of a 16-Bit Self-Timed ALU for Asynchronous Microprocessors%适用于异步微处理器的1 6位自定时ALU

    Institute of Scientific and Technical Information of China (English)

    管超; 葛元庆; 吴瑞; 周润德

    2001-01-01

    针对嵌入式微处理器设计中提出的高性能,低功耗的要求,提出了一种面向异步微处理器的由动态电压级联逻辑电路(DCVS)构成的16位自定时ALU.在综合考虑面积、速度、功耗及指令的统计分布情况下,该ALU具有优异的性能.

  2. Replication of cloned DNA containing the Alu family sequence during cell extract-promoting simian virus 40 DNA synthesis.

    OpenAIRE

    Ariga, H

    1984-01-01

    The replicating activity of several cloned DNAs containing putative origin sequences was examined in a cell-free extract that absolutely depends on simian virus 40 (SV40) T antigen promoting initiation of SV40 DNA replication in vitro. Of the three DNAs containing the human Alu family sequence (BLUR8), the origin of (Saccharomyces cerevisiae plasmid 2 micron DNA (pJD29), and the yeast autonomous replicating sequence (YRp7), only BLUR8 was active as a template. Replication in a reaction mixtur...

  3. The CRISPR/Cas genome-editing tool: application in improvement of crops

    Directory of Open Access Journals (Sweden)

    SURENDER eKHATODIA

    2016-04-01

    Full Text Available The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR associated Cas9/sgRNA system is a novel fledgling targeted genome-editing technique from bacterial immune system, which is a cheap, easy and most rapidly adopted genome editing tool transforming to revolutionary paradigm. Cas9 protein is an RNA guided endonuclease utilized for creating targeted double stranded breaks with only a short RNA sequence to confer recognition of the target in animals and plants. Development of genetically edited (GE crops similar to those developed by conventional or mutation breeding using this potential technique makes it a promising and extremely versatile tool for providing sustainable productive agriculture for better feeding of rapidly growing population in changing climate. The emerging areas of research for the genome editing in plants are like, interrogating gene function, rewiring the regulatory signaling networks, sgRNA library for high-throughput loss-of-function screening. In this review, we will discuss the broad applicability of the Cas9 nuclease mediated targeted plant genome editing for development of designer crops. The regulatory uncertainty and social acceptance of plant breeding by Cas9 genome editing have also been discussed. The non-GM designer genetically edited plants could prospect climate resilient and sustainable energy agriculture in coming future for maximizing the yield by combating abiotic and biotic stresses with this new innovative plant breeding technique.

  4. 苯乙酸抑制胰腺癌BXPC-3细胞增殖及RNA编辑酶表达的研究%Effects of phenylacetate on proliferation and RNA editing deaminase expression of human pancreatic carcinoma BXPC-3 cell lines

    Institute of Scientific and Technical Information of China (English)

    姜涛; 王岩; 任辉; 张广; 刘安安; 田宇

    2010-01-01

    Objective To evaluate the differentiation-inducing effect of phenylacetate on pancreatic carcinoma cells and underlying mechanism of RNA editing deaminase in cell proliferation and differentiation. Methods The effect of phenylacetate on cell proliferation and cell cycle were investigated in cultured pancreatic carcinoma BXPC-3 cell lines by methylthiazoletetrazolium( MTT) assay, and flow cytometry. The effect of phenylacetate on the expression of RNA editing deaminase (ADAR2 mRNA) in BXPC-3 cells and fresh pancreatic carcinoma specimen was evaluated by RT-PCR. Results ADAR1 mRNA expression was positive in human pancreatic carcinoma tissues and BXPC-3 cell lines. After application of 1.0 and 2.0 mmol/L phenylacetate for 24 h and 72 h, BXPC-3 cell growth inhibition rate increased, G0/G1 phase cells decreased, S phase cells increased, ADAR2 mRNA expression decreased ( P < 0.01 ). Conclusion ADAR2 mRNA played a vital role of regulation in the process of pancreatic carcinoma cells growth and differentiation. Phenylacetate could regulate the proliferation and differentiation of pancreatic carcinoma cells through the regulation of ADAR2 mRNA expression.%目的 探讨苯乙酸对胰腺癌诱导分化作用及RNA编辑在胰腺癌细胞增殖分化过程中的作用机理.方法 采用MTT比色法和流式细胞术检测苯乙酸对胰腺癌BXPC-3细胞系增殖抑制作用及对BXPC-3细胞周期的影响.RT-PCR方法检测RNA编辑酶ADAR mRNA在胰腺癌细胞系和胰腺癌组织中的表达以及应用苯乙酸后ADAR mRNA表达的变化.结果 人胰腺癌组织及胰腺癌BXPC-3细胞中均可检测到编辑酶ADAR2.应用1.0及2.0mmol/L苯乙酸后24h及72 h,BXPC-3细胞增殖抑制率增加,G0/G1期比例下降,S期比例显著升高.ADAR2 mRNA表达降低,差异均有统计学意义(P<0.01).结论 ADAR2可能在胰腺细胞增殖分化过程中发挥作用,且苯乙酸可能通过调控ADAR2 mRNA的表达来发挥诱导分化作用.

  5. Psychology. 5th edition

    OpenAIRE

    Martin, G. Neil; Carlson, Neil R.; Buskist, William

    2013-01-01

    A comprehensive, lively and engaging introduction to the fascinating study of the subject. The fifth edition of the best-selling Psychology is a contemporary text that will captivate all psychology students. The authors describe and explore every major area of psychology and present the latest findings, along with clear evaluation of controversial theories and models, to give a rigorous and critical grounding in the subject. Over 420 new references in this thoroughly updated fifth editi...

  6. Interactive Metro Map Editing.

    Science.gov (United States)

    Wang, Yu-Shuen; Peng, Wan-Yu

    2016-02-01

    Manual editing of a metro map is essential because many aesthetic and readability demands in map generation cannot be achieved by using a fully automatic method. In addition, a metro map should be updated when new metro lines are developed in a city. Considering that manually designing a metro map is time-consuming and requires expert skills, we present an interactive editing system that considers human knowledge and adjusts the layout to make it consistent with user expectations. In other words, only a few stations are controlled and the remaining stations are relocated by our system. Our system supports both curvilinear and octilinear layouts when creating metro maps. It solves an optimization problem, in which even spaces, route straightness, and maximum included angles at junctions are considered to obtain a curvilinear result. The system then rotates each edge to extend either vertically, horizontally, or diagonally while approximating the station positions provided by users to generate an octilinear layout. Experimental results, quantitative and qualitative evaluations, and user studies show that our editing system is easy to use and allows even non-professionals to design a metro map.

  7. Itella logistiikan yrityskaupan yhteydessä ilmenneet päällekkäisyydet ja ongelmat Oulusta ajettavissa alue- ja runkokuljetuksissa

    OpenAIRE

    Niemelä, Hannes

    2014-01-01

    Työn aiheena on Itella Logistics Oyj:n ja VR Transtpointin yhdistymisestä aiheutuneiden ongelmien ja päällekkäisyyksien selvittäminen Oulusta ajettaviin alue- ja runkovuoroihin. Työn tavoitteena oli selvittää syntyneet ongelmat ja niihin jo tehdyt ratkaisut sekä miettiä mahdollisia ratkaisuehdotuksia selvittämättömiin ongelmiin. Työssä esitellään myös yksityiskohtaisesti Itella Logistiikan Oulusta ajettavat alue- ja runkovuorot. Työ suoritettiin teoreettisen tutkimuksen ja pääasiassa työss...

  8. Genome Editing in Human Pluripotent Stem Cells.

    Science.gov (United States)

    Smith, Cory; Ye, Zhaohui; Cheng, Linzhao

    2016-01-01

    Pluripotent stem cells (PSCs), defined by their capacity for self-renewal and differentiation into all cell types, are an integral tool for basic biological research and disease modeling. However, full use of PSCs for research and regenerative medicine requires the ability to precisely edit their DNA to correct disease-causing mutations and for functional analysis of genetic variations. Recent advances in DNA editing of human stem cells (including PSCs) have benefited from the use of designer nucleases capable of making double-strand breaks (DSBs) at specific sequences that stimulate endogenous DNA repair. The clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has become the preferred designer nuclease for genome editing in human PSCs and other cell types. Here we describe the principles for designing a single guide RNA to uniquely target a gene of interest and describe strategies for disrupting, inserting, or replacing a specific DNA sequence in human PSCs. The improvements in efficiency and ease provided by these techniques allow individuals to precisely engineer PSCs in a way previously limited to large institutes and core facilities. PMID:27037079

  9. 稳定高效表达正反义Alu-Sx细胞系的建立%Establishment of HEK293 cell line expressing sense and antisense Alu-Sx stably and efficiently

    Institute of Scientific and Technical Information of China (English)

    彭亮; 吴刚; 蒋继志; 靳风烁; 丁瑞

    2006-01-01

    目的建立稳定高效表达正反义Alu-Sx的细胞系.方法根据Alu亚家族Sx序列合成两对两端带有限制性酶切位点的引物,提取HEK293细胞的总DNA后PCR扩增,产物连接到真核表达载体pcDNA3.1/myc-His A中构建成重组体.经酶切及测序鉴定后,阳离子脂质体转染法转染HEK293细胞后G418筛选稳定表达的细胞克隆,Northern杂交检测并挑选出表达最强的亚克隆.结果成功构建Alu亚家族Sx的正反义真核表达载体并获得高效稳定表达的HEK293细胞亚克隆.结论高效稳定表达正反义Alu Sx的HEK293细胞亚克隆可用于下一步研究.

  10. Alu-alu recombination results in a duplication of seven exons in the lysyl hydroxylase gene in a patient with the type VI variant of Ethlers-Danlos syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Pousi, B.; Hautala, T.; Heikkinen, J.; Pajunen, L.; Kivirikko, K.I.; Myllylae, R. [Univ. of Oulu (Finland)

    1994-11-01

    The type VI variant of the Ethlers-Danlos syndrome (EDS) is a recessively inherited connective-tissue disorder. The characteristic features of the variant are muscular hyptonia, kyphoscoliosis, ocular manifestations, joint hypermobility, skin fragility and hyperextensibility, and other signs of connective-tissue involvement. The biochemical defect in most but not all patients is a deficiency in lysyl hydroxylase activity. Lysyl hydroxylase is an enzyme that catalyzes the formation of hydroxylysine in collagens and other proteins with collagen-like amino acid sequences. We have recently reported an apparently homozygous large-duplication rearrangement in the gene for lysyl hydroxylase, leading to the type VI variant of EDS in two siblings. We now report an identical, apparently homozygous large duplication in an unrelated 49-year-old female originally analyzed by Sussman et al. Our simple-sequence-repeat-polymorphism analysis does not support uniparental isodisomy inheritance for either of the two duplications. Furthermore, we indicate in this study that the duplication in the lysyl hydroxylase gene is caused by an Alu-Alu recombination in both families. Cloning of the junction fragment of the duplication has allowed synthesis of appropriate primers for rapid screening for this rearrangement in other families with the type VI variant of EDS. 38 refs., 6 figs.

  11. Efficient Mitochondrial Genome Editing by CRISPR/Cas9

    Directory of Open Access Journals (Sweden)

    Areum Jo

    2015-01-01

    Full Text Available The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9 system has been widely used for nuclear DNA editing to generate mutations or correct specific disease alleles. Despite its flexible application, it has not been determined if CRISPR/Cas9, originally identified as a bacterial defense system against virus, can be targeted to mitochondria for mtDNA editing. Here, we show that regular FLAG-Cas9 can localize to mitochondria to edit mitochondrial DNA with sgRNAs targeting specific loci of the mitochondrial genome. Expression of FLAG-Cas9 together with gRNA targeting Cox1 and Cox3 leads to cleavage of the specific mtDNA loci. In addition, we observed disruption of mitochondrial protein homeostasis following mtDNA truncation or cleavage by CRISPR/Cas9. To overcome nonspecific distribution of FLAG-Cas9, we also created a mitochondria-targeted Cas9 (mitoCas9. This new version of Cas9 localizes only to mitochondria; together with expression of gRNA targeting mtDNA, there is specific cleavage of mtDNA. MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition. This mitoCas9 could be applied to edit mtDNA together with gRNA expression vectors without affecting genomic DNA. In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9. Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing.

  12. Revising and editing for translators

    CERN Document Server

    Mossop, Brian

    2014-01-01

    Revising and Editing for Translators provides guidance and learning materials for translation students learning to edit texts written by others, and professional translators wishing to improve their self-revision ability or learning to revise the work of others. Editing is understood as making corrections and improvements to texts, with particular attention to tailoring them to the given readership. Revising is this same task applied to draft translations. The linguistic work of editors and revisers is related to the professional situations in which they work. Mossop offers in-depth coverage of a wide range of topics, including copyediting, style editing, structural editing, checking for consistency, revising procedures and principles, and translation quality assessment. This third edition provides extended coverage of computer aids for revisers, and of the different degrees of revision suited to different texts. The inclusion of suggested activities and exercises, numerous real-world examples, a proposed gra...

  13. Optimization of genome editing through CRISPR-Cas9 engineering.

    Science.gov (United States)

    Zhang, Jian-Hua; Adikaram, Poorni; Pandey, Mritunjay; Genis, Allison; Simonds, William F

    2016-04-01

    CRISPR (Clustered Regularly-Interspaced Short Palindromic Repeats)-Cas9 (CRISPR associated protein 9) has rapidly become the most promising genome editing tool with great potential to revolutionize medicine. Through guidance of a 20 nucleotide RNA (gRNA), CRISPR-Cas9 finds and cuts target protospacer DNA precisely 3 base pairs upstream of a PAM (Protospacer Adjacent Motif). The broken DNA ends are repaired by either NHEJ (Non-Homologous End Joining) resulting in small indels, or by HDR (Homology Directed Repair) for precise gene or nucleotide replacement. Theoretically, CRISPR-Cas9 could be used to modify any genomic sequences, thereby providing a simple, easy, and cost effective means of genome wide gene editing. However, the off-target activity of CRISPR-Cas9 that cuts DNA sites with imperfect matches with gRNA have been of significant concern because clinical applications require 100% accuracy. Additionally, CRISPR-Cas9 has unpredictable efficiency among different DNA target sites and the PAM requirements greatly restrict its genome editing frequency. A large number of efforts have been made to address these impeding issues, but much more is needed to fully realize the medical potential of CRISPR-Cas9. In this article, we summarize the existing problems and current advances of the CRISPR-Cas9 technology and provide perspectives for the ultimate perfection of Cas9-mediated genome editing.

  14. Editing at the crossroad of innate and adaptive immunity.

    Science.gov (United States)

    Turelli, Priscilla; Trono, Didier

    2005-02-18

    Genetic information can be altered through the enzymatic modification of nucleotide sequences. This process, known as editing, was originally identified in the mitochondrial RNA of trypanosomes and later found to condition events as diverse as neurotransmission and lipid metabolism in mammals. Recent evidence reveals that editing enzymes may fulfill one of their most essential roles in the defense against infectious agents: first, as the mediators of antibody diversification, a step crucial for building adaptive immunity, and second, as potent intracellular poisons for the replication of viruses. Exciting questions are raised, which take us to the depth of the intimate relations between vertebrates and the microbial underworld.

  15. In vivo genome editing using Staphylococcus aureus Cas9

    OpenAIRE

    Ran, F Ann; Cong, Le; Yan, Winston X.; Scott, David A.; Gootenberg, Jonathan S.; Kriz, Andrea J.; Zetsche, Bernd; Shalem, Ophir; Wu, Xuebing; Makarova, Kira S.; Koonin, Eugene; Sharp, Phillip A.; Zhang, Feng

    2015-01-01

    The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that employ the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologs and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being >1...

  16. Contribution of Large Genomic Rearrangements in Italian Lynch Syndrome Patients: Characterization of a Novel Alu-Mediated Deletion

    Directory of Open Access Journals (Sweden)

    Francesca Duraturo

    2013-01-01

    Full Text Available Lynch syndrome is associated with germ-line mutations in the DNA mismatch repair (MMR genes, mainly MLH1 and MSH2. Most of the mutations reported in these genes to date are point mutations, small deletions, and insertions. Large genomic rearrangements in the MMR genes predisposing to Lynch syndrome also occur, but the frequency varies depending on the population studied on average from 5 to 20%. The aim of this study was to examine the contribution of large rearrangements in the MLH1 and MSH2 genes in a well-characterised series of 63 unrelated Southern Italian Lynch syndrome patients who were negative for pathogenic point mutations in the MLH1, MSH2, and MSH6 genes. We identified a large novel deletion in the MSH2 gene, including exon 6 in one of the patients analysed (1.6% frequency. This deletion was confirmed and localised by long-range PCR. The breakpoints of this rearrangement were characterised by sequencing. Further analysis of the breakpoints revealed that this rearrangement was a product of Alu-mediated recombination. Our findings identified a novel Alu-mediated rearrangement within MSH2 gene and showed that large deletions or duplications in MLH1 and MSH2 genes are low-frequency mutational events in Southern Italian patients with an inherited predisposition to colon cancer.

  17. Research of the origin of a particular Tunisian group using a physical marker and Alu insertion polymorphisms

    Directory of Open Access Journals (Sweden)

    Wifak El Moncer

    2011-01-01

    Full Text Available The aim of this study was to show how, in some particular circumstances, a physical marker can be used along with molecular markers in the research of an ancient people movement. A set of five Alu insertions was analysed in 42 subjects from a particular Tunisian group (El Hamma that has, unlike most of the Tunisian population, a very dark skin, similar to that of sub-Saharans, and in 114 Tunisian subjects (Gabes sample from the same governorate, but outside the group. Our results showed that the El Hamma group is genetically midway between sub-Saharan populations and North Africans, whereas the Gabes sample is clustered among North Africans. In addition, The A25 Alu insertion, considered characteristic to sub-Saharan Africans, was present in the El Hamma group at a relatively high frequency. This frequency was similar to that found in sub-Saharans from Nigeria, but significantly different from those found in the Gabes sample and in other North African populations. Our molecular results, consistent with the skin color status, suggest a sub-Saharan origin of this particular Tunisian group.

  18. Structural organization of glycophorin A and B genes: Glycophorin B gene evolved by homologous recombination at Alu repeat sequences

    International Nuclear Information System (INIS)

    Glycophorins A (GPA) and B (GPB) are two major sialoglycoproteins of the human erythrocyte membrane. Here the authors present a comparison of the genomic structures of GPA and GPB developed by analyzing DNA clones isolated from a K562 genomic library. Nucleotide sequences of exon-intron junctions and 5' and 3' flanking sequences revealed that the GPA and GPB genes consist of 7 and 5 exons, respectively, and both genes have >95% identical sequence from the 5' flanking region to the region ∼ 1 kilobase downstream from the exon encoding the transmembrane regions. In this homologous part of the genes, GPB lacks one exon due to a point mutation at the 5' splicing site of the third intron, which inactivates the 5' cleavage event of splicing and leads to ligation of the second to the fourth exon. Following these very homologous sequences, the genomic sequences for GPA and GPB diverge significantly and no homology can be detected in their 3' end sequences. The analysis of the Alu sequences and their flanking direct repeat sequences suggest that an ancestral genomic structure has been maintained in the GPA gene, whereas the GPB gene has arisen from the acquisition of 3' sequences different from those of the GPA gene by homologous recombination at the Alu repeats during or after gene duplication

  19. Edit Distance with Block Deletions

    OpenAIRE

    Dana Shapira; Storer, James A.

    2011-01-01

    Several variants of the edit distance problem with block deletions are considered. Polynomial time optimal algorithms are presented for the edit distance with block deletions allowing character insertions and character moves, but without block moves. We show that the edit distance with block moves and block deletions is NP-complete (Nondeterministic Polynomial time problems in which any given solution to such problem can be verified in polynomial time, and any NP problem can be converted into...

  20. ADAR2 editing activity in newly diagnosed versus relapsed pediatric high-grade astrocytomas

    International Nuclear Information System (INIS)

    High-grade (WHO grade III and IV) astrocytomas are aggressive malignant brain tumors affecting humans with a high risk of recurrence in both children and adults. To date, limited information is available on the genetic and molecular alterations important in the onset and progression of pediatric high-grade astrocytomas and, even less, on the prognostic factors that influence long-term outcome in children with recurrence. A-to-I RNA editing is an essential post-transcriptional mechanism that can alter the nucleotide sequence of several RNAs and is mediated by the ADAR enzymes. ADAR2 editing activity is particularly important in mammalian brain and is impaired in both adult and pediatric high-grade astrocytomas. Moreover, we have recently shown that the recovered ADAR2 activity in high-grade astrocytomas inhibits in vivo tumor growth. The aim of the present study is to investigate whether changes may occur in ADAR2-mediated RNA editing profiles of relapsed high-grade astrocytomas compared to their respective specimens collected at diagnosis, in four pediatric patients. Total RNAs extracted from all tumor samples and controls were tested for RNA editing levels (by direct sequencing on cDNA pools) and for ADAR2 mRNA expression (by qRT-PCR). A significant loss of ADAR2-editing activity was observed in the newly diagnosed and recurrent astrocytomas in comparison to normal brain. Surprisingly, we found a substantial rescue of ADAR2 editing activity in the relapsed tumor of the only patient showing prolonged survival. High-grade astrocytomas display a generalized loss of ADAR2-mediated RNA editing at both diagnosis and relapse. However, a peculiar Case, in complete remission of disease, displayed a total rescue of RNA editing at relapse, intriguingly suggesting ADAR2 activity/expression as a possible marker for long-term survival of patients with high-grade astrocytomas

  1. A contextual normalised edit distance

    OpenAIRE

    Higuera, Colin de la; Micó Andrés, Luisa

    2008-01-01

    In order to better fit a variety of pattern recognition problems over strings, using a normalised version of the edit or Levenshtein distance is considered to be an appropriate approach. The goal of normalisation is to take into account the lengths of the strings. We define a new normalisation, contextual, where each edit operation is divided by the length of the string on which the edit operation takes place. We prove that this contextual edit distance is a metric and that it can be...

  2. A clique-based method for the edit distance between unordered trees and its application to analysis of glycan structures

    OpenAIRE

    2011-01-01

    Background Measuring similarities between tree structured data is important for analysis of RNA secondary structures, phylogenetic trees, glycan structures, and vascular trees. The edit distance is one of the most widely used measures for comparison of tree structured data. However, it is known that computation of the edit distance for rooted unordered trees is NP-hard. Furthermore, there is almost no available software tool that can compute the exact edit distance for unordered trees. Result...

  3. Studies of the Haplotypes of CTG Triplet Repeat and Alu±1kb in DMPK Gene of Myotonic Dystrophy%强直性肌营养不良症DMPK基因CTG重复序列与Alu±1kb单倍型研究

    Institute of Scientific and Technical Information of China (English)

    肖翠英; 武辉; 潘阿根; 张思仲

    2000-01-01

    强直性肌营养不良(myotonic dystrophy,DM)是由于DMPK基因3′非翻译区CTG重复序列异常扩展所致的、主要累及神经肌肉系统的常染色体显性遗传病.在该基因的第8内含子中还存在一个Alu重复序列的1kb插入/缺失多态性,即Alu±1kb多态性.为了帮助阐明汉族人群中DM突变的起源,并为解释DM在不同群体中发病率的差异提供更多依据,本文从300例已知CTG拷贝数的正常汉族群体中随机挑选60例,首先通过PCR扩增确定其Alu±1kb多态性,然后对Alu±1kb和CTG双杂合的标本,采用长PCR方法先行扩增含Alu±1kb和CTG重复序列的DNA片段,再分别对含Alu(+)和Alu(-)的DNA片段中的CTG拷贝数进行常规PCR分析,以确定二位点的单倍型.结果表明60例正常人中二位点间呈连锁不平衡.其单倍型为:(CTG)5均与Alu(+)连锁;多数(CTG)11~14与Alu(-)连锁;在两个(CTG)≥19的等位基因中一个与Alu(+)连锁,另一个与Alu(-)连锁.各民族相关资料的比较提示,汉族人群中(CTG)11~14与非洲黑人的起源可能不同;(CTG)19~30/Alu-1kb在汉族人群中的频率远比欧洲人群的高;(CTG)19~30/Alu-1kb与(CTG)19~30/Alu+1kb在汉族人群中是以一定比例共存的;(CTG)19~30在不同民族间的起源不尽相同;如果从(CTG)5到(CTG)19~30的假设成立的话,则很可能是一个较为复杂的过程.

  4. Natural Hazards, Second Edition

    Science.gov (United States)

    Rouhban, Badaoui

    Natural disaster loss is on the rise, and the vulnerability of the human and physical environment to the violent forces of nature is increasing. In many parts of the world, disasters caused by natural hazards such as earthquakes, floods, landslides, drought, wildfires, intense windstorms, tsunami, and volcanic eruptions have caused the loss of human lives, injury, homelessness, and the destruction of economic and social infrastructure. Over the last few years, there has been an increase in the occurrence, severity, and intensity of disasters, culminating with the devastating tsunami of 26 December 2004 in South East Asia.Natural hazards are often unexpected or uncontrollable natural events of varying magnitude. Understanding their mechanisms and assessing their distribution in time and space are necessary for refining risk mitigation measures. This second edition of Natural Hazards, (following a first edition published in 1991 by Cambridge University Press), written by Edward Bryant, associate dean of science at Wollongong University, Australia, grapples with this crucial issue, aspects of hazard prediction, and other issues. The book presents a comprehensive analysis of different categories of hazards of climatic and geological origin.

  5. HMMEditor: a visual editing tool for profile hidden Markov model

    Directory of Open Access Journals (Sweden)

    Cheng Jianlin

    2008-03-01

    Full Text Available Abstract Background Profile Hidden Markov Model (HMM is a powerful statistical model to represent a family of DNA, RNA, and protein sequences. Profile HMM has been widely used in bioinformatics research such as sequence alignment, gene structure prediction, motif identification, protein structure prediction, and biological database search. However, few comprehensive, visual editing tools for profile HMM are publicly available. Results We develop a visual editor for profile Hidden Markov Models (HMMEditor. HMMEditor can visualize the profile HMM architecture, transition probabilities, and emission probabilities. Moreover, it provides functions to edit and save HMM and parameters. Furthermore, HMMEditor allows users to align a sequence against the profile HMM and to visualize the corresponding Viterbi path. Conclusion HMMEditor provides a set of unique functions to visualize and edit a profile HMM. It is a useful tool for biological sequence analysis and modeling. Both HMMEditor software and web service are freely available.

  6. Advances in therapeutic CRISPR/Cas9 genome editing.

    Science.gov (United States)

    Savić, Nataša; Schwank, Gerald

    2016-02-01

    Targeted nucleases are widely used as tools for genome editing. Two years ago the clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas9 nuclease was used for the first time, and since then has largely revolutionized the field. The tremendous success of the CRISPR/Cas9 genome editing tool is powered by the ease design principle of the guide RNA that targets Cas9 to the desired DNA locus, and by the high specificity and efficiency of CRISPR/Cas9-generated DNA breaks. Several studies recently used CRISPR/Cas9 to successfully modulate disease-causing alleles in vivo in animal models and ex vivo in somatic and induced pluripotent stem cells, raising hope for therapeutic genome editing in the clinics. In this review, we will summarize and discuss such preclinical CRISPR/Cas9 gene therapy reports.

  7. Effective Alu Repeat Based RT-Qpcr Normalization in Cancer Cell Perturbation Experiments

    OpenAIRE

    Ali Rihani; Tom Van Maerken; Filip Pattyn; Gert Van Peer; Anneleen Beckers; Sara De Brouwer; Candy Kumps; Evelien Mets; Joni Van der Meulen; Pieter Rondou; Carina Leonelli; Pieter Mestdagh; Frank Speleman; Jo Vandesompele

    2013-01-01

    BACKGROUND: Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations. These manipulations may result in dramatic alterations in ...

  8. Introduction to Surgical Technology. Third Edition. Teacher Edition [and] Student Edition.

    Science.gov (United States)

    Bushey, Vicki; Hildebrand, Bob; Hildebrand, Dinah; Johnson, Dave; Sikes, John; Tahah, Ann; Walker, Susan; Zielsdorf, Lani

    These teacher and student editions provide instructional materials for an introduction to surgical technology course. Introductory materials in the teacher edition include information on use, instructional/task analysis, academic and workplace skill classifications and definitions, related academic and workplace skill list, and crosswalk to…

  9. Residential and Light Commercial HVAC. Teacher Edition and Student Edition. Second Edition.

    Science.gov (United States)

    Stephenson, David

    This package contains teacher and student editions of a residential and light commercial heating, ventilation, and air conditioning (HVAC) course of study. The teacher edition contains information on the following: using the publication; national competencies; competency profile; related academic and workplace skills list; tools, equipment, and…

  10. Oxyacetylene Welding and Oxyfuel Cutting. Third Edition. Teacher Edition [and] Student Edition [and] Student Workbook.

    Science.gov (United States)

    Knapp, John; Harper, Eddie

    This Oklahoma curriculum guide, which includes a teacher edition, a student edition, and a student workbook, provides three units for a course on oxyacetylene welding, oxyfuel cutting, and cutting done with alternative fuels such as MAPP, propane, and natural gas. The three units are: "Oxyacetylene Welding"; "Oxyfuel Cutting"; and "Oxyacetylene…

  11. Fundamentals of Welding. Teacher Edition [and] Student Edition [and] Student Workbook. Second Edition.

    Science.gov (United States)

    Fortney, Clarence; Gregory, Mike; New, Larry

    Teacher and student editions and a student workbook for fundamentals of welding comprise the first of six in a series of competency-based instructional materials for welding programs. Introductory pages in the teacher edition are training and competency profile, instructional/task analysis, basic skills icons and classifications, basic skills…

  12. Map Edit Distance vs Graph Edit Distance for Matching Images

    OpenAIRE

    Combier, Camille; Damiand, Guillaume; Solnon, Christine

    2013-01-01

    Generalized maps are widely used to model the topology of nD objects (such as 2D or 3D images) by means of incidence and adjacency relationships between cells (0D vertices, 1D edges, 2D faces, 3D volumes, ...). Recently, we have introduced a map edit distance. This distance compares maps by means of a minimum cost sequence of edit operations that should be performed to transform a map into another map. In this paper, we introduce labelled maps and we show how the map edit distance may be exte...

  13. The potential role of Alu Y in the development of resistance to SN38 (Irinotecan) or oxaliplatin in colorectal cancer

    DEFF Research Database (Denmark)

    Lin, Xue; Stenvang, Jan; Rasmussen, Mads Heilskov;

    2015-01-01

    Background: Irinotecan (SN38) and oxaliplatin are chemotherapeutic agents used in the treatment of colorectal cancer. However, the frequent development of resistance to these drugs represents a considerable challenge in the clinic. Alus as retrotransposons comprise 11% of the human genome. Genomic...... toxicity induced by carcinogens or drugs can reactivate Alus by altering DNA methylation. Whether or not reactivation of Alus occurs in SN38 and oxaliplatin resistance remains unknown. Results: We applied reduced representation bisulfite sequencing (RRBS) to investigate the DNA methylome in SN38......' to estimate the diversity of DNA methylation states of the identified resistance phenotype-associated methylation loci observed in the cell line models. We identified different loci being characteristic for the different resistant cell lines. Interestingly, 53% of the identified loci were Alu sequences...

  14. Design of an ALU Compatible with MCS-96%兼容MCS-96指令集的ALU设计

    Institute of Scientific and Technical Information of China (English)

    梁圃; 王道富; 毛志刚

    2008-01-01

    设计了一款能够完全兼容MCS-96系列单片机指令集的ALU.在设计中使用了经过逻辑简化的运算单元和改进的T型进位链,有效缩短了关键路径的延迟.采用硬件资源共享的策略进行运算单元和移位单元的结构组织设计,在不增加指令执行周期的前提下,最大限度地减小了电路面积.

  15. Design and Analysis of a High Speed, Power Efficient 8 Bit ALU Based on SOI / SON MOSFET Technology

    Directory of Open Access Journals (Sweden)

    Subhramita Basak

    2014-01-01

    Full Text Available This paper shows an overall performance comparative analysis in terms of Average Power Consumption, Average Delay and Power-Delay Product for an 8 bit Arithmetic Logic Unit (ALU using bulk MOS, Silicon-on-Insulator (SOI and Silicon-on-Nothing (SON technology. The entire design is done in 32nm technology for all the three cases (Bulk, SOI & SON and then compared. The comparisons have been carried out with the help of the simulation runs on Synopsys HSpice tool, and that clearly indicates, for lower Supply Voltages (Vdd, SOI / SON technology provides a significant reduction in Average Power Consumption, Average Delay and Power-Delay Product compared to that of Bulk MOS technology.

  16. Alu Sx repeat-induced homozygous deletion of the StAR gene causes lipoid congenital adrenal hyperplasia.

    Science.gov (United States)

    Eiden-Plach, Antje; Nguyen, Huy-Hoang; Schneider, Ursula; Hartmann, Michaela F; Bernhardt, Rita; Hannemann, Frank; Wudy, Stefan A

    2012-05-01

    Lipoid congenital adrenal hyperplasia (Lipoid CAH) is the most severe form of the autosomal recessive disorder CAH. A general loss of the steroid biosynthetic activity caused by defects in the StAR gene manifests as life-threatening primary adrenal insufficiency. We report a case of Lipoid CAH caused by a so far not described homozygous deletion of the complete StAR gene and provide diagnostic results based on a GC-MS steroid metabolomics and molecular genetic analysis. The patient presented with postnatal hypoglycemia, vomiting, adynamia, increasing pigmentation and hyponatremia. The constellation of urinary steroid metabolites suggested Lipoid CAH and ruled out all other forms of CAH or defects of aldosterone biosynthesis. After treatment with sodium supplementation, hydrocortisone and fludrocortisone the child fully recovered. Molecular genetic analysis demonstrated a homozygous 12.1 kb deletion in the StAR gene locus. The breakpoints of the deletion are embedded into two typical genomic repetitive Alu Sx elements upstream and downstream of the gene leading to the loss of all exons and regulatory elements. We established deletion-specific and intact allele-specific PCR methods and determined the StAR gene status of all available family members over three generations. This analysis revealed that one of the siblings, who died a few weeks after birth, carried the same genetic defect. Since several Alu repeats at the StAR gene locus increase the probability of deletions, patients with typical symptoms of lipoid CAH lacking evidence for the presence of both StAR alleles should be analyzed carefully for this kind of disorder.

  17. CrisprGE: a central hub of CRISPR/Cas-based genome editing

    OpenAIRE

    Kaur, Karambir; Tandon, Himani; Gupta, Amit Kumar; Kumar, Manoj

    2015-01-01

    CRISPR system is a powerful defense mechanism in bacteria and archaea to provide immunity against viruses. Recently, this process found a new application in intended targeting of the genomes. CRISPR-mediated genome editing is performed by two main components namely single guide RNA and Cas9 protein. Despite the enormous data generated in this area, there is a dearth of high throughput resource. Therefore, we have developed CrisprGE, a central hub of CRISPR/Cas-based genome editing. Presently,...

  18. Wilson's disease caused by alternative splicing and Alu exonization due to a homozygous 3039-bp deletion spanning from intron 1 to exon 2 of the ATP7B gene.

    Science.gov (United States)

    Mameli, Eva; Lepori, Maria Barbara; Chiappe, Francesca; Ranucci, Giusy; Di Dato, Fabiola; Iorio, Raffaele; Loudianos, Georgios

    2015-09-15

    We describe a case of Wilson's disease (WD) diagnosed at 5 years after routine biochemical test showed increased aminotransferases. Mutation analysis of the ATP7B gene revealed a 3039-bp deletion in the homozygous state spanning from the terminal part of intron 1 to nt position 368 of exon 2. This deletion results in the activation of 3 cryptic splice sites: an AG acceptor splice site in nt positions 578-579 producing a different breakpoint and removing the first 577 nts of exon 2, an acceptor and a donor splice site in nt positions 20363-4 and 20456-7, respectively, in intron 1, resulting in the activation of a 94-bp cryptic Alu exon being incorporated into the mature transcript. The resulting alternative transcript contains a TAG stop codon in the first amino acid position of the cryptic exon, likely producing a truncated, non-functional protein. This study shows that intron exonization can also occur in humans through naturally occurring gross deletions. The results suggest that the combination of DNA and RNA analyses can be used for molecular characterization of gross ATP7B deletions, thus improving genetic counseling and diagnosis of WD. Moreover these studies help to better establish new molecular mechanisms producing Wilson's disease.

  19. PAM multiplicity marks genomic target sites as inhibitory to CRISPR-Cas9 editing

    OpenAIRE

    Malina, Abba; Cameron, Christopher J. F.; Robert, Francis; Blanchette, Mathieu; Dostie, Josée; Pelletier, Jerry

    2015-01-01

    In CRISPR-Cas9 genome editing, the underlying principles for selecting guide RNA (gRNA) sequences that would ensure for efficient target site modification remain poorly understood. Here we show that target sites harbouring multiple protospacer adjacent motifs (PAMs) are refractory to Cas9-mediated repair in situ. Thus we refine which substrates should be avoided in gRNA design, implicating PAM density as a novel sequence-specific feature that inhibits in vivo Cas9-driven DNA modification.

  20. A non-inheritable maternal Cas9-based multiple-gene editing system in mice

    OpenAIRE

    Takayuki Sakurai; Akiko Kamiyoshi; Hisaka Kawate; Chie Mori; Satoshi Watanabe; Megumu Tanaka; Ryuichi Uetake; Masahiro Sato; Takayuki Shindo

    2016-01-01

    The CRISPR/Cas9 system is capable of editing multiple genes through one-step zygote injection. The preexisting method is largely based on the co-injection of Cas9 DNA (or mRNA) and guide RNAs (gRNAs); however, it is unclear how many genes can be simultaneously edited by this method, and a reliable means to generate transgenic (Tg) animals with multiple gene editing has yet to be developed. Here, we employed non-inheritable maternal Cas9 (maCas9) protein derived from Tg mice with systemic Cas9...

  1. Edit while watching: home video editing made easy

    Science.gov (United States)

    Campanella, Marco; Weda, Hans; Barbieri, Mauro

    2007-01-01

    In recent years, more and more people capture their experiences in home videos. However, home video editing still is a difficult and time-consuming task. We present the Edit While Watching system that allows users to automatically create and change a summary of a home video in an easy, intuitive and lean-back way. Based on content analysis, video is indexed, segmented, and combined with proper music and editing effects. The result is an automatically generated home video summary that is shown to the user. While watching it, users can indicate whether they like certain content, so that the system will adapt the summary to contain more content that is similar or related to the displayed content. During the video playback users can also modify and enrich the content, seeing immediately the effects of their changes. Edit While Watching does not require a complex user interface: a TV and a few keys of a remote control are sufficient. A user study has shown that it is easy to learn and to use, even if users expressed the need for more control in the editing operations and in the editing process.

  2. RNA-SSPT: RNA Secondary Structure Prediction Tools.

    Science.gov (United States)

    Ahmad, Freed; Mahboob, Shahid; Gulzar, Tahsin; Din, Salah U; Hanif, Tanzeela; Ahmad, Hifza; Afzal, Muhammad

    2013-01-01

    The prediction of RNA structure is useful for understanding evolution for both in silico and in vitro studies. Physical methods like NMR studies to predict RNA secondary structure are expensive and difficult. Computational RNA secondary structure prediction is easier. Comparative sequence analysis provides the best solution. But secondary structure prediction of a single RNA sequence is challenging. RNA-SSPT is a tool that computationally predicts secondary structure of a single RNA sequence. Most of the RNA secondary structure prediction tools do not allow pseudoknots in the structure or are unable to locate them. Nussinov dynamic programming algorithm has been implemented in RNA-SSPT. The current studies shows only energetically most favorable secondary structure is required and the algorithm modification is also available that produces base pairs to lower the total free energy of the secondary structure. For visualization of RNA secondary structure, NAVIEW in C language is used and modified in C# for tool requirement. RNA-SSPT is built in C# using Dot Net 2.0 in Microsoft Visual Studio 2005 Professional edition. The accuracy of RNA-SSPT is tested in terms of Sensitivity and Positive Predicted Value. It is a tool which serves both secondary structure prediction and secondary structure visualization purposes. PMID:24250115

  3. Marine botany. Second edition

    Energy Technology Data Exchange (ETDEWEB)

    Dawes, C.J. [Univ. of South Florida, Tampa, FL (United States)

    1998-12-01

    Marine plants are a diverse group that include unicellular algae, seaweeds, seagrasses, salt marshes, and mangrove forests. They carry out a variety of ecological functions and serve as the primary producers in coastal wetlands and oceanic waters. The theme that connects such a wide variety of plants is their ecology, which was also emphasized in the 1981 edition. The goal of this revision is to present taxonomic, physiological, chemical, and ecological aspects of marine plants, their adaptations, and how abiotic and biotic factors interact in their communities. The data are presented in a concise, comparative manner in order to identify similarities and differences between communities such as salt marsh and mangroves or subtidal seaweeds and seagrasses. To accomplish this, the text is organized into five chapters that introduce the marine habitats, consider abiotic and biotic factors, and anthropogenic influences on the communities followed by seven chapters that deal with microalgae, seaweeds, salt marshes, mangroves, seagrasses, and coral reefs. Two appendixes are included; one presents simple field techniques and the other is a summary of seaweed uses.

  4. Benthic habitat map of U.S. Coral Reef Task Force Faga‘alu Bay priority study area, Tutuila, American Samoa

    Science.gov (United States)

    Cochran, Susan A.; Gibbs, Ann E.; D'Antonio, Nicole L.; Storlazzi, Curt D.

    2016-01-01

    The coral reef in Faga‘alu Bay, Tutuila, American Samoa, has suffered numerous natural and anthropogenic stresses. Areas once dominated by live coral are now mostly rubble surfaces covered with turf or macroalgae. In an effort to improve the health and resilience of the coral reef system, the U.S. Coral Reef Task Force selected Faga‘alu Bay as a priority study area. To support these efforts, the U.S. Geological Survey mapped nearly 1 km2 of seafloor to depths of about 60 m. Unconsolidated sediment (predominantly sand) constitutes slightly greater than 50 percent of the seafloor in the mapped area; reef and other hardbottom potentially available for coral recruitment constitute nearly 50 percent of the mapped area. Of this potentially available hardbottom, only slightly greater than 37 percent is covered with at least 10 percent coral, which is fairly evenly distributed between the reef flat, fore reef, and offshore bank/shelf. 

  5. Design of an efficient ALU based on reused logic structure%基于功能复用的高性能ALU设计

    Institute of Scientific and Technical Information of China (English)

    张嘉琛; 蒋剑飞; 毛志刚

    2010-01-01

    算术逻辑单元(ALU)是处理器中不可或缺的重要部分,可以进行两输入逻辑和加减法运算.设计了一款通用数字信号处理器中使用的高性能ALU.提出了一种高效的逻辑与算术运算复用的电路结构,提高复用度的同时,减少了ALU的面积.并提出一种融合进位选择和超前进位加法器结构的优化进位链设计,该进位链可以提高加法器的速度,并同时支持数字信号处理器的双16位运算.

  6. Benthic habitat map of U.S. Coral Reef Task Force Faga‘alu Bay priority study area, Tutuila, American Samoa

    Science.gov (United States)

    Cochran, Susan A.; Gibbs, Ann E.; D'Antonio, Nicole L.; Storlazzi, Curt D.

    2016-05-18

    The coral reef in Faga‘alu Bay, Tutuila, American Samoa, has suffered numerous natural and anthropogenic stresses. Areas once dominated by live coral are now mostly rubble surfaces covered with turf or macroalgae. In an effort to improve the health and resilience of the coral reef system, the U.S. Coral Reef Task Force selected Faga‘alu Bay as a priority study area. To support these efforts, the U.S. Geological Survey mapped nearly 1 km2 of seafloor to depths of about 60 m. Unconsolidated sediment (predominantly sand) constitutes slightly greater than 50 percent of the seafloor in the mapped area; reef and other hardbottom potentially available for coral recruitment constitute nearly 50 percent of the mapped area. Of this potentially available hardbottom, only slightly greater than 37 percent is covered with at least 10 percent coral, which is fairly evenly distributed between the reef flat, fore reef, and offshore bank/shelf. 

  7. Detection of Human Hepatocarcinoma Cell Line PLC/PRF/5 Genome Hepatitis b Virus DNA Integration with Alu-PCR%Alu-PCR检测人肝癌细胞株PLC/PRF/5基因组中乙型肝炎病毒DNA的整合

    Institute of Scientific and Technical Information of China (English)

    邱爽; 张会英

    2015-01-01

    目的 应用Alu-PCR方法检测人肝癌细胞株PLC/PRF/5 基因组中乙型肝炎病毒( HBV) DNA的整合. 方法 提取经培养扩增的人肝癌细胞株PLC/PRF/5基因组DNA,根据Alu-PCR方法经过3轮PCR反应扩增潜在的HBV DNA和人基因组DNA整合片段. 琼脂糖凝胶电泳观察PCR扩增产物片段,切取并纯化整合阳性的电泳条带,对纯化产物进行核酸测序,得到整合片段的核苷酸序列. 结果 经琼脂糖凝胶电泳检测,用Alu-PCR方法能够从PLC/PRF/5 细胞株中扩增得到4 条HBV DNA整合序列,经测序后与比对其中3条整合序列能够定位于人染色体03p21.31、05p15.33、12q13.12~q14.1. 结论 Alu-PCR可以准确测定肝细胞中HBV DNA的整合,为研究HBV DNA在肝细胞中的整合研究提供了一个简单、经济的方法.%Objective In this research with the method of Alu -PCR we investigate the integration of hepatitis B virus ( HBV) DNA in human hepatocarcinoma cell line (PLC/PRF/5) genome DNA.Methods We at first extracted the genome DNA from PLC/PRF/5 cells, and then the potential integration fragments of HBV DNA and human genome DNA were amplified with according to the Alu -PCR after three rounds PCR .The Alu-PCR amplification products were observated with agarose gel electrophoresis , then integration positive electrophoresis bandings were chipped and purified for nucleic acid sequencing .At last he bioinformatics information was acquired by blast online.Results Through agarose gel electrophoresis after Alu -PCR amplification, we got four potential integration bindings , among which we got three integration sequences of HBV DNA in human genome DNA .These integration sequences could be individually located in the human chromosome of 03p21.31, 05p15.33, 12q13 and 12-q14.1.Conclusion With Alu-PCR we can accurately measure the integration of HBV DNA in human genome DNA , and Alu-PCR can be a a convenience and economic method in the study of HBV DNA ′s integration in human genome

  8. CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells.

    Science.gov (United States)

    Yuen, Kit-San; Chan, Chi-Ping; Wong, Nok-Hei Mickey; Ho, Chau-Ha; Ho, Ting-Hin; Lei, Ting; Deng, Wen; Tsao, Sai Wah; Chen, Honglin; Kok, Kin-Hang; Jin, Dong-Yan

    2015-03-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep sequencing. The loss of BART miRNA expression and activity was verified, supporting the BART promoter as the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses could be recovered and introduced into other cells at low m.o.i. Recombinant viruses with an edited genome could be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provided not only the first genetic evidence that the BART promoter drives the expression of the BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells.

  9. Calcitonin Receptor AluI (rs1801197) and Taq1 Calcitonin Genes Polymorphism in 45-and Over 45-year-old Women and their Association with Bone Density

    Science.gov (United States)

    Dehghan, Morteza; Pourahmad-Jaktaji, Razieh; Farzaneh, Zarghampoor

    2016-01-01

    Purpose: Calcitonin receptor gene has also a polymorphism which is associated with bone mass density. This study evaluates the association between calcitonin receptor AluI (rs1801197) and Taq1 calcitonin genes polymorphism with bone density rate. Methods: In this descriptive-analytical study in 2013 in southwestern Iran, 200 blood samples, per the Cochran sample size formula, were taken from women aged 45 and older. DNA was extracted from the samples using the phenol– chloroform method and the genomic fragments in question were proliferated using the polymerase chain reaction (PCR) method. Results: The genotypic distribution of polymorphism AluI for TT, TC, and CC genotypes in control group was 31.4%, 38.6%, and 30% and in patients 25.4%, 55.4%, and 19.2%, respectively. There was no significant difference in polymorphism AluI between patients and control group and no significant association was found between this gene and bone density rate (P > 0.05). All patients and the individuals in the control group exhibited tt genotype for TaqI calcitonin gene and no significant association was found between these participants and osteoporosis. Conclusion: There was no association between two polymorphisms and osteoporosis, and between polymorphism of these two genes and osteoporosis development rate in the participants. PMID:27708484

  10. DETECTION OF STRAND BREAKS OF DNA IN HUMAN EARLY CHORIONIC VILLUS CELLS INDUCED BY DIAGNOSTIC ULTRASOUND USING 32p-LABELED ALU HYBRIDIZATION

    Institute of Scientific and Technical Information of China (English)

    Wang Caifeng; Li Xu; Zhang Yunjing

    2006-01-01

    Objective To explore if strand breaks of DNA in human early chorionic villus cells in uterus were induced by diagnostic ultrasound and to evaluate the method used for detection of single-stranded breaks and doublestranded breaks in human DNA. Methods 60 normal pregnant women aged 20-30, who underwent artificial abortion during 6-8 weeks of gestation, were randomly divided into 2 experimental groups: All 30 cases were exposed to diagnostic ultrasound in uterus for 10 minutes, and 24 hours later chorionic villi were extracted; the other 30 cases were taken as the control group. Single-stranded DNA and double-stranded DNA in villus cells in all cases were isolated by the alkaline unwinding combined with hydroxylapatite chromatography, and were quantitatively detected using32 P-labeled Alu probe for dot-blotting hybridization. Results There was no significant difference in quantity and percentage in single-stranded DNA and double-stranded DNA between 2 groups (P>0.05). 32 P-Alu probe could only hybridize with human DNA, and could detect DNA isolated from as few as 2.5 × 103 chorionic villus cells and 0.45 ng DNA in human leukocytes. Conclusion The results suggested that there were no DNA strand damages in human chorionic villus cells when the uterus was exposed to diagnostic ultrasound for 10 minutes. The method, 32P-Alu probe for dot-blotting hybridization, was even more specific, sensitive and accurate than conventional approaches.

  11. CRISPR/Cas9-Mediated Genome Editing in Soybean Hairy Roots

    OpenAIRE

    Yupeng Cai; Li Chen; Xiujie Liu; Shi Sun; Cunxiang Wu; Bingjun Jiang; Tianfu Han; Wensheng Hou

    2015-01-01

    As a new technology for gene editing, the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been rapidly and widely used for genome engineering in various organisms. In the present study, we successfully applied type II CRISPR/Cas9 system to generate and estimate genome editing in the desired target genes in soybean (Glycine max (L.) Merrill.). The single-guide RNA (sgRNA) and Cas9 cassettes were assembled on one vector to improve transformat...

  12. Circadian RNA expression elicited by 3’-UTR IRAlu-paraspeckle associated elements

    Science.gov (United States)

    Torres, Manon; Becquet, Denis; Blanchard, Marie-Pierre; Guillen, Séverine; Boyer, Bénédicte; Moreno, Mathias; Franc, Jean-Louis; François-Bellan, Anne-Marie

    2016-01-01

    Paraspeckles are nuclear bodies form around the long non-coding RNA, Neat1, and RNA-binding proteins. While their role is not fully understood, they are believed to control gene expression at a post-transcriptional level by means of the nuclear retention of mRNA containing in their 3’-UTR inverted repeats of Alu sequences (IRAlu). In this study, we found that, in pituitary cells, all components of paraspeckles including four major proteins and Neat1 displayed a circadian expression pattern. Furthermore the insertion of IRAlu at the 3’-UTR of the EGFP cDNA led to a rhythmic circadian nuclear retention of the egfp mRNA that was lost when paraspeckles were disrupted whereas insertion of a single antisense Alu had only a weak effect. Using real-time video-microscopy, these IRAlu were further shown to drive a circadian expression of EGFP protein. This study shows that paraspeckles, thanks to their circadian expression, control circadian gene expression at a post-transcriptional level. DOI: http://dx.doi.org/10.7554/eLife.14837.001 PMID:27441387

  13. RNA-Mediated Gene Duplication and Retroposons: Retrogenes, LINEs, SINEs, and Sequence Specificity

    Directory of Open Access Journals (Sweden)

    Kazuhiko Ohshima

    2013-01-01

    Full Text Available A substantial number of “retrogenes” that are derived from the mRNA of various intron-containing genes have been reported. A class of mammalian retroposons, long interspersed element-1 (LINE1, L1, has been shown to be involved in the reverse transcription of retrogenes (or processed pseudogenes and non-autonomous short interspersed elements (SINEs. The -end sequences of various SINEs originated from a corresponding LINE. As the -untranslated regions of several LINEs are essential for retroposition, these LINEs presumably require “stringent” recognition of the -end sequence of the RNA template. However, the -ends of mammalian L1s do not exhibit any similarity to SINEs, except for the presence of -poly(A repeats. Since the -poly(A repeats of L1 and Alu SINE are critical for their retroposition, L1 probably recognizes the poly(A repeats, thereby mobilizing not only Alu SINE but also cytosolic mRNA. Many flowering plants only harbor L1-clade LINEs and a significant number of SINEs with poly(A repeats, but no homology to the LINEs. Moreover, processed pseudogenes have also been found in flowering plants. I propose that the ancestral L1-clade LINE in the common ancestor of green plants may have recognized a specific RNA template, with stringent recognition then becoming relaxed during the course of plant evolution.

  14. Sill emplacement and corresponding ground deformation processes at the Alu-Dalafilla volcanic centre in the Danakil Depression, Ethiopia

    Science.gov (United States)

    Magee, Craig; Bastow, Ian; Hetherington, Rachel; van Wyk de Vries, Ben; Jackson, Christopher

    2016-04-01

    A consensus has emerged from a variety of disciplines over the past 15 years that Quaternary magmatism in Ethiopia is almost entirely dominated by dike intrusion. Focused dike intrusion within 60 km long, 20 km wide, rift zones is considered to mark the present day locus of extension in Ethiopia, and represent the proto-ridge axis location of an incipient ocean spreading centre. However, it has been suggested on the strength of Moho depths and Quaternary eruptive volumes in northernmost Ethiopia, that the final transition from continental rifting to incipient oceanic spreading may instead be characterised by an abrupt, rheologically driven, late-phase of crustal thinning. Development of a sedimentary basin and mantle decompression melting occurring in the Danakil Depression, driven by this late-phase crustal thinning, should result in a markedly different style of magmatism in the upper crust: i.e. field observations, high-resolution seismic reflection studies, and experimental modelling suggest that interconnected networks of sill intrusions dominate in sedimentary basins. Here, we present the first evidence from the Danakil Depression that links surficial structures, observed at the Alu-Dalafilla volcanic centre, to the ongoing emplacement of an underlying sill. In particular, we use satellite imagery to examine a dome-shaped fold, associated fracture patterns, and surrounding lava flows, which we suggest likely formed in response to roof uplift above and extrusion from a saucer-shaped sill; i.e. a sub-horizontal inner sill encircled by an inward-dipping, transgressive inclined rim. InSAR observations by Pagli et al. (2012) of ground uplift and deflation occurring during the eruption of basaltic lava at Alu-Dalafilla in 2008 capture what we believe to be the first real-time evidence for intrusion-induced forced folding dynamics above a saucer-shaped sill. InSAR data further suggest that intrusion occurred at a depth of ~1 km, likely placing the sill within an

  15. GluA2 is rapidly edited at the Q/R site during neural differentiation in vitro

    Directory of Open Access Journals (Sweden)

    Svenja ePachernegg

    2015-03-01

    Full Text Available The majority of AMPA receptors in the adult brain contain GluA2 subunits, which can be edited at the Q/R site, changing a glutamine to an arginine within the ion pore. Q/R editing renders AMPARs virtually Ca2+-impermeable, which is important for normal AMPA receptor function. Thus, all GluA2 subunits are Q/R-edited in the adult brain. However, it has remained controversial precisely when editing sets in during development. In the present study, we show that GluA2 mRNA is very rapidly Q/R-edited immediately after its appearance, which is after 4.5 days of differentiation from 46C embryonic stem cells (ESCs to neuroepithelial precursor cells (NEPs. At this time point, most of the GluA2 transcripts were already edited, with only a small fraction remaining unedited, and half a day later all GluA2 transcripts were edited. This can be explained by the observation that the enzyme that Q/R-edits GluA2 transcripts, ADAR2, is already expressed in the cell well before GluA2 transcription starts, and later is not significantly upregulated any more. Editing at another site works differently: The R/G site within the ligand-binding domain was never completely edited at any of the developmental stages tested, and the enzyme that performs this editing, ADAR1, was significantly upregulated during neural differentiation. This confirms previous data suggesting that R/G editing, in contrast to Q/R editing, progresses gradually during development.

  16. Connectivity editing for quadrilateral meshes

    KAUST Repository

    Peng, Chihan

    2011-12-12

    We propose new connectivity editing operations for quadrilateral meshes with the unique ability to explicitly control the location, orientation, type, and number of the irregular vertices (valence not equal to four) in the mesh while preserving sharp edges. We provide theoretical analysis on what editing operations are possible and impossible and introduce three fundamental operations to move and re-orient a pair of irregular vertices. We argue that our editing operations are fundamental, because they only change the quad mesh in the smallest possible region and involve the fewest irregular vertices (i.e., two). The irregular vertex movement operations are supplemented by operations for the splitting, merging, canceling, and aligning of irregular vertices. We explain how the proposed highlevel operations are realized through graph-level editing operations such as quad collapses, edge flips, and edge splits. The utility of these mesh editing operations are demonstrated by improving the connectivity of quad meshes generated from state-of-art quadrangulation techniques. © 2011 ACM.

  17. Connectivity editing for quadrilateral meshes

    KAUST Repository

    Peng, Chihan

    2011-12-01

    We propose new connectivity editing operations for quadrilateral meshes with the unique ability to explicitly control the location, orientation, type, and number of the irregular vertices (valence not equal to four) in the mesh while preserving sharp edges. We provide theoretical analysis on what editing operations are possible and impossible and introduce three fundamental operations to move and re-orient a pair of irregular vertices. We argue that our editing operations are fundamental, because they only change the quad mesh in the smallest possible region and involve the fewest irregular vertices (i.e., two). The irregular vertex movement operations are supplemented by operations for the splitting, merging, canceling, and aligning of irregular vertices. We explain how the proposed high-level operations are realized through graph-level editing operations such as quad collapses, edge flips, and edge splits. The utility of these mesh editing operations are demonstrated by improving the connectivity of quad meshes generated from state-of-art quadrangulation techniques.

  18. CTD writing and editing standards

    Science.gov (United States)

    Caruthers, C. M.

    1991-03-01

    The Computer and Telecommunication Division (CTD) recognizes that the communication of clear, accurate, reasonably complete information is essential to the success of its Laboratory mission. CTD therefore encourages all Division personnel to adhere to the principles of good writing and to the standards for grammar, usage, style, formats, and publication procedures that are described in CTD Writing and Editing Standards. We encourage CTD personnel to read CTD Writing and Editing Standards and to use it continually as a desktop reference. It will help CTD writers to produce better documents consistent with CTD standards in less time. Applying the principles specified in this document on how to write and organize technical information will speed up the editing, review, and revision processes. CTD Writing and Editing Standards complements the Argonne National Laboratory Technical Publications Guide, which serves as the basic Argonne documentation reference on issues concerning DOE orders and guidelines, NRC directives, and other sponsor requirements. However, this Laboratory-wide document does not address matters of grammar or style. Documents recommended in CTD Writing and Editing Standards are usually available for purchase at the Document Distribution Counter (Building 221, Room A-134) or through the mail (by calling extension 2-5405 and ordering copies).

  19. Leucyl-tRNA synthetase from the ancestral bacterium Aquifex aeolicus contains relics of synthetase evolution

    OpenAIRE

    Zhao, Ming-Wei; Zhu, Bin; Hao, Rui; Xu, Min-Gang; Eriani, Gilbert; Wang, En-Duo

    2005-01-01

    The editing reactions catalyzed by aminoacyl-tRNA synthetases are critical for the faithful protein synthesis by correcting misactivated amino acids and misaminoacylated tRNAs. We report that the isolated editing domain of leucyl-tRNA synthetase from the deep-rooted bacterium Aquifex aeolicus (αβ-LeuRS) catalyzes the hydrolytic editing of both mischarged tRNALeu and minihelixLeu. Within the domain, we have identified a crucial 20-amino-acid peptide that confers editing capacity when transplan...

  20. Newer Gene Editing Technologies toward HIV Gene Therapy

    Directory of Open Access Journals (Sweden)

    Premlata Shankar

    2013-11-01

    Full Text Available Despite the great success of highly active antiretroviral therapy (HAART in ameliorating the course of HIV infection, alternative therapeutic approaches are being pursued because of practical problems associated with life-long therapy. The eradication of HIV in the so-called “Berlin patient” who received a bone marrow transplant from a CCR5-negative donor has rekindled interest in genome engineering strategies to achieve the same effect. Precise gene editing within the cells is now a realistic possibility with recent advances in understanding the DNA repair mechanisms, DNA interaction with transcription factors and bacterial defense mechanisms. Within the past few years, four novel technologies have emerged that can be engineered for recognition of specific DNA target sequences to enable site-specific gene editing: Homing Endonuclease, ZFN, TALEN, and CRISPR/Cas9 system. The most recent CRISPR/Cas9 system uses a short stretch of complementary RNA bound to Cas9 nuclease to recognize and cleave target DNA, as opposed to the previous technologies that use DNA binding motifs of either zinc finger proteins or transcription activator-like effector molecules fused to an endonuclease to mediate sequence-specific DNA cleavage. Unlike RNA interference, which requires the continued presence of effector moieties to maintain gene silencing, the newer technologies allow permanent disruption of the targeted gene after a single treatment. Here, we review the applications, limitations and future prospects of novel gene-editing strategies for use as HIV therapy.

  1. Understanding Editing Behaviors in Multilingual Wikipedia.

    Science.gov (United States)

    Kim, Suin; Park, Sungjoon; Hale, Scott A; Kim, Sooyoung; Byun, Jeongmin; Oh, Alice H

    2016-01-01

    Multilingualism is common offline, but we have a more limited understanding of the ways multilingualism is displayed online and the roles that multilinguals play in the spread of content between speakers of different languages. We take a computational approach to studying multilingualism using one of the largest user-generated content platforms, Wikipedia. We study multilingualism by collecting and analyzing a large dataset of the content written by multilingual editors of the English, German, and Spanish editions of Wikipedia. This dataset contains over two million paragraphs edited by over 15,000 multilingual users from July 8 to August 9, 2013. We analyze these multilingual editors in terms of their engagement, interests, and language proficiency in their primary and non-primary (secondary) languages and find that the English edition of Wikipedia displays different dynamics from the Spanish and German editions. Users primarily editing the Spanish and German editions make more complex edits than users who edit these editions as a second language. In contrast, users editing the English edition as a second language make edits that are just as complex as the edits by users who primarily edit the English edition. In this way, English serves a special role bringing together content written by multilinguals from many language editions. Nonetheless, language remains a formidable hurdle to the spread of content: we find evidence for a complexity barrier whereby editors are less likely to edit complex content in a second language. In addition, we find that multilinguals are less engaged and show lower levels of language proficiency in their second languages. We also examine the topical interests of multilingual editors and find that there is no significant difference between primary and non-primary editors in each language. PMID:27171158

  2. Understanding Editing Behaviors in Multilingual Wikipedia

    Science.gov (United States)

    Hale, Scott A.; Kim, Sooyoung; Byun, Jeongmin; Oh, Alice H.

    2016-01-01

    Multilingualism is common offline, but we have a more limited understanding of the ways multilingualism is displayed online and the roles that multilinguals play in the spread of content between speakers of different languages. We take a computational approach to studying multilingualism using one of the largest user-generated content platforms, Wikipedia. We study multilingualism by collecting and analyzing a large dataset of the content written by multilingual editors of the English, German, and Spanish editions of Wikipedia. This dataset contains over two million paragraphs edited by over 15,000 multilingual users from July 8 to August 9, 2013. We analyze these multilingual editors in terms of their engagement, interests, and language proficiency in their primary and non-primary (secondary) languages and find that the English edition of Wikipedia displays different dynamics from the Spanish and German editions. Users primarily editing the Spanish and German editions make more complex edits than users who edit these editions as a second language. In contrast, users editing the English edition as a second language make edits that are just as complex as the edits by users who primarily edit the English edition. In this way, English serves a special role bringing together content written by multilinguals from many language editions. Nonetheless, language remains a formidable hurdle to the spread of content: we find evidence for a complexity barrier whereby editors are less likely to edit complex content in a second language. In addition, we find that multilinguals are less engaged and show lower levels of language proficiency in their second languages. We also examine the topical interests of multilingual editors and find that there is no significant difference between primary and non-primary editors in each language. PMID:27171158

  3. Understanding Editing Behaviors in Multilingual Wikipedia.

    Directory of Open Access Journals (Sweden)

    Suin Kim

    Full Text Available Multilingualism is common offline, but we have a more limited understanding of the ways multilingualism is displayed online and the roles that multilinguals play in the spread of content between speakers of different languages. We take a computational approach to studying multilingualism using one of the largest user-generated content platforms, Wikipedia. We study multilingualism by collecting and analyzing a large dataset of the content written by multilingual editors of the English, German, and Spanish editions of Wikipedia. This dataset contains over two million paragraphs edited by over 15,000 multilingual users from July 8 to August 9, 2013. We analyze these multilingual editors in terms of their engagement, interests, and language proficiency in their primary and non-primary (secondary languages and find that the English edition of Wikipedia displays different dynamics from the Spanish and German editions. Users primarily editing the Spanish and German editions make more complex edits than users who edit these editions as a second language. In contrast, users editing the English edition as a second language make edits that are just as complex as the edits by users who primarily edit the English edition. In this way, English serves a special role bringing together content written by multilinguals from many language editions. Nonetheless, language remains a formidable hurdle to the spread of content: we find evidence for a complexity barrier whereby editors are less likely to edit complex content in a second language. In addition, we find that multilinguals are less engaged and show lower levels of language proficiency in their second languages. We also examine the topical interests of multilingual editors and find that there is no significant difference between primary and non-primary editors in each language.

  4. Understanding Editing Behaviors in Multilingual Wikipedia.

    Science.gov (United States)

    Kim, Suin; Park, Sungjoon; Hale, Scott A; Kim, Sooyoung; Byun, Jeongmin; Oh, Alice H

    2016-01-01

    Multilingualism is common offline, but we have a more limited understanding of the ways multilingualism is displayed online and the roles that multilinguals play in the spread of content between speakers of different languages. We take a computational approach to studying multilingualism using one of the largest user-generated content platforms, Wikipedia. We study multilingualism by collecting and analyzing a large dataset of the content written by multilingual editors of the English, German, and Spanish editions of Wikipedia. This dataset contains over two million paragraphs edited by over 15,000 multilingual users from July 8 to August 9, 2013. We analyze these multilingual editors in terms of their engagement, interests, and language proficiency in their primary and non-primary (secondary) languages and find that the English edition of Wikipedia displays different dynamics from the Spanish and German editions. Users primarily editing the Spanish and German editions make more complex edits than users who edit these editions as a second language. In contrast, users editing the English edition as a second language make edits that are just as complex as the edits by users who primarily edit the English edition. In this way, English serves a special role bringing together content written by multilinguals from many language editions. Nonetheless, language remains a formidable hurdle to the spread of content: we find evidence for a complexity barrier whereby editors are less likely to edit complex content in a second language. In addition, we find that multilinguals are less engaged and show lower levels of language proficiency in their second languages. We also examine the topical interests of multilingual editors and find that there is no significant difference between primary and non-primary editors in each language.

  5. Description scheme for video editing work

    Science.gov (United States)

    Ruiloba, Rosa I.; Joly, Philippe

    2001-03-01

    This article presents a Description Scheme (DS) to describe the audio-visual documents from the video editing work point of view. This DS is based on edition techniques used in the video edition domain. The main objective of this DS is to provide a complete, modular and extensible description of the structure of the video documents based on editing process. This VideoEditing DS is generic in the sense that it may be used in a large number of applications such as video document indexing and analysis, description of Edit Decision List and elaboration of editing patterns. It is based on accurate and complete definitions of shots and transition effects required for video document analysis applications. The VideoEditing DS allows three levels of description : analytic, synthetic and semantic. In the DS, the higher (resp. the lower) is the element of description, the more analytic (resp. synthetic) is the information. %Phil This DS allows describing the editing work made by editing boards, using more detailed descriptors of Shots and Transition DSs. These elements are provided to define editing patterns that allow several possible reconstructions of movies depending on, for example, the target audience. A part of the video description made with this DS may be automatically produced by the video to shots segmentation algorithms (analytic DSs ) or by editing software, at the same time the edition work is made. This DS gives an answer to the needs related to the exchange of editing work descriptions between editing softwares. At the same time, the same DS provide an analytic description of editing work which is complementary to existing standards for Edit Decision Lists like SMPTE or AAF.

  6. Boneless Pose Editing and Animation

    DEFF Research Database (Denmark)

    Bærentzen, Jakob Andreas; Hansen, Kristian Evers; Erleben, Kenny

    2007-01-01

    In this paper, we propose a pose editing and animation method for triangulated surfaces based on a user controlled partitioning of the model into deformable parts and rigid parts which are denoted handles. In our pose editing system, the user can sculpt a set of poses simply by transforming...... the handles for each pose. Using Laplacian editing, the deformable parts are deformed to match the handles. In our animation system the user can constrain one or several handles in order to define a new pose. New poses are interpolated from the examples poses, by solving a small non-linear optimization...... problem in order to obtain the interpolation weights. While the system can be used simply for building poses, it is also an animation system. The user can specify a path for a given constraint and the model is animated correspondingly....

  7. Modulation of LINE-1 and Alu/SVA Retrotransposition by Aicardi-Goutières Syndrome-Related SAMHD1

    Directory of Open Access Journals (Sweden)

    Ke Zhao

    2013-09-01

    Full Text Available Long interspersed elements 1 (LINE-1 occupy at least 17% of the human genome and are its only active autonomous retrotransposons. However, the host factors that regulate LINE-1 retrotransposition are not fully understood. Here, we demonstrate that the Aicardi-Goutières syndrome gene product SAMHD1, recently revealed to be an inhibitor of HIV/simian immunodeficiency virus (SIV infectivity and neutralized by the viral Vpx protein, is also a potent regulator of LINE-1 and LINE-1-mediated Alu/SVA retrotransposition. We also found that mutant SAMHD1s of Aicardi-Goutières syndrome patients are defective in LINE-1 inhibition. Several domains of SAMHD1 are critical for LINE-1 regulation. SAMHD1 inhibits LINE-1 retrotransposition in dividing cells. An enzymatic active site mutant SAMHD1 maintained substantial anti-LINE-1 activity. SAMHD1 inhibits ORF2p-mediated LINE-1 reverse transcription in isolated LINE-1 ribonucleoproteins by reducing ORF2p level. Thus, SAMHD1 may be a cellular regulator of LINE-1 activity that is conserved in mammals.

  8. VLSI System Implementation of 200 MHz, 8-bit, 90nm CMOS Arithmetic and Logic Unit (ALU Processor Controller

    Directory of Open Access Journals (Sweden)

    Fazal NOORBASHA

    2012-08-01

    Full Text Available In this present study includes the Very Large Scale Integration (VLSI system implementation of 200MHz, 8-bit, 90nm Complementary Metal Oxide Semiconductor (CMOS Arithmetic and Logic Unit (ALU processor control with logic gate design style and 0.12µm six metal 90nm CMOS fabrication technology. The system blocks and the behaviour are defined and the logical design is implemented in gate level in the design phase. Then, the logic circuits are simulated and the subunits are converted in to 90nm CMOS layout. Finally, in order to construct the VLSI system these units are placed in the floor plan and simulated with analog and digital, logic and switch level simulators. The results of the simulations indicates that the VLSI system can control different instructions which can divided into sub groups: transfer instructions, arithmetic and logic instructions, rotate and shift instructions, branch instructions, input/output instructions, control instructions. The data bus of the system is 16-bit. It runs at 200MHz, and operating power is 1.2V. In this paper, the parametric analysis of the system, the design steps and obtained results are explained.

  9. CTG repeats distribution and Alu insertion polymorphism at myotonic dystrophy (DM) gene in Amhara and Oromo populations of Ethiopia.

    Science.gov (United States)

    Gennarelli, M; Pavoni, M; Cruciani, F; De Stefano, G; Dallapiccola, B; Novelli, G

    1999-01-01

    Myotonic dystrophy (DM) is a dominantly inherited neuromuscular disease, highly variable and multisystemic, which is caused by the expansion of a CTG repeat located in the 3' untranslated region of the DMPK gene. Normal alleles show a copy number of 5-37 repeats on normal chromosomes, amplified to 50-3000 copies on DM chromosomes. The trinucleotide repeat shows a trimodal allele distribution in the majority of the examined population. The first class includes alleles carrying (CTG)5, the second class, alleles in the range 7-18 repeats, and the third class, alleles (CTG) > or =19. The frequency of this third class is directly related to the prevalence of DM in different populations, suggesting that normal large-sized alleles predispose toward DM. We studied CTG repeat allele distribution and Alu insertion and/or deletion polymorphism at the myotonic dystrophy locus in two major Ethiopian populations, the Amhara and Oromo. CTG allele distribution and haplotype analysis on a total of 224 normal chromosomes showed significant differences between the two ethnic groups. These differences have a bearing on the out-of-Africa hypothesis for the origin of the DM mutation. In addition, (CTG) > or =19 were exclusively detected in the Amhara population, confirming the predisposing role of these alleles compared with the DM expansion-mutation.

  10. Beginning XML, 5th Edition

    CERN Document Server

    Fawcett, Joe; Quin, Liam R E

    2012-01-01

    A complete update covering the many advances to the XML language The XML language has become the standard for writing documents on the Internet and is constantly improving and evolving. This new edition covers all the many new XML-based technologies that have appeared since the previous edition four years ago, providing you with an up-to-date introductory guide and reference. Packed with real-world code examples, best practices, and in-depth coverage of the most important and relevant topics, this authoritative resource explores both the advantages and disadvantages of XML and addresses the mo

  11. Isotope-edited infrared spectroscopy.

    Science.gov (United States)

    Buchner, Ginka S; Kubelka, Jan

    2012-01-01

    Isotope-edited infrared (IR) spectroscopy is a powerful tool for studying structural and dynamical properties of peptides and proteins with site-specific resolution. Labeling of selected amide carbonyls with (13)C results in detectable sidebands of amide I' vibrations, which provide information about local conformation and/or solvent exposure without structural perturbation to the protein. Incorporation of isotopically labeled amino acids at specific positions is achieved by the chemical synthesis of the studied proteins. We describe the basic procedures for synthesis of (13)C isotopically edited protein samples, experimental IR spectroscopic measurements, and analysis of the site-specific structural changes from the thermal unfolding IR data.

  12. Introducing ZBrush 3rd Edition

    CERN Document Server

    Keller, Eric

    2012-01-01

    Learn ZBrush inside and out with this updated new edition Get totally comfortable sculpting in a digital environment with the latest edition of this bestselling beginner's guide to ZBrush. Fully updated for the newest version of the software, ZBrush 4R3, this book dispels any fears you might have about the difficulty of using ZBrush and soon has you creating realistic, cartoon, and organic models with flair. Learn all the essentials, as you complete fun tutorials on painting, meshes, organic scripting, hard surface sculpting, lighting, rendering, and more. Introduces you to ZBrush, the sculpt

  13. Medical writing, revising and editing

    DEFF Research Database (Denmark)

    Pilegaard, Morten

    2006-01-01

    The globalization of science makes medical writing, editing and revision a rapidly growing field of linguistic study and practice. Medical science texts are written according to uniform, general guidelines and medical genres have become highly conventionalized in terms of structure and linguistic...... form. Medical editing often takes the form of peer review and mainly addresses issues of contents and overall validity. Medical revision incorporates the checking of the macrostructure and the microstructure of the text, its language and style and its suitability for the target reader or client...

  14. Gas Metal Arc Welding and Flux-Cored Arc Welding. Third Edition. Teacher Edition [and] Student Edition [and] Student Workbook.

    Science.gov (United States)

    Knapp, John; Harper, Eddie

    This packet, containing a teacher's edition, a student edition, and a student workbook, introduces students to high deposition welding and processes for "shielding" a weld. In addition to general information, the teacher edition consists of introductory pages and teacher pages, as well as unit information that corresponds to the materials in the…

  15. Civil Technology Applications. Teacher Edition [and] Student Edition.

    Science.gov (United States)

    Schertz, Karen

    Teacher and student editions of Civil Technology Applications are one in a series of competency-based instructional materials for drafting and civil technology programs. It includes the technical content and tasks necessary for a student to be employed as a drafter or civil technician in a civil engineering firm. Introductory pages in the teacher…

  16. A non-inheritable maternal Cas9-based multiple-gene editing system in mice.

    Science.gov (United States)

    Sakurai, Takayuki; Kamiyoshi, Akiko; Kawate, Hisaka; Mori, Chie; Watanabe, Satoshi; Tanaka, Megumu; Uetake, Ryuichi; Sato, Masahiro; Shindo, Takayuki

    2016-01-01

    The CRISPR/Cas9 system is capable of editing multiple genes through one-step zygote injection. The preexisting method is largely based on the co-injection of Cas9 DNA (or mRNA) and guide RNAs (gRNAs); however, it is unclear how many genes can be simultaneously edited by this method, and a reliable means to generate transgenic (Tg) animals with multiple gene editing has yet to be developed. Here, we employed non-inheritable maternal Cas9 (maCas9) protein derived from Tg mice with systemic Cas9 overexpression (Cas9 mice). The maCas9 protein in zygotes derived from mating or in vitro fertilization of Tg/+ oocytes and +/+ sperm could successfully edit the target genome. The efficiency of such maCas9-based genome editing was comparable to that of zygote microinjection-based genome editing widely used at present. Furthermore, we demonstrated a novel approach to create "Cas9 transgene-free" gene-modified mice using non-Tg (+/+) zygotes carrying maCas9. The maCas9 protein in mouse zygotes edited nine target loci simultaneously after injection with nine different gRNAs alone. Cas9 mouse-derived zygotes have the potential to facilitate the creation of genetically modified animals carrying the Cas9 transgene, enabling repeatable genome engineering and the production of Cas9 transgene-free mice. PMID:26817415

  17. Controlled flexibility in technical editing - The levels-of-edit concept at JPL

    Science.gov (United States)

    Buehler, M. F.

    1977-01-01

    The levels-of-edit concept, which can be used to specify the amount of editorial effort involved in the preparation of a manuscript for publication, is discussed. Nine types of editing are identified and described. These include coordination edit (preparing estimates, gathering cost data, monitoring production processes), policy edit, integrity edit (making sure that parts of a publication match in a physical or numerical sense), screening edit (ensuring that the quality of camera-ready copy is sufficient for external publication), copy clarification edit, format edit, mechanical style edit, language edit, and substantive edit (reviewing the manuscript for content coherence, emphasis, subordination and parallelism). These functions are grouped into five levels of edit. An edit-level number is assigned to each manuscript, providing a quantitative and qualitative indicator of the editing to be done which is clearly understood by authors, managers, and editors alike. In addition, clear boundaries are drawn between normal and extraordinary editing tasks. Individual organizations will group various edits in different ways to reflect their needs and priorities; the essential element of the system is unambiguous definition and coding of the types and amount of work to be done.

  18. Parameter-Free Extended Edit Distance

    OpenAIRE

    Muhammad Fuad, Muhammad Marwan

    2014-01-01

    The edit distance is the most famous distance to compute the similarity between two strings of characters. The main drawback of the edit distance is that it is based on local procedures which reflect only a local view of similarity. To remedy this problem we presented in a previous work the extended edit distance, which adds a global view of similarity between two strings. However, the extended edit distance includes a parameter whose computation requires a long training time. In this paper w...

  19. DNA Editing by APOBECs: A Genomic Preserver and Transformer.

    Science.gov (United States)

    Knisbacher, Binyamin A; Gerber, Doron; Levanon, Erez Y

    2016-01-01

    Information warfare is not limited to the cyber world because it is waged within our cells as well. The unique AID (activation-induced cytidine deaminase)/APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) family comprises proteins that alter DNA sequences by converting deoxycytidines to deoxyuridines through deamination. This C-to-U DNA editing enables them to inhibit parasitic viruses and retrotransposons by disrupting their genomic content. In addition to attacking genomic invaders, APOBECs can target their host genome, which can be beneficial by initiating processes that create antibody diversity needed for the immune system or by accelerating the rate of evolution. AID can also alter gene regulation by removing epigenetic modifications from genomic DNA. However, when uncontrolled, these powerful agents of change can threaten genome stability and eventually lead to cancer. PMID:26608778

  20. Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity.

    Science.gov (United States)

    Tycko, Josh; Myer, Vic E; Hsu, Patrick D

    2016-08-01

    Advances in the development of delivery, repair, and specificity strategies for the CRISPR-Cas9 genome engineering toolbox are helping researchers understand gene function with unprecedented precision and sensitivity. CRISPR-Cas9 also holds enormous therapeutic potential for the treatment of genetic disorders by directly correcting disease-causing mutations. Although the Cas9 protein has been shown to bind and cleave DNA at off-target sites, the field of Cas9 specificity is rapidly progressing, with marked improvements in guide RNA selection, protein and guide engineering, novel enzymes, and off-target detection methods. We review important challenges and breakthroughs in the field as a comprehensive practical guide to interested users of genome editing technologies, highlighting key tools and strategies for optimizing specificity. The genome editing community should now strive to standardize such methods for measuring and reporting off-target activity, while keeping in mind that the goal for specificity should be continued improvement and vigilance. PMID:27494557

  1. [An introduction on the editions of Zhang Gao's Yi shuo (Medical Narrations)].

    Science.gov (United States)

    Wang, Xuguang; Lu, Xiang

    2014-11-01

    Zhang Gao's Yi shuo (Medical Narrations) of the Southern Song Dynasty had 2 kinds of editions: domestic editions and foreign editions. The former includes 1 Song edition, 14 Ming editions, 3 Qing editions and 25 editions after the Republic of China. The latter, mainly 2 classes, the Japanese edition and Korean printing type edition. In the Ming Dynasty, the editions of Yi shuo generated 2 branches: inherited edition and supplementary edition. The inherited editions include Gu Dingfang's edition, Zhang Yaode's edition, Wu Mianxue's edition, Wu Zhongheng's edition, Wang Kentang's edition, editions from Si ku quan shu (Imperial Collection of Four), stereotype edition of the 3th year of Xuantong reign (1911) from Shanghai Civilization Bookstore, edition of the 2(nd) year of Manji of Japan etc. The supplemental editions include Zhang Zili's edition, Shen Fan's edition, Fu Feng'ao's edition, transcript of the late Ming Dynasty preserved in the Library of Peking University, and Korean printing type edition etc.

  2. [An introduction on the editions of Zhang Gao's Yi shuo (Medical Narrations)].

    Science.gov (United States)

    Wang, Xuguang; Lu, Xiang

    2014-11-01

    Zhang Gao's Yi shuo (Medical Narrations) of the Southern Song Dynasty had 2 kinds of editions: domestic editions and foreign editions. The former includes 1 Song edition, 14 Ming editions, 3 Qing editions and 25 editions after the Republic of China. The latter, mainly 2 classes, the Japanese edition and Korean printing type edition. In the Ming Dynasty, the editions of Yi shuo generated 2 branches: inherited edition and supplementary edition. The inherited editions include Gu Dingfang's edition, Zhang Yaode's edition, Wu Mianxue's edition, Wu Zhongheng's edition, Wang Kentang's edition, editions from Si ku quan shu (Imperial Collection of Four), stereotype edition of the 3th year of Xuantong reign (1911) from Shanghai Civilization Bookstore, edition of the 2(nd) year of Manji of Japan etc. The supplemental editions include Zhang Zili's edition, Shen Fan's edition, Fu Feng'ao's edition, transcript of the late Ming Dynasty preserved in the Library of Peking University, and Korean printing type edition etc. PMID:25620361

  3. Editing plant genomes with CRISPR/Cas9.

    Science.gov (United States)

    Belhaj, Khaoula; Chaparro-Garcia, Angela; Kamoun, Sophien; Patron, Nicola J; Nekrasov, Vladimir

    2015-04-01

    CRISPR/Cas9 is a rapidly developing genome editing technology that has been successfully applied in many organisms, including model and crop plants. Cas9, an RNA-guided DNA endonuclease, can be targeted to specific genomic sequences by engineering a separately encoded guide RNA with which it forms a complex. As only a short RNA sequence must be synthesized to confer recognition of a new target, CRISPR/Cas9 is a relatively cheap and easy to implement technology that has proven to be extremely versatile. Remarkably, in some plant species, homozygous knockout mutants can be produced in a single generation. Together with other sequence-specific nucleases, CRISPR/Cas9 is a game-changing technology that is poised to revolutionise basic research and plant breeding.

  4. Strategies of Qualitative Inquiry. Third Edition

    Science.gov (United States)

    Denzin, Norman K., Ed.; Lincoln, Yvonna S., Ed.

    2007-01-01

    "Strategies of Qualitative Inquiry, Third Edition," the second volume in the paperback version of "The SAGE Handbook of Qualitative Research, 3rd Edition," consists of Part III of the handbook ("Strategies of Inquiry"). "Strategies of Qualitative Inquiry, Third Edition" presents the major tactics--historically, the research methods--that…

  5. Teaching Students to Self-Edit.

    Science.gov (United States)

    Ferris, Dana

    1995-01-01

    Emphasizes the need for students to develop their editing skills. This article suggests that teachers and students should concentrate on major error patterns, and teachers should personalize editing instruction. Attention should also be given to the most frequent and glaring errors. Students who followed this editing approach significantly reduced…

  6. Testing post-editing guidelines

    DEFF Research Database (Denmark)

    Flanagan, Marian; Christensen, Tina Paulsen

    2014-01-01

    There is a growing interest in machine translation (MT) and post-editing (PE). MT has been around for decades, but the use of the technology has grown significantly in the language industry in recent years, while PE is still a relatively new task. Consequently, there are currently no standard PE...

  7. Veterinary Microbiology, 3rd Edition

    Science.gov (United States)

    Veterinary Microbiology, Third Edition is organized into four sections and begins with an updated and expanded introductory section on infectious disease pathogenesis, diagnosis and clinical management. The second section covers bacterial and fungal pathogens, and the third section describes viral d...

  8. Money and Schools. Fifth Edition

    Science.gov (United States)

    Thompson, David C.; Crampton, Faith E.; Wood, R. Craig

    2012-01-01

    In the new edition of this essential, all-inclusive text, the authors provide more important research for future principals and others enrolled in graduate-level school finance courses. Written in a style that is highly readable, the book offers strong connections to real-world experiences. Readers get both a broad overview of funding concepts and…

  9. New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.

    Science.gov (United States)

    Laughery, Marian F; Hunter, Tierra; Brown, Alexander; Hoopes, James; Ostbye, Travis; Shumaker, Taven; Wyrick, John J

    2015-12-01

    Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology is an important tool for genome editing because the Cas9 endonuclease can induce targeted DNA double-strand breaks. Targeting of the DNA break is typically controlled by a single-guide RNA (sgRNA), a chimeric RNA containing a structural segment important for Cas9 binding and a 20mer guide sequence that hybridizes to the genomic DNA target. Previous studies have demonstrated that CRISPR-Cas9 technology can be used for efficient, marker-free genome editing in Saccharomyces cerevisiae. However, introducing the 20mer guide sequence into yeast sgRNA expression vectors often requires cloning procedures that are complex, time-consuming and/or expensive. To simplify this process, we have developed a new sgRNA expression cassette with internal restriction enzyme sites that permit rapid, directional cloning of 20mer guide sequences. Here we describe a flexible set of vectors based on this design for cloning and expressing sgRNAs (and Cas9) in yeast using different selectable markers. We anticipate that the Cas9-sgRNA expression vector with the URA3 selectable marker (pML104) will be particularly useful for genome editing in yeast, since the Cas9 machinery can be easily removed by counter-selection using 5-fluoro-orotic acid (5-FOA) following successful genome editing. The availability of new vectors that simplify and streamline the technical steps required for guide sequence cloning should help accelerate the use of CRISPR-Cas9 technology in yeast genome editing.

  10. Efficient CRISPR/Cas9 Plasmids for Rapid and Versatile Genome Editing in Drosophila

    OpenAIRE

    Gokcezade, Joseph; Sienski, Grzegorz; Duchek, Peter

    2014-01-01

    The CRISPR-associated RNA-guided nuclease Cas9 has emerged as a powerful tool for genome engineering in a variety of organisms. To achieve efficient gene targeting rates in Drosophila, current approaches require either injection of in vitro transcribed RNAs or injection into transgenic Cas9-expressing embryos. We report a simple and versatile alternative method for CRISPR-mediated genome editing in Drosophila using bicistronic Cas9/sgRNA expression vectors. Gene targeting with this single-pla...

  11. Intronic Alus influence alternative splicing.

    Directory of Open Access Journals (Sweden)

    Galit Lev-Maor

    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.

  12. Polymorphic Alu insertions and their associations with HLA Ⅰ alleles in Yugu and Zhuang ethnic populations%中国壮族和裕固族群体HLAⅠ类区域Alu插入多态性及其与HLA-A位点的相关性

    Institute of Scientific and Technical Information of China (English)

    史磊; 杨昭庆; 褚嘉祐; 姚宇峰; 史荔; 陶玉芬; 于亮; 黄小琴; 林克勤; 易文; 孙浩

    2011-01-01

    Many studies have show that the structurally polymorphic Alu insertion within HLA class I region are useful tools for investigating the origin, evolmion and recombination of HLA class Ⅰ progenitor haplotypes and gene diversity in different ethnic populations.In the present study, we determined the frequencies of HLA-Alus (i.e., AluMICB, AluTF,AluHJ, AluHG, and AluHF) in Zhuang and Yugu ethnic populations at first.Then combined with HLA genotyping data.we studied associations between HLA-Alus and HLA-A alleles in Zhuang, Yugu Bulang, Dai, and Hani ethnic populations.Our results showed that (l) the frequencies of five HLA-Alus were 1.5%~ 35.8% and 9.2%~34.8% in Zhuang and Yugu, respectively: and (2) the results of association between HLA-A alleles and HLA-Alu showed strong association between AluHG insalion and HLA-A *02 subtypes in all populations, association between AluHJ insertion and HLA-A *2402 in all populations, and association between AluHJ inseflion and HLA-A V/O/, -A *2407 in Bulang.The present study suggested that the distribution of HLA-Alus as well as the associations between HLA-Alus and HLA class I alleles are variable in different ethnic populations.HLA Alus alone or together with the HLA classⅠ alieles are informative genetic markers for the identification of HLA class I allele and variation of haplotype lineages in different populations.%近年来研究发现:位于HLAⅠ类基因区域的Alu插入是研究不同群体HLAⅠ类基因区域祖先单倍型和HLAⅠ类基因多样性产生、进化和重组的理想工具.文章对中国壮族和裕固族群体HLAⅠ类基因区域5个Alu插入多态性(AluMICB、AluTF、AluHJ、AluHG和AluHF)进行研究,结合HLA基因分型数据,分析壮族、裕固族、哈尼族、布朗族和傣族5个民族群体中Alu插入与HLA-A等位基因的关系.研究结果显示:(1)壮族和裕固族人群中5个Alu插入频率范围分别为1.5%~35.8%和9.2~34.8%,AluMICB、AluTF和Alu,HF插入

  13. Transgene-Free Genome Editing in Caenorhabditis elegans Using CRISPR-Cas

    OpenAIRE

    Chiu, Hui; Schwartz, Hillel T.; Antoshechkin, Igor; Paul W Sternberg

    2013-01-01

    CRISPR-Cas is an efficient method for genome editing in organisms from bacteria to human cells. We describe a transgene-free method for CRISPR-Cas-mediated cleavage in nematodes, enabling RNA-homology-targeted deletions that cause loss of gene function; analysis of whole-genome sequencing indicates that the nuclease activity is highly specific.

  14. Characterizing the transcriptome upon depletion of RNA processing factors

    DEFF Research Database (Denmark)

    Herudek, Jan

    2016-01-01

    The human genome is pervasively transcribed and produces an enormous amount of non-coding RNA (ncRNA). Compared to protein-coding transcripts, many classes of ncRNAs are very unstable and rapidly degraded by the RNA decay machinery. The RNA exosome complex is a main RNA ‘degrader’ in the human nu...... this method with CRISPR/Cas9 genome editing to pursue rapid depletion of endogenous protein. Applying this technology, I aim to study dynamics of the RNA decay machinery and obtain a deeper understanding of recently characterized ncRNAs....

  15. RNA-DNA Differences Are Generated in Human Cells within Seconds after RNA Exits Polymerase II

    Directory of Open Access Journals (Sweden)

    Isabel X. Wang

    2014-03-01

    Full Text Available RNA sequences are expected to be identical to their corresponding DNA sequences. Here, we found all 12 types of RNA-DNA sequence differences (RDDs in nascent RNA. Our results show that RDDs begin to occur in RNA chains ∼55 nt from the RNA polymerase II (Pol II active site. These RDDs occur so soon after transcription that they are incompatible with known deaminase-mediated RNA-editing mechanisms. Moreover, the 55 nt delay in appearance indicates that they do not arise during RNA synthesis by Pol II or as a direct consequence of modified base incorporation. Preliminary data suggest that RDD and R-loop formations may be coupled. These findings identify sequence substitution as an early step in cotranscriptional RNA processing.

  16. RNA Crystallization

    Science.gov (United States)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  17. Correlation between GH gene polymorphism of the 5th exon Alu Ⅰ site and early growth traits in Xinjiang Brown Cattle%新疆褐牛GH基因第5外显子Alu Ⅰ位点多态性与早期生长性状的相关性

    Institute of Scientific and Technical Information of China (English)

    牛志刚; 史洪才; 刘明军; 周振勇; 张扬

    2012-01-01

    [目的]探讨生长激素(CH)基因对新疆褐牛早期生长性能的影响,为新疆褐牛选育提高奠定分子遗传基础.[方法]以116头份新疆褐牛血样为研究对象,利用PCR-RFLP技术检测CH基因第5外显子上Alu Ⅰ突变位点(CH/Alu Ⅰ位点)的多态性,并分析不同基因型与新疆褐牛早期生长性状的相关性.[结果]GH/Alu Ⅰ位点在新疆褐牛群体中表现为VV、LV和LL3种基因型,其基因频率分别为0.138、0.397和0.465;新疆褐牛GH/Alu Ⅰ位点的杂合度为0.4408,多态信息含量为0.3437;不同基因型与早期生长发育性状的关系为:1~4月龄的LV和LL基因型个体体重、体长均较VV基因型个体的优,且差异显著(P<0.05),而出生体重、5~6月龄体重、体长差异不显著(P>0.05).[结论]GH/Alu Ⅰ位点的L等位基因对新疆褐牛早期生长发育性状有正向选择作用,可作为新疆褐牛辅助选择的遗传标记.%[Objective]Effects of GH gene on early growth traits of Xinjiang Brown Cattle were investigatool in order to provide molecular genetic information for breeding Xinjiang Brown Cattle. [ Method ]Blood samples of total 116 Xinjiang Brown Cattle were collected to detect polymorphism of Alu I mutational site on 5th exon of CH using PCR-RFLP method. Correlation between genotype polymorphism and early growth traits was also assayed. [ Result ]GH/Alu I site was expressed in Xinjiang Brown Cattle as VV, LV, LL genetype with gene frequencies 0.138, 0.397 and 0.465, respectively. Helernzygosity and polymorphic information of GH/Alu I were 0.4408 and 0.3437, respectively. Relations between various genotypes and early growth traits were as follows: for I -4 months old cattle, body weight and length of LV and LL genotypes were superior to VV genotype, and the difference was significant (P<0.05). However, no significant differences were observed in VV, LV, LL genotypes for birth weight and body weight and length of 5-6 months old cattle. [Conclusion

  18. CRISPR/Cas9 in Genome Editing and Beyond.

    Science.gov (United States)

    Wang, Haifeng; La Russa, Marie; Qi, Lei S

    2016-06-01

    The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers.

  19. A novel web-based TinT application and the chronology of the Primate Alu retroposon activity

    Directory of Open Access Journals (Sweden)

    Makałowski Wojciech

    2010-12-01

    Full Text Available Abstract Background DNA sequences afford access to the evolutionary pathways of life. Particularly mobile elements that constantly co-evolve in genomes encrypt recent and ancient information of their host's history. In mammals there is an extraordinarily abundant activity of mobile elements that occurs in a dynamic succession of active families, subfamilies, types, and subtypes of retroposed elements. The high frequency of retroposons in mammals implies that, by chance, such elements also insert into each other. While inactive elements are no longer able to retropose, active elements retropose by chance into other active and inactive elements. Thousands of such directional, element-in-element insertions are found in present-day genomes. To help analyze these events, we developed a computational algorithm (Transpositions in Transpositions, or TinT that examines the different frequencies of nested transpositions and reconstructs the chronological order of retroposon activities. Results By examining the different frequencies of such nested transpositions, the TinT application reconstructs the chronological order of retroposon activities. We use such activity patterns as a comparative tool to (1 delineate the historical rise and fall of retroposons and their relations to each other, (2 understand the retroposon-induced complexity of recent genomes, and (3 find selective informative homoplasy-free markers of phylogeny. The efficiency of the new application is demonstrated by applying it to dimeric Alu Short INterspersed Elements (SINE to derive a complete chronology of such elements in primates. Conclusion The user-friendly, web-based TinT interface presented here affords an easy, automated screening for nested transpositions from genome assemblies or trace data, assembles them in a frequency-matrix, and schematically displays their chronological activity history.

  20. Maternal phthalate exposure during pregnancy is associated with DNA methylation of LINE-1 and Alu repetitive elements in Mexican-American children.

    Science.gov (United States)

    Huen, Karen; Calafat, Antonia M; Bradman, Asa; Yousefi, Paul; Eskenazi, Brenda; Holland, Nina

    2016-07-01

    Phthalates are frequently used in personal care products and plasticizers and phthalate exposure is ubiquitous in the US population. Exposure to phthalates during critical periods in utero has been associated with a variety of adverse health outcomes but the biological mechanisms linking these exposures with disease are not well characterized. In this study, we examined the relationship of in utero phthalate exposure with repetitive element DNA methylation, an epigenetic marker of genome instability, in children from the longitudinal birth cohort CHAMACOS. Methylation of Alu and long interspersed nucleotide elements (LINE-1) was determined using pyrosequencing of bisulfite-treated DNA isolated from whole blood samples collected from newborns and 9 year old children (n=355). Concentrations of eleven phthalate metabolites were measured in urine collected from pregnant mothers at 13 and 26 weeks gestation. We found a consistent inverse association between prenatal concentrations of monoethyl phthalate, the most frequently detected urinary metabolite, with cord blood methylation of Alu repeats (β(95%CI): -0.14 (-0.28,0.00) and -0.16 (-0.31, -0.02)) for early and late pregnancy, respectively, and a similar but weaker association with LINE-1 methylation. Additionally, increases in urinary concentrations of di-(2-ethylhexyl) phthalate metabolites during late pregnancy were associated with lower levels of methylation of Alu repeats in 9 year old blood (significant p-values ranged from 0.003 to 0.03). Our findings suggest that prenatal exposure to some phthalates may influence differences in repetitive element methylation, highlighting epigenetics as a plausible biological mechanism through which phthalates may affect health. PMID:27019040

  1. Feedback inhibition of L1 and alu retrotransposition through altered double strand break repair kinetics

    Directory of Open Access Journals (Sweden)

    Wallace Nicholas A

    2010-10-01

    Full Text Available Abstract Background Cells adapt to various chronic toxic exposures in a multitude of ways to minimize further damage and maximize their growth potential. Expression of L1 elements in the human genome can be greatly deleterious to cells, generating numerous double strand breaks (DSBs. Cells have been reported to respond to chronic DSBs by altering the repair of these breaks, including increasing the rate of homology independent DSB repair. Retrotransposition is strongly affected by proteins involved in DSB repair. Therefore, L1 expression has the potential to be a source of chronic DSBs and thus bring about the changes in cellular environment that could ultimately restrict its own retrotransposition. Results We demonstrate that constitutive L1 expression leads to quicker DSB repair and decreases in the retrotransposition potential of L1 and other retrotransposons dependent on L1 expression for their mobility. This cellular adaptation results in reduced sensitivity to L1 induced toxicity. These effects can be induced by constitutive expression of the functional L1 ORF2 alone, but not by the constitutive expression of an L1 open reading frame 2 with mutations to its endonuclease and reverse transcriptase domains. This adaptation correlates with the relative activity of the L1 introduced into the cells. Conclusions The increased number of DSBs resulting from constitutive expression of L1 results in a more rapid rate of repair. The cellular response to this L1 expression also results in attenuation of retrotransposition and reduced sensitivity of the cells to negative consequences of L1 ORF2 expression. The influence does not appear to be through RNA interference. We believe that the increased rate of DSB repair is the most likely cause of the attenuation of retrotransposition. These alterations act as a fail safe mechanism that allows cells to escape the toxicity associated with the unchecked L1 expression. This gives cells that overexpress L1, such

  2. Recent Advances in Genome Editing Using CRISPR/Cas9

    Directory of Open Access Journals (Sweden)

    Yuduan eDing

    2016-05-01

    Full Text Available AbstractThe CRISPR (clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-associated nuclease 9 system is a versatile tool for genome engineering that uses a guide RNA (gRNA to target Cas9 to a specific sequence. This simple RNA-guided genome-editing technology has become a revolutionary tool in biology and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing method, summarize the recent advances in CRISPR/Cas9 technology, and discuss their implications for plant research. To date, targeted gene knockout using the Cas9/gRNA system has been established in many plant species, and the targeting efficiency and capacity of Cas9 has been improved by optimizing its expression and that of its gRNA. The CRISPR/Cas9 system can also be used for sequence-specific mutagenesis/integration and transcriptional control of target genes. We also discuss off-target effects and the constraint that the protospacer-adjacent motif (PAM puts on CRISPR/Cas9 genome engineering. To address these problems, a number of bioinformatic tools are available to help design specific gRNAs, and new Cas9 variants and orthologs with high fidelity and alternative PAM specificities have been engineered. Owing to these recent efforts, the CRISPR/Cas9 system is becoming a revolutionary and flexible tool for genome engineering. Adoption of the CRISPR/Cas9 technology in plant research would enable the investigation of plant biology at an unprecedented depth and create innovative applications in precise crop breeding.

  3. Edition des Corpus areopagiticum slavicum

    Directory of Open Access Journals (Sweden)

    Dieter Fahl

    2005-12-01

    Full Text Available An Edition of the Corpus areopagiticum slavicum In the fourteenth century, the monk Isaiah of the holy Mount Athos translated the writings of pseudo-Dionysius the Areopagite (c. end of the 5th century, core texts for Eastern and Western European theological and philosophical thought, from Greek into Church Slavonic. This first Slavic translation of Dionysius’ oeuvre (“De Coelesti Hierarchia,” “De Ecclesiastica Hierarchia,” “De Divinis Nominibus,” “De Mystica Theologia,” the epistles and scholia, which played a significant role in the development of Slavic culture, Orthodox Slavic socio-political theory and praxis, is still central to the study of Slavia Orthodoxa. A working group of German and Russian scholars has completed an edition of the translator’s Church Slavonic autograph with an en face reconstruction of the Greek text used by the translator and philological commentary. A Church Slavonic-Greek and Greek-Church Slavonic dictionary of this edition, currently in preparation, plans to make the terminology used in this influential translation accessible to interdisciplinary researchers. For the first time, the Church Slavonic lexica of this corpus, a substantial part of which was coined by the translator, will be registered in an index of words and forms.

  4. Coupling and Coordination in Gene Expression Processes with Pre-mRNA Splicing

    OpenAIRE

    Kewu Pan; Jimmy Tsz Hang Lee; Zhe Huang; Chi-Ming Wong

    2015-01-01

    RNA processing is a tightly regulated and highly complex pathway which includes transcription, splicing, editing, transportation, translation and degradation. It has been well-documented that splicing of RNA polymerase II medicated nascent transcripts occurs co-transcriptionally and is functionally coupled to other RNA processing. Recently, increasing experimental evidence indicated that pre-mRNA splicing influences RNA degradation and vice versa. In this review, we summarized the recent find...

  5. The emerging role of viral vectors as vehicles for DMD gene editing.

    Science.gov (United States)

    Maggio, Ignazio; Chen, Xiaoyu; Gonçalves, Manuel A F V

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a genetic disorder caused by mutations in the dystrophin-encoding DMD gene. The DMD gene, spanning over 2.4 megabases along the short arm of the X chromosome (Xp21.2), is the largest genetic locus known in the human genome. The size of DMD, combined with the complexity of the DMD phenotype and the extent of the affected tissues, begs for the development of novel, ideally complementary, therapeutic approaches. Genome editing based on the delivery of sequence-specific programmable nucleases into dystrophin-defective cells has recently enriched the portfolio of potential therapies under investigation. Experiments involving different programmable nuclease platforms and target cell types have established that the application of genome-editing principles to the targeted manipulation of defective DMD loci can result in the rescue of dystrophin protein synthesis in gene-edited cells. Looking towards translation into the clinic, these proof-of-principle experiments have been swiftly followed by the conversion of well-established viral vector systems into delivery agents for DMD editing. These gene-editing tools consist of zinc-finger nucleases (ZFNs), engineered homing endoculeases (HEs), transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases (RGNs) based on clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 systems. Here, we succinctly review these fast-paced developments and technologies, highlighting their relative merits and potential bottlenecks, when used as part of in vivo and ex vivo gene-editing strategies. PMID:27215286

  6. Quantifying Genome-Editing Outcomes at Endogenous Loci with SMRT Sequencing

    Directory of Open Access Journals (Sweden)

    Ayal Hendel

    2014-04-01

    Full Text Available Targeted genome editing with engineered nucleases has transformed the ability to introduce precise sequence modifications at almost any site within the genome. A major obstacle to probing the efficiency and consequences of genome editing is that no existing method enables the frequency of different editing events to be simultaneously measured across a cell population at any endogenous genomic locus. We have developed a method for quantifying individual genome-editing outcomes at any site of interest with single-molecule real-time (SMRT DNA sequencing. We show that this approach can be applied at various loci using multiple engineered nuclease platforms, including transcription-activator-like effector nucleases (TALENs, RNA-guided endonucleases (CRISPR/Cas9, and zinc finger nucleases (ZFNs, and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred. This approach offers a technique for studying double-strand break repair, facilitates the evaluation of gene-editing technologies, and permits sensitive quantification of editing outcomes in almost every experimental system used.

  7. CRISPR/Cas9 for genome editing: progress, implications and challenges.

    Science.gov (United States)

    Zhang, Feng; Wen, Yan; Guo, Xiong

    2014-09-15

    Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 system provides a robust and multiplexable genome editing tool, enabling researchers to precisely manipulate specific genomic elements, and facilitating the elucidation of target gene function in biology and diseases. CRISPR/Cas9 comprises of a nonspecific Cas9 nuclease and a set of programmable sequence-specific CRISPR RNA (crRNA), which can guide Cas9 to cleave DNA and generate double-strand breaks at target sites. Subsequent cellular DNA repair process leads to desired insertions, deletions or substitutions at target sites. The specificity of CRISPR/Cas9-mediated DNA cleavage requires target sequences matching crRNA and a protospacer adjacent motif locating at downstream of target sequences. Here, we review the molecular mechanism, applications and challenges of CRISPR/Cas9-mediated genome editing and clinical therapeutic potential of CRISPR/Cas9 in future.

  8. Connectivity editing for quad-dominant meshes

    KAUST Repository

    Peng, Chihan

    2013-08-01

    We propose a connectivity editing framework for quad-dominant meshes. In our framework, the user can edit the mesh connectivity to control the location, type, and number of irregular vertices (with more or fewer than four neighbors) and irregular faces (non-quads). We provide a theoretical analysis of the problem, discuss what edits are possible and impossible, and describe how to implement an editing framework that realizes all possible editing operations. In the results, we show example edits and illustrate the advantages and disadvantages of different strategies for quad-dominant mesh design. © 2013 The Author(s) Computer Graphics Forum © 2013 The Eurographics Association and John Wiley & Sons Ltd.

  9. Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system.

    Science.gov (United States)

    Cheong, Taek-Chin; Compagno, Mara; Chiarle, Roberto

    2016-03-09

    Applications of the CRISPR-Cas9 system to edit the genome have widely expanded to include DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings. Here we show the application of CRISPR-Cas9 technology to edit the mouse and human immunoglobulin (Ig) genes. By delivering Cas9 and guide-RNA (gRNA) with retro- or lenti-virus to IgM(+) mouse B cells and hybridomas, we induce class-switch recombination (CSR) of the IgH chain to the desired subclass. Similarly, we induce CSR in all human B cell lines tested with high efficiency to targeted IgH subclass. Finally, we engineer mouse hybridomas to secrete Fab' fragments instead of the whole Ig. Our results indicate that Ig genes in mouse and human cells can be edited to obtain any desired IgH switching helpful to study the biology of normal and lymphoma B cells. We also propose applications that could transform the technology of antibody production.

  10. Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system.

    Science.gov (United States)

    Cheong, Taek-Chin; Compagno, Mara; Chiarle, Roberto

    2016-01-01

    Applications of the CRISPR-Cas9 system to edit the genome have widely expanded to include DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings. Here we show the application of CRISPR-Cas9 technology to edit the mouse and human immunoglobulin (Ig) genes. By delivering Cas9 and guide-RNA (gRNA) with retro- or lenti-virus to IgM(+) mouse B cells and hybridomas, we induce class-switch recombination (CSR) of the IgH chain to the desired subclass. Similarly, we induce CSR in all human B cell lines tested with high efficiency to targeted IgH subclass. Finally, we engineer mouse hybridomas to secrete Fab' fragments instead of the whole Ig. Our results indicate that Ig genes in mouse and human cells can be edited to obtain any desired IgH switching helpful to study the biology of normal and lymphoma B cells. We also propose applications that could transform the technology of antibody production. PMID:26956543

  11. Growth and carcass traits associated with GH1/Alu I and POU1F1/Hinf I gene polymorphisms in Zebu and crossbred beef cattle

    Directory of Open Access Journals (Sweden)

    Rogério A. Curi

    2006-01-01

    Full Text Available The objectives of the present study were to estimate the allele and genotype frequencies of the GH1/Alu I and POU1F1/Hinf I polymorphisms in beef cattle belonging to different genetic groups and to determine the effects of these polymorphisms on growth and carcass traits in cattle submitted to feedlot management, an intensive production model. Genotyping was performed on 384 animals, including 79 Nellore, 30 Canchim (5/8 Charolais + 3/8 Zebu, 30 Simmental x Nellore crossbred and 245 Angus x Nellore crossbred cattle. Body weight, weight gain, dressing percentage, Longissimus dorsi area and backfat thickness were fitted using the General Linear Model (GLM procedure of the SAS program and the least square means of the genotypes were compared using the F test. The results showed significant associations between the LL genotype of the GH1/Alu I polymorphism and higher weight gain and body weight at slaughter (p < 0.05. The POU1F1/Hinf I polymorphism did not have any effect on the growth and carcass traits analyzed.

  12. Branched DNA-based Alu quantitative assay for cell-free plasma DNA levels in patients with sepsis or systemic inflammatory response syndrome.

    Science.gov (United States)

    Hou, Yan-Qiang; Liang, Dong-Yu; Lou, Xiao-Li; Zhang, Mei; Zhang, Zhen-huan; Zhang, Lu-rong

    2016-02-01

    Cell-free circulating DNA (cf-DNA) can be detected by various of laboratory techniques. We described a branched DNA-based Alu assay for measuring cf-DNA in septic patients. Compared to healthy controls and systemic inflammatory response syndrome (SIRS) patients, serum cf-DNA levels were significantly higher in septic patients (1426.54 ± 863.79 vs 692.02 ± 703.06 and 69.66 ± 24.66 ng/mL). The areas under the receiver operating characteristic curve of cf-DNA for normal vs sepsis and SIRS vs sepsis were 0.955 (0.884-1.025), and 0.856 (0.749-0.929), respectively. There was a positive correlation between cf-DNA and interleukin 6 or procalcitonin or Acute Physiology and Chronic Health Evaluation II. The cf-DNA concentration was higher in intensive care unit nonsurviving patients compared to surviving patients (2183.33 ± 615.26 vs 972.46 ± 648.36 ng/mL; P DNA-based Alu assays are feasible and useful to quantify serum cf-DNA levels. Increased cf-DNA levels in septic patients might complement C-reactive protein and procalcitonin in a multiple marker format. Cell-free circulating DNA might be a new marker in discrimination of sepsis and SIRS.

  13. Application of the genome editing tool CRISPR/Cas9 in non-human primates.

    Science.gov (United States)

    Luo, Xin; Li, Min; Su, Bing

    2016-07-18

    In the past three years, RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system has been used to facilitate efficient genome editing in many model and non-model animals. However, its application in nonhuman primates is still at the early stage, though in view of the similarities in anatomy, physiology, behavior and genetics, closely related nonhuman primates serve as optimal models for human biology and disease studies. In this review, we summarize the current proceedings of gene editing using CRISPR/Cas9 in nonhuman primates.

  14. Application of the genome editing tool CRISPR/Cas9 in non-human primates

    Science.gov (United States)

    LUO, Xin; LI, Min; SU, Bing

    2016-01-01

    In the past three years, RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system has been used to facilitate efficient genome editing in many model and non-model animals. However, its application in nonhuman primates is still at the early stage, though in view of the similarities in anatomy, physiology, behavior and genetics, closely related nonhuman primates serve as optimal models for human biology and disease studies. In this review, we summarize the current proceedings of gene editing using CRISPR/Cas9 in nonhuman primates. PMID:27469252

  15. The Landscape of A-to-I RNA Editome Is Shaped by Both Positive and Purifying Selection

    Science.gov (United States)

    Kong, Yimeng; Pan, Bohu; Chen, Longxian; Wang, Hongbing; Hao, Pei; Li, Xuan

    2016-01-01

    The hydrolytic deamination of adenosine to inosine (A-to-I editing) in precursor mRNA induces variable gene products at the post-transcription level. How and to what extent A-to-I RNA editing diversifies transcriptome is not fully characterized in the evolution, and very little is known about the selective constraints that drive the evolution of RNA editing events. Here we present a study on A-to-I RNA editing, by generating a global profile of A-to-I editing for a phylogeny of seven Drosophila species, a model system spanning an evolutionary timeframe of approximately 45 million years. Of totally 9281 editing events identified, 5150 (55.5%) are located in the coding sequences (CDS) of 2734 genes. Phylogenetic analysis places these genes into 1,526 homologous families, about 5% of total gene families in the fly lineages. Based on conservation of the editing sites, the editing events in CDS are categorized into three distinct types, representing events on singleton genes (type I), and events not conserved (type II) or conserved (type III) within multi-gene families. While both type I and II events are subject to purifying selection, notably type III events are positively selected, and highly enriched in the components and functions of the nervous system. The tissue profiles are documented for three editing types, and their critical roles are further implicated by their shifting patterns during holometabolous development and in post-mating response. In conclusion, three A-to-I RNA editing types are found to have distinct evolutionary dynamics. It appears that nervous system functions are mainly tested to determine if an A-to-I editing is beneficial for an organism. The coding plasticity enabled by A-to-I editing creates a new class of binary variations, which is a superior alternative to maintain heterozygosity of expressed genes in a diploid mating system. PMID:27467689

  16. Operational Transformation In Co-Operative Editing

    Directory of Open Access Journals (Sweden)

    Mandeep Kaur

    2015-08-01

    Full Text Available Cooperative Editing Systems in real-time allows a virtual team to view and edit a shared document at the same time. The document shared must be synchronized in order to ensure consistency for all the participants. This paper describes the Operational Transformation the evolution of its techniques its various applications major issues and achievements. In addition this paper will present working of a platform where two users can edit a code programming file at the same time.

  17. Edit Distance between Unlabeled Ordered Trees

    OpenAIRE

    Micheli, Anne; Rossin, Dominique

    2005-01-01

    There exists a bijection between one stack sortable permutations --permutations which avoid the pattern $231$-- and planar trees. We define an edit distance between permutations which is coherent with the standard edit distance between trees. This one-to-one correspondence yields a polynomial algorithm for the subpermutation problem for $(231)$ avoiding permutations. Moreover, we obtain the generating function of the edit distance between ordered trees and some special ones. For the general c...

  18. Electronic editions – possibilities, limitations, perspectives

    OpenAIRE

    Mertens, Martin

    2010-01-01

    The thesis "Electronic Editions – Possibilities, Limitations, Perspectives" presents concepts and technologies for electronic editions in the field of German language and literature. The thesis focuses on describing forms of information contained by written texts that extend beyond the literal information content, and on the possibilities that exist for encoding this information in electronic editions and thus opening it up to electronic evaluation. It begins with a survey of existing electro...

  19. Potential pitfalls of CRISPR/Cas9-mediated genome editing.

    Science.gov (United States)

    Peng, Rongxue; Lin, Guigao; Li, Jinming

    2016-04-01

    Recently, a novel technique named the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)9 system has been rapidly developed. This genome editing tool has improved our ability tremendously with respect to exploring the pathogenesis of diseases and correcting disease mutations, as well as phenotypes. With a short guide RNA, Cas9 can be precisely directed to target sites, and functions as an endonuclease to efficiently produce breaks in DNA double strands. Over the past 30 years, CRISPR has evolved from the 'curious sequences of unknown biological function' into a promising genome editing tool. As a result of the incessant development in the CRISPR/Cas9 system, Cas9 co-expressed with custom guide RNAs has been successfully used in a variety of cells and organisms. This genome editing technology can also be applied to synthetic biology, functional genomic screening, transcriptional modulation and gene therapy. However, although CRISPR/Cas9 has a broad range of action in science, there are several aspects that affect its efficiency and specificity, including Cas9 activity, target site selection and short guide RNA design, delivery methods, off-target effects and the incidence of homology-directed repair. In the present review, we highlight the factors that affect the utilization of CRISPR/Cas9, as well as possible strategies for handling any problems. Addressing these issues will allow us to take better advantage of this technique. In addition, we also review the history and rapid development of the CRISPR/Cas system from the time of its initial discovery in 2012. PMID:26535798

  20. Fuel Cell Handbook, Fourth Edition

    Energy Technology Data Exchange (ETDEWEB)

    Stauffer, D.B; Hirschenhofer, J.H.; Klett, M.G.; Engleman, R.R.

    1998-11-01

    Robust progress has been made in fuel cell technology since the previous edition of the Fuel Cell Handbook was published in January 1994. This Handbook provides a foundation in fuel cells for persons wanting a better understanding of the technology, its benefits, and the systems issues that influence its application. Trends in technology are discussed, including next-generation concepts that promise ultra high efficiency and low cost, while providing exceptionally clean power plant systems. Section 1 summarizes fuel cell progress since the last edition and includes existing power plant nameplate data. Section 2 addresses the thermodynamics of fuel cells to provide an understanding of fuel cell operation at two levels (basic and advanced). Sections 3 through 6 describe the four major fuel cell types and their performance based on cell operating conditions. The section on polymer electrolyte membrane fuel cells has been added to reflect their emergence as a significant fuel cell technology. Phosphoric acid, molten carbonate, and solid oxide fuel cell technology description sections have been updated from the previous edition. New information indicates that manufacturers have stayed with proven cell designs, focusing instead on advancing the system surrounding the fuel cell to lower life cycle costs. Section 7, Fuel Cell Systems, has been significantly revised to characterize near-term and next-generation fuel cell power plant systems at a conceptual level of detail. Section 8 provides examples of practical fuel cell system calculations. A list of fuel cell URLs is included in the Appendix. A new index assists the reader in locating specific information quickly.

  1. Short RNA guides cleavage by eukaryotic RNase III.

    Directory of Open Access Journals (Sweden)

    Bruno Lamontagne

    Full Text Available In eukaryotes, short RNAs guide a variety of enzymatic activities that range from RNA editing to translation repression. It is hypothesized that pre-existing proteins evolved to bind and use guide RNA during evolution. However, the capacity of modern proteins to adopt new RNA guides has never been demonstrated. Here we show that Rnt1p, the yeast orthologue of the bacterial dsRNA-specific RNase III, can bind short RNA transcripts and use them as guides for sequence-specific cleavage. Target cleavage occurred at a constant distance from the Rnt1p binding site, leaving the guide RNA intact for subsequent cleavage. Our results indicate that RNase III may trigger sequence-specific RNA degradation independent of the RNAi machinery, and they open the road for a new generation of precise RNA silencing tools that do not trigger a dsRNA-mediated immune response.

  2. Biocommunication and natural genome editing

    Institute of Scientific and Technical Information of China (English)

    Guenther; Witzany

    2010-01-01

    The biocommunicative approach investigates communication processes within and among cells,tissues,organs and organisms as sign-mediated interactions,and nucleotide sequences as code,i.e.language-like text,which follows in parallel three kinds of rules:combinatorial (syntactic),context-sensitive(pragmatic),and contentspecific(semantic).Natural genome editing from a bio-communicative perspective is competent agent-driven generation and integration of meaningful nucleotide sequences into pre-existing genomic content arrangements and the ability to(re-)combine and(re-)regulate them according to context-dependent(i.e.adaptational) purposes of the host organism.

  3. APP Snrinn Edition Kicks Off

    Institute of Scientific and Technical Information of China (English)

    Tang Rong

    2012-01-01

    When European textile insiders still take delight in talking about the last apparel sourcing show in September 2011, the debut of APP Spring edition bring people back to the Le Bourget Expo Center, with entire passion. The 7th APP Show, sponsored by China National Textile and Apparel Council (CNTAC), organized by the Sub-Council of Textile Industry, China Council for the Promotion of International Trade (CCPIT TEX), China National Garment Association and Messe Frankfurt French Subsidiary, was on display from Feb 13th to 16th 2012,

  4. 6. Electronic Editions for Everyone

    OpenAIRE

    Robinson, Peter

    2013-01-01

    1. Books Defy the Digital Revolution In January 2004, I gave a lecture on electronic scholarly editing at the University of Virginia. At the beginning of the lecture I asked the audience, of around 60 people, three questions. The first question was: who among them had bought a movie on DVD in the last year; who had bought a piece of music on CD-ROM or by download in the last year; who had taken digital photographs? Almost everyone in the audience had done all three. The second question: who i...

  5. GPU Computing Gems Emerald Edition

    CERN Document Server

    Hwu, Wen-mei W

    2011-01-01

    ".the perfect companion to Programming Massively Parallel Processors by Hwu & Kirk." -Nicolas Pinto, Research Scientist at Harvard & MIT, NVIDIA Fellow 2009-2010 Graphics processing units (GPUs) can do much more than render graphics. Scientists and researchers increasingly look to GPUs to improve the efficiency and performance of computationally-intensive experiments across a range of disciplines. GPU Computing Gems: Emerald Edition brings their techniques to you, showcasing GPU-based solutions including: Black hole simulations with CUDA GPU-accelerated computation and interactive display of

  6. Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing.

    Science.gov (United States)

    Kelley, Melissa L; Strezoska, Žaklina; He, Kaizhang; Vermeulen, Annaleen; Smith, Anja van Brabant

    2016-09-10

    The CRISPR-Cas9 system has become the most popular and efficient method for genome engineering in mammalian cells. The Streptococcus pyogenes Cas9 nuclease can function with two types of guide RNAs: the native dual crRNA and tracrRNA (crRNA:tracrRNA) or a chimeric single guide RNA (sgRNA). Although sgRNAs expressed from a DNA vector are predominant in the literature, guide RNAs can be rapidly generated by chemical synthesis and provide equivalent functionality in gene editing experiments. This review highlights the attributes and advantages of chemically synthesized guide RNAs including the incorporation of chemical modifications to enhance gene editing efficiencies in certain applications. The use of synthetic guide RNAs is also uniquely suited to genome-scale high throughput arrayed screening, particularly when using complex phenotypic assays for functional genomics studies. Finally, the use of synthetic guide RNAs along with DNA-free sources of Cas9 (mRNA or protein) allows for transient CRISPR-Cas9 presence in the cell, thereby resulting in a decreased probability of off-target events. PMID:27374403

  7. Induction of inosine containing mRNA during the inflammatory stress in C57BL/6 mouse

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A-to-I RNA editing is a recently discovered process of post-transcription modification of pre-mRNA by adenosine deamination that results in the production of proteins not inherent in the genome. The present study aimed to identify a role for A-to-I RNA editing in the acute inflammation. It was found that A-to-I edtiting activities of RNA editases, ADARs, were upregulated in lung, spleen, thymus and lymph node of mouse during endotoxin stimulation. Importantly, the number of inosine in poly(A) RNA isolated from mouse lung and spleen was significantly increased in correlating with the induction of ADARs' editing activity. The in vitro synthesized RNA which did not contain inosine was edited by thymus extracts and the generation of inosine was greatly increased after editing in LPS treated thymus extract. Take together, these data suggest that A-to-I RNA editing by ADARs may play an important role in pathogenesis of inflammation. The existence of high level of I-mRNA also suggests that more protein isoforms might be generated from a single gene via adenosine deamination by ADARs during inflammatory stress.

  8. Sex Differences in Cognitive Abilities. Fourth Edition

    Science.gov (United States)

    Halpern, Diane F.

    2011-01-01

    The fourth edition of "Sex Differences in Cognitive Abilities" critically examines the breadth of research on this complex and controversial topic, with the principal aim of helping the reader to understand where sex differences are found--and where they are not. Since the publication of the third edition, there have been many exciting and…

  9. Edit Distance to Monotonicity in Sliding Windows

    DEFF Research Database (Denmark)

    Chan, Ho-Leung; Lam, Tak-Wah; Lee, Lap Kei;

    2011-01-01

    Given a stream of items each associated with a numerical value, its edit distance to monotonicity is the minimum number of items to remove so that the remaining items are non-decreasing with respect to the numerical value. The space complexity of estimating the edit distance to monotonicity of a ...

  10. Books in Action: The Armed Services Editions.

    Science.gov (United States)

    Cole, John Y., Ed.

    In an effort to reach a wide audience, the Center for the Book in the Library of Congress presents this book in honor of the 40th anniversary celebration of the Armed Services Editions (ASE), the paperback books distributed during World War II. The titles of the essays and their authors are as follows: "The Armed Services Editions: An…

  11. Applications of genome editing in insects

    Science.gov (United States)

    Insect genome editing was first reported 1991 in Drosophila melanogaster but the technology used was not portable to other species. Not until the recent development of facile, engineered DNA endonuclease systems has gene editing become widely available to insect scientists. Most applications in inse...

  12. Profs, Professionals Agree about Students' Editing Skills.

    Science.gov (United States)

    Fee, Frank; Russial, John; Auman Ann

    2003-01-01

    Considers where journalism educators should focus when they design editing curriculum. Examines what professors say is important for students to know about editing. Compares what professors at accredited programs say about necessary skills with what professional copy editors say is important. Concludes that professors and professionals are largely…

  13. Accounting for Independent Schools. Second Edition.

    Science.gov (United States)

    National Association of Independent Schools, Boston, MA.

    This is a thoroughly revised edition of the 1969 publication, "Accounting for Independent Schools," a guide that attempted to codify basic accounting principles and practices for specific application to independent schools. The focus of the second edition is more on refining practices than on initiating them, and more on extending the managerial…

  14. The Aldine Edition of Aristotle's De Sensu

    DEFF Research Database (Denmark)

    Bloch, David Kristian

    2006-01-01

    This small article examines the quality of and the textual foundation for the først printed edition ever of Aristotle's De Sensu et Sensibilibus, that is, Aldus Manutius' (1497).......This small article examines the quality of and the textual foundation for the først printed edition ever of Aristotle's De Sensu et Sensibilibus, that is, Aldus Manutius' (1497)....

  15. Editing Technical Proposals (The Friendly Editor).

    Science.gov (United States)

    Bush, Don

    1994-01-01

    Makes suggestions for editing technical proposals. Discusses the marketeers, the hierarchy of hype, how to save days, managing story boards, expediting a laborious process, teaching engineers to write, writing incrementally, the art group, and the editing task. Argues that the best proposals come from starting to write early. (SR)

  16. Efficient Communication Protocols for Deciding Edit Distance

    DEFF Research Database (Denmark)

    Jowhari, Hossein

    2012-01-01

    Parsing method of [5,6] in combination with the Karp-Rabin fingerprints. In addition to yielding non-trivial bounds for the edit distance problem, this paper suggest a new conceptual framework and raises new questions regarding the embeddability of edit distance into the Hamming cube which might...

  17. Lentivirus pre-packed with Cas9 protein for safer gene editing.

    Science.gov (United States)

    Choi, J G; Dang, Y; Abraham, S; Ma, H; Zhang, J; Guo, H; Cai, Y; Mikkelsen, J G; Wu, H; Shankar, P; Manjunath, N

    2016-07-01

    The CRISPR/Cas9 system provides an easy way to edit specific site/s in the genome and thus offers tremendous opportunity for human gene therapy for a wide range of diseases. However, one major concern is off-target effects, particularly with long-term expression of Cas9 nuclease when traditional expression methods such as via plasmid/viral vectors are used. To overcome this limitation, we pre-packaged Cas9 protein (Cas9P LV) in lentiviral particles for transient exposure and showed its effectiveness for gene disruption in cells, including primary T cells expressing specific single guide RNAs (sgRNAs). We then constructed an 'all in one virus' to express sgRNAs in association with pre-packaged Cas9 protein (sgRNA/Cas9P LV). We successfully edited CCR5 in TZM-bl cells by this approach. Using an sgRNA-targeting HIV long terminal repeat, we also were able to disrupt HIV provirus in the J-LAT model of viral latency. Moreover, we also found that pre-packaging Cas9 protein in LV particle reduced off-target editing of chromosome 4:-29134166 locus by CCR5 sgRNA, compared with continued expression from the vector. These results show that sgRNA/Cas9P LV can be used as a safer approach for human gene therapy applications. PMID:27052803

  18. CRISPR/Cas9-Mediated Genome Editing in Soybean Hairy Roots.

    Directory of Open Access Journals (Sweden)

    Yupeng Cai

    Full Text Available As a new technology for gene editing, the CRISPR (clustered regularly interspaced short palindromic repeat/Cas (CRISPR-associated system has been rapidly and widely used for genome engineering in various organisms. In the present study, we successfully applied type II CRISPR/Cas9 system to generate and estimate genome editing in the desired target genes in soybean (Glycine max (L. Merrill.. The single-guide RNA (sgRNA and Cas9 cassettes were assembled on one vector to improve transformation efficiency, and we designed a sgRNA that targeted a transgene (bar and six sgRNAs that targeted different sites of two endogenous soybean genes (GmFEI2 and GmSHR. The targeted DNA mutations were detected in soybean hairy roots. The results demonstrated that this customized CRISPR/Cas9 system shared the same efficiency for both endogenous and exogenous genes in soybean hairy roots. We also performed experiments to detect the potential of CRISPR/Cas9 system to simultaneously edit two endogenous soybean genes using only one customized sgRNA. Overall, generating and detecting the CRISPR/Cas9-mediated genome modifications in target genes of soybean hairy roots could rapidly assess the efficiency of each target loci. The target sites with higher efficiencies can be used for regular soybean transformation. Furthermore, this method provides a powerful tool for root-specific functional genomics studies in soybean.

  19. Adaptive sampling for mesh spectrum editing

    Institute of Scientific and Technical Information of China (English)

    ZHAO Xiang-jun; ZHANG Hong-xin; BAO Hu-jun

    2006-01-01

    A mesh editing framework is presented in this paper, which integrates Free-Form Deformation (FFD) and geometry signal processing. By using simplified model from original mesh, the editing task can be accomplished with a few operations. We take the deformation of the proxy and the position coordinates of the mesh models as geometry signal. Wavelet analysis is employed to separate local detail information gracefully. The crucial innovation of this paper is a new adaptive regular sampling approach for our signal analysis based editing framework. In our approach, an original mesh is resampled and then refined iteratively which reflects optimization of our proposed spectrum preserving energy. As an extension of our spectrum editing scheme,the editing principle is applied to geometry details transferring, which brings satisfying results.

  20. One-step high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces.

    Science.gov (United States)

    Huang, He; Zheng, Guosong; Jiang, Weihong; Hu, Haifeng; Lu, Yinhua

    2015-04-01

    The RNA-guided DNA editing technology CRISPRs (clustered regularly interspaced short palindromic repeats)/Cas9 had been used to introduce double-stranded breaks into genomes and to direct subsequent site-specific insertions/deletions or the replacement of genetic material in bacteria, such as Escherichia coli, Streptococcus pneumonia, and Lactobacillus reuteri. In this study, we established a high-efficiency CRISPR/Cas9 genome editing plasmid pKCcas9dO for use in Streptomyces genetic manipulation, which comprises a target-specific guide RNA, a codon-optimized cas9, and two homology-directed repair templates. By delivering pKCcas9dO series editing plasmids into the model strain Streptomyces coelicolor M145, through one-step intergeneric transfer, we achieved the genome editing at different levels with high efficiencies of 60%-100%, including single gene deletion, such as actII-orf4, redD, and glnR, and single large-size gene cluster deletion, such as the antibiotic biosynthetic clusters of actinorhodin (ACT) (21.3 kb), undecylprodigiosin (RED) (31.6 kb), and Ca(2+)-dependent antibiotic (82.8 kb). Furthermore, we also realized simultaneous deletions of actII-orf4 and redD, and of the ACT and RED biosynthetic gene clusters with high efficiencies of 54% and 45%, respectively. Finally, we applied this system to introduce nucleotide point mutations into the rpsL gene, which conferred the mutants with resistance to streptomycin. Notably, using this system, the time required for one round of genome modification is reduced by one-third or one-half of those for conventional methods. These results clearly indicate that the established CRISPR/Cas9 genome editing system substantially improves the genome editing efficiency compared with the currently existing methods in Streptomyces, and it has promise for application to genome modification in other Actinomyces species.

  1. Evaluation of microRNA alignment techniques.

    Science.gov (United States)

    Ziemann, Mark; Kaspi, Antony; El-Osta, Assam

    2016-08-01

    Genomic alignment of small RNA (smRNA) sequences such as microRNAs poses considerable challenges due to their short length (∼21 nucleotides [nt]) as well as the large size and complexity of plant and animal genomes. While several tools have been developed for high-throughput mapping of longer mRNA-seq reads (>30 nt), there are few that are specifically designed for mapping of smRNA reads including microRNAs. The accuracy of these mappers has not been systematically determined in the case of smRNA-seq. In addition, it is unknown whether these aligners accurately map smRNA reads containing sequence errors and polymorphisms. By using simulated read sets, we determine the alignment sensitivity and accuracy of 16 short-read mappers and quantify their robustness to mismatches, indels, and nontemplated nucleotide additions. These were explored in the context of a plant genome (Oryza sativa, ∼500 Mbp) and a mammalian genome (Homo sapiens, ∼3.1 Gbp). Analysis of simulated and real smRNA-seq data demonstrates that mapper selection impacts differential expression results and interpretation. These results will inform on best practice for smRNA mapping and enable more accurate smRNA detection and quantification of expression and RNA editing. PMID:27284164

  2. Fuel Cell Handbook, Fifth Edition

    Energy Technology Data Exchange (ETDEWEB)

    Energy and Environmental Solutions

    2000-10-31

    Progress continues in fuel cell technology since the previous edition of the Fuel Cell Handbook was published in November 1998. Uppermost, polymer electrolyte fuel cells, molten carbonate fuel cells, and solid oxide fuel cells have been demonstrated at commercial size in power plants. The previously demonstrated phosphoric acid fuel cells have entered the marketplace with more than 220 power plants delivered. Highlighting this commercial entry, the phosphoric acid power plant fleet has demonstrated 95+% availability and several units have passed 40,000 hours of operation. One unit has operated over 49,000 hours. Early expectations of very low emissions and relatively high efficiencies have been met in power plants with each type of fuel cell. Fuel flexibility has been demonstrated using natural gas, propane, landfill gas, anaerobic digester gas, military logistic fuels, and coal gas, greatly expanding market opportunities. Transportation markets worldwide have shown remarkable interest in fuel cells; nearly every major vehicle manufacturer in the U.S., Europe, and the Far East is supporting development. This Handbook provides a foundation in fuel cells for persons wanting a better understanding of the technology, its benefits, and the systems issues that influence its application. Trends in technology are discussed, including next-generation concepts that promise ultrahigh efficiency and low cost, while providing exceptionally clean power plant systems. Section 1 summarizes fuel cell progress since the last edition and includes existing power plant nameplate data. Section 2 addresses the thermodynamics of fuel cells to provide an understanding of fuel cell operation at two levels (basic and advanced). Sections 3 through 8 describe the six major fuel cell types and their performance based on cell operating conditions. Alkaline and intermediate solid state fuel cells were added to this edition of the Handbook. New information indicates that manufacturers have stayed

  3. Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing.

    Science.gov (United States)

    Sun, Wujin; Ji, Wenyan; Hall, Jordan M; Hu, Quanyin; Wang, Chao; Beisel, Chase L; Gu, Zhen

    2015-10-01

    CRISPR-Cas9 represents a promising platform for genome editing, yet means for its safe and efficient delivery remain to be fully realized. A novel vehicle that simultaneously delivers the Cas9 protein and single guide RNA (sgRNA) is based on DNA nanoclews, yarn-like DNA nanoparticles that are synthesized by rolling circle amplification. The biologically inspired vehicles were efficiently loaded with Cas9/sgRNA complexes and delivered the complexes to the nuclei of human cells, thus enabling targeted gene disruption while maintaining cell viability. Editing was most efficient when the DNA nanoclew sequence and the sgRNA guide sequence were partially complementary, offering a design rule for enhancing delivery. Overall, this strategy provides a versatile method that could be adapted for delivering other DNA-binding proteins or functional nucleic acids.

  4. RNA oxidation

    DEFF Research Database (Denmark)

    Kjaer, L. K.; Cejvanovic, V.; Henriken, T.;

    2015-01-01

    RNA modification has attracted increasing interest as it is realized that epitranscriptomics is important in disease development. In type 2 diabetes we have suggested that high urinary excretion of 8-oxo-2'-Guanosine (8oxoGuo), as a measure of global RNA oxidation, is associated with poor survival.......9 significant hazard ratio for death compared with the quartile with the lowest 8oxoGuo excretion when adjusted for age, sex, BMI, smoker status, s-HbA1c, urine protein excretion and s-cholesterol. We conclude that it is now established that RNA oxidation is an independent risk factor for death in type 2...... diabetes. In agreement with our previous finding, DNA oxidation did not show any prognostic value. RNA oxidation represents oxidative stress intracellularly, presumably predominantly in the cytosol. The mechanism of RNA oxidation is not clear, but hypothesized to result from mitochondrial dysfunction...

  5. Design and implementation of ALU and shifter in X-DSP%X-DSP ALU与移位部件的设计与实现

    Institute of Scientific and Technical Information of China (English)

    彭元喜; 邹佳骏

    2010-01-01

    针对DSP CPU的算术运算逻辑单元(ALU)与移位部件在性能、功耗与面积上面临的挑战,研究了X型DSP的CPU体系结构,在对X型DSP ALU部件和移位器部件相关指令进行归类分析的基础上,设计实现了ALU部件和移位器部件.采用Design Compiler综合工具,基于SMIC公司0.13μm CMOS工艺库对ALU移位部件进行了逻辑综合,电路功耗共为4.2821mW,电路面积为71042.9804μm2,工作频率达到250MHz.

  6. 32位同时多线程微处理器的ALU设计%Design of ALU of 32-bit simultaneous multithreading processor

    Institute of Scientific and Technical Information of China (English)

    刘权胜; 杨洪斌; 吴悦

    2008-01-01

    针对传统ALU存在较大硬件资源浪费的缺点,提出了一种指令执行并行度宽,资源利用率高的同时多线程ALU.同时多线程ALU由7个并行的部件组成.每个部件高效的执行两个线程的指令.这种由7个部分组成的分布式ALU提高了指令并行执行的宽度,大大降低了水平浪费和垂直浪费.对微处理器ALU进行功能验证与仿真,并用综合工具完成逻辑综合.

  7. AAO Observer - August 2011 Edition

    CERN Document Server

    Brough, Sarah

    2011-01-01

    This edition of the Australian Astronomical Observatory Observer contains articles on the commissioning of the new SAMI instrument giving the first hexabundle galaxy spectra; galaxy parameter variations across and through the 6dFGS Fundamental Plane; an introduction to the new Dragonfly stellar interferometer; an update on the RAdial VElocity (RAVE) survey at half a million spectra; the Magellanic Quasars Survey; the Integrated Photonic Spectrograph's first look at the heart of the Scorpion; using AAOMega to measure the age of the young open cluster IC2602; making MANIFEST fibres for the Giant Magellan Telescope and a Voyage through Filaments of Galaxies. The Observer also contains thoughts on diversity in the astronomy community and reports on the recent Supernovae and their Host Galaxies conference and the 2011 Science Meets Parliament. In addition there are the usual features of the AUSGO Corner, Epping News and Letter from Coona.

  8. Exploiting CRISPR-Cas immune systems for genome editing in bacteria.

    Science.gov (United States)

    Barrangou, Rodolphe; van Pijkeren, Jan-Peter

    2016-02-01

    The CRISPR-Cas immune system is a DNA-encoded, RNA-mediated, DNA-targeting defense mechanism, which provides sequence-specific targeting of DNA. This molecular machinery can be engineered into the sgRNA:Cas9 technology, for programmable cleavage of DNA. Following the genesis of double-stranded DNA breaks, the DNA repair machinery generates mutations at the cleavage site using various pathways. This technology has revolutionized eukaryotic genome editing, and we are at the cusp of full exploitation in bacteria. Here, we discuss the potential of CRISPR-based technologies for use in bacteria, and highlight the application of single stranded DNA recombineering combined with CRISPR-Cas selection to edit the genome of a probiotic organism. We envision that CRISPR-Cas technologies will play a key role in the development of next-generation industrial bacteria. PMID:26629846

  9. Targeted plant genome editing via the CRISPR/Cas9 technology.

    Science.gov (United States)

    Li, Jian-Feng; Zhang, Dandan; Sheen, Jen

    2015-01-01

    Targeted modification of plant genome is key for elucidating and manipulating gene functions in basic and applied plant research. The CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated protein (Cas) technology is emerging as a powerful genome editing tool in diverse organisms. This technology utilizes an easily reprogrammable guide RNA (gRNA) to guide Streptococcus pyogenes Cas9 endonuclease to generate a DNA double-strand break (DSB) within an intended genomic sequence and subsequently stimulate chromosomal mutagenesis or homologous recombination near the DSB site through cellular DNA repair machineries. In this chapter, we describe the detailed procedure to design, construct, and evaluate dual gRNAs for plant codon-optimized Cas9 (pcoCas9)-mediated genome editing using Arabidopsis thaliana and Nicotiana benthamiana protoplasts as model cellular systems. We also discuss strategies to apply the CRISPR/Cas9 system to generating targeted genome modifications in whole plants.

  10. Targeted viral-mediated plant genome editing using crispr/cas9

    KAUST Repository

    Mahfouz, Magdy M.

    2015-12-17

    The present disclosure provides a viral-mediated genome-editing platform that facilitates multiplexing, obviates stable transformation, and is applicable across plant species. The RNA2 genome of the tobacco rattle virus (TRV) was engineered to carry and systemically deliver a guide RNA molecules into plants overexpressing Cas9 endonuclease. High genomic modification frequencies were observed in inoculated as well as systemic leaves including the plant growing points. This system facilitates multiplexing and can lead to germinal transmission of the genomic modifications in the progeny, thereby obviating the requirements of repeated transformations and tissue culture. The editing platform of the disclosure is useful in plant genome engineering and applicable across plant species amenable to viral infections for agricultural biotechnology applications.

  11. NRP 7th Edition: Are You Prepared?

    Science.gov (United States)

    Zaichkin, Jeanette; Mccarney, Linda; Weiner, Gary

    2016-01-01

    The seventh edition of the American Academy of Pediatrics/American Heart Association Neonatal Resuscitation Program (NRP) materials must be in use by January 1, 2017. As in previous editions, changes in resuscitation science are based on an international review and consensus of current resuscitation science. The seventh edition NRP materials also include enhancements to training materials aimed at improving the quality of NRP instruction and providing the opportunity for ongoing education. A standardized approach to instructor training, an online Instructor Toolkit, eSim cases, and a new learning management system are among the new resources. PMID:27461196

  12. Graph edit distance from spectral seriation

    OpenAIRE

    Robles-Kelly, A; Hancock, E.R.

    2005-01-01

    This paper is concerned with computing graph edit distance. One of the criticisms that can be leveled at existing methods for computing graph edit distance is that they lack some of the formality and rigor of the computation of string edit distance. Hence, our aim is to convert graphs to string sequences so that string matching techniques can be used. To do this, we use a graph spectral seriation method to convert the adjacency matrix into a string or sequence order. We show how the serial or...

  13. On the computation of edit distance functions

    OpenAIRE

    Martin, Ryan R.

    2010-01-01

    The edit distance between two graphs on the same labeled vertex set is the size of the symmetric difference of the edge sets. The edit distance function of the hereditary property, $\\mathcal{H}$, is a function of $p\\in[0,1]$ and is the limit of the maximum normalized distance between a graph of density $p$ and $\\mathcal{H}$. This paper uses the symmetrization method of Sidorenko in order to compute the edit distance function of various hereditary properties. For any graph $H$, ${\\rm Forb}(H)$...

  14. Efficient CRISPR-Cas9–mediated genome editing in Plasmodium falciparum

    OpenAIRE

    Wagner, Jeffrey C; Platt, Randall J.; Goldfless, Stephen J.; ZHANG Feng; Niles, Jacquin C.

    2014-01-01

    Malaria is a major cause of global morbidity and mortality, and new strategies for treating and preventing this disease are needed. Here we show that the Streptococcus pyogenes Cas9 DNA endonuclease and single guide RNAs (sgRNAs) produced using T7 RNA polymerase (T7 RNAP) efficiently edit the Plasmodium falciparum genome. Targeting the genes encoding native knob-associated histidine-rich protein (kahrp) and erythrocyte binding antigen 175 (eba-175), we achieved high (≥50–100%) gene disruption...

  15. A redesigned CRISPR/Cas9 system for marker-free genome editing in Plasmodium falciparum

    OpenAIRE

    Lu, Junnan; TONG, Ying; Pan, Jiaqiang; Yang, Yijun; Liu, Quan; Tan, Xuefang; Zhao, Siting; Qin, Li; Chen, Xiaoping

    2016-01-01

    Background A highly efficient CRISPR/Cas9-based marker-free genome editing system has been established in Plasmodium falciparum (Pf). However, with the current methods, two drug-selectable markers are needed for episome retention, which may present hurdles for consecutive genome manipulations due to the limited number of available selectable markers. The loading capacity of donor DNA is also unsatisfactory due to the large size of the Cas9 nuclease and sgRNA co-expression system, which limits...

  16. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins.

    Science.gov (United States)

    Woo, Je Wook; Kim, Jungeun; Kwon, Soon Il; Corvalán, Claudia; Cho, Seung Woo; Kim, Hyeran; Kim, Sang-Gyu; Kim, Sang-Tae; Choe, Sunghwa; Kim, Jin-Soo

    2015-11-01

    Editing plant genomes without introducing foreign DNA into cells may alleviate regulatory concerns related to genetically modified plants. We transfected preassembled complexes of purified Cas9 protein and guide RNA into plant protoplasts of Arabidopsis thaliana, tobacco, lettuce and rice and achieved targeted mutagenesis in regenerated plants at frequencies of up to 46%. The targeted sites contained germline-transmissible small insertions or deletions that are indistinguishable from naturally occurring genetic variation.

  17. Creation of a split-cas9 system for effective plant genome editing

    OpenAIRE

    Costa Carrió, Pedro

    2015-01-01

    Precise and efficient genome-targeting technologies are needed to enable both systematic genome editing and interrogation of gene function. Although technologies such as designer zinc fingers (ZFs), transcription activator-like effectors (TALENs), and homing meganucleases have begun to enable targeted genome modifications, there is a need for new technologies that are scalable, affordable, and easy to engineer. Easy programmability of the RNA-guided DNA-endonuclease Cas9 of the type II CRISPR...

  18. The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells

    OpenAIRE

    Lee, Ciaran M; Cradick, Thomas J.; Bao, Gang

    2016-01-01

    The clustered regularly-interspaced short palindromic repeats (CRISPR)—CRISPR-associated (Cas) system from Streptococcus pyogenes (Spy) has been successfully adapted for RNA-guided genome editing in a wide range of organisms. However, numerous reports have indicated that Spy CRISPR-Cas9 systems may have significant off-target cleavage of genomic DNA sequences differing from the intended on-target site. Here, we report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that...

  19. Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination

    OpenAIRE

    Chen, C.; Fenk, L. A.; Bono, M.

    2013-01-01

    Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR–Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR–CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand bre...

  20. Endonuclease V cleaves at inosines in RNA.

    Science.gov (United States)

    Vik, Erik Sebastian; Nawaz, Meh Sameen; Strøm Andersen, Pernille; Fladeby, Cathrine; Bjørås, Magnar; Dalhus, Bjørn; Alseth, Ingrun

    2013-01-01

    Endonuclease V orthologues are highly conserved proteins found in all kingdoms of life. While the prokaryotic enzymes are DNA repair proteins for removal of deaminated adenosine (inosine) from the genome, no clear role for the eukaryotic counterparts has hitherto been described. Here we report that human endonuclease V (ENDOV) and also Escherichia coli endonuclease V are highly active ribonucleases specific for inosine in RNA. Inosines are normal residues in certain RNAs introduced by specific deaminases. Adenosine-to-inosine editing is essential for proper function of these transcripts and defects are linked to various human disease. Here we show that human ENDOV cleaves an RNA substrate containing inosine in a position corresponding to a biologically important site for deamination in the Gabra-3 transcript of the GABA(A) neurotransmitter. Further, human ENDOV specifically incises transfer RNAs with inosine in the wobble position. This previously unknown RNA incision activity may suggest a role for endonuclease V in normal RNA metabolism. PMID:23912683

  1. Transfer RNA-derived small RNAs in the cancer transcriptome

    OpenAIRE

    Green, Darrell; Fraser, William; Dalmay, Tamas

    2016-01-01

    The cellular lifetime includes stages such as differentiation, proliferation, division, senescence and apoptosis.These stages are driven by a strictly ordered process of transcription dynamics. Molecular disruption to RNA polymerase assembly, chromatin remodelling and transcription factor binding through to RNA editing, splicing, post-transcriptional regulation and ribosome scanning can result in significant costs arising from genome instability. Cancer development is one example of when such...

  2. CRISPR-Cas systems for editing, regulating and targeting genomes.

    Science.gov (United States)

    Sander, Jeffry D; Joung, J Keith

    2014-04-01

    Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.

  3. Negative Feedback Regulation of HIV-1 by Gene Editing Strategy

    Science.gov (United States)

    Kaminski, Rafal; Chen, Yilan; Salkind, Julian; Bella, Ramona; Young, Won-bin; Ferrante, Pasquale; Karn, Jonathan; Malcolm, Thomas; Hu, Wenhui; Khalili, Kamel

    2016-01-01

    The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells. PMID:27528385

  4. Negative Feedback Regulation of HIV-1 by Gene Editing Strategy.

    Science.gov (United States)

    Kaminski, Rafal; Chen, Yilan; Salkind, Julian; Bella, Ramona; Young, Won-Bin; Ferrante, Pasquale; Karn, Jonathan; Malcolm, Thomas; Hu, Wenhui; Khalili, Kamel

    2016-01-01

    The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells. PMID:27528385

  5. Getting Started with Isabelle/jEdit

    OpenAIRE

    Sternagel, Christian

    2012-01-01

    We give a beginner-oriented introduction to Isabelle/jEdit, providing motivation for using it as well as pointing at some differences to the traditional Proof General interface and current limitations.

  6. Specification Editing and Discovery Assistant Project

    Data.gov (United States)

    National Aeronautics and Space Administration — The project will prototype a specification editing and discovery tool (SPEEDY) for C/C++ that will assist software developers with modular formal verification tasks...

  7. Prescott’s Microbiology, Eighth Edition

    OpenAIRE

    Joanne J. Dobbins

    2010-01-01

    Review of: Prescott’s Microbiology, Eighth Edition. Joanne M. Willey, Linda M. Sherwood, and Christopher J. Woolverton. 2011. McGraw-Hill Higher Education, NewYork, NY. 1070 pages. ISBN- 978-0-07-337526-7.

  8. KWIC Index of nuclear codes (1975 edition)

    International Nuclear Information System (INIS)

    It is a KWIC Index for 254 nuclear codes in the Nuclear Code Abstracts (1975 edition). The classification of nuclear codes and the form of index are the same as those in the Computer Programme Library at Ispra, Italy. (auth.)

  9. LabVIEW 8 student edition

    CERN Document Server

    Bishop, Robert H

    2007-01-01

    For courses in Measurement and Instrumentation, Electrical Engineering lab, and Physics and Chemistry lab. This revised printing has been updated to include new LabVIEW 8.2 Student Edition. National Instruments' LabVIEW is the defacto industry standard for test, measurement, and automation software solutions. With the Student Edition of LabVIEW, students can design graphical programming solutions to their classroom problems and laboratory experiments with software that delivers the graphical programming capabilites of the LabVIEW professional version. . The Student Edition is also compatible with all National Instruments data acquisition and instrument control hardware. Note: The LabVIEW Student Edition is available to students, faculty, and staff for personal educational use only. It is not intended for research, institutional, or commercial use. For more information about these licensing options, please visit the National Instruments website at (http:www.ni.com/academic/)

  10. CRISPR/Cas9 for plant genome editing: accomplishments, problems and prospects.

    Science.gov (United States)

    Paul, Joseph W; Qi, Yiping

    2016-07-01

    The increasing burden of the world population on agriculture requires the development of more robust crops. Dissecting the basic biology that underlies plant development and stress responses will inform the design of better crops. One powerful tool for studying plants at the molecular level is the RNA-programmed genome editing system composed of a clustered regularly interspaced short palindromic repeats (CRISPR)-encoded guide RNA and the nuclease Cas9. Here, some of the recent advances in CRISPR/Cas9 technology that have profound implications for improving the study of plant biology are described. These tools are also paving the way towards new horizons for biotechnologies and crop development. PMID:27114166

  11. Inosine-mediated modulation of RNA sensing by Toll-like receptor 7 (TLR7) and TLR8.

    Science.gov (United States)

    Sarvestani, Soroush T; Tate, Michelle D; Moffat, Jessica M; Jacobi, Ashley M; Behlke, Mark A; Miller, Alistair R; Beckham, Simone A; McCoy, Claire E; Chen, Weisan; Mintern, Justine D; O'Keeffe, Meredith; John, Matthias; Williams, Bryan R G; Gantier, Michael P

    2014-01-01

    RNA-specific adenosine deaminase (ADAR)-mediated adenosine-to-inosine (A-to-I) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of nonself RNA by innate immune sensors and accordingly investigated the impact of inosine-modified RNA on Toll-like receptor 7 and 8 (TLR7/8) sensing. Inosine incorporation into synthetic single-stranded RNA (ssRNA) potentiated tumor necrosis factor alpha (TNF-α) or alpha interferon (IFN-α) production in human peripheral blood mononuclear cells (PBMCs) in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs and was not seen with inosine-containing short double-stranded RNAs or with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated IFN-α production in human PBMCs through TLR7 recruitment, as established through the use of a TLR7 antagonist and Tlr7-deficient cells. There was a correlation between hyperediting of influenza A viral ssRNA and its ability to stimulate TNF-α, independent of 5'-triphosphate residues, and involving Adar-1. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus, we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate TLR7/8 sensing of phagocytosed viral RNA. PMID:24227841

  12. Good edit similarity learning by loss minimization

    OpenAIRE

    Bellet, Aurélien; Habrard, Amaury; Sebban, Marc

    2012-01-01

    Similarity functions are a fundamental component of many learning algorithms. When dealing with string or tree-structured data, edit distancebased measures are widely used, and there exists a few methods for learning them from data. However, these methods offer no theoretical guarantee as to the generalization ability and discriminative power of the learned similarities. In this paper, we propose a loss minimization-based edit similarity learning approach, called GESL. It is driven by the not...

  13. Nuclear electronics laboratory manual 1989 edition

    International Nuclear Information System (INIS)

    This manual is a joint product of several electronics experts who have been associated with IAEA activity in this field for many years. It is based on the experience of conducting twenty-three training courses on nuclear electronics. Compared with the first edition, published 1984, this edition contains many new experiments, mainly on the advanced technical level. The total number of experiments and special projects is 58. Tabs and figs

  14. Applications of genome editing in insects.

    Science.gov (United States)

    Reid, William; O'Brochta, David A

    2016-02-01

    Insect genome editing was first reported 1991 in Drosophila melanogaster but the technology used was not portable to other species. Not until the recent development of facile, engineered DNA endonuclease systems has gene editing become widely available to insect scientists. Most applications in insects to date have been technical in nature but this is rapidly changing. Functional genomics and genetics-based insect control efforts will be major beneficiaries of the application of contemporary gene editing technologies. Engineered endonucleases like Cas9 make it possible to create powerful and effective gene drive systems that could be used to reduce or even eradicate specific insect populations. 'Best practices' for using Cas9-based editing are beginning to emerge making it easier and more effective to design and use but gene editing technologies still require traditional means of delivery in order to introduce them into somatic and germ cells of insects-microinjection of developing embryos. This constrains the use of these technologies by insect scientists. Insects created using editing technologies challenge existing governmental regulatory structures designed to manage genetically modified organisms. PMID:27436552

  15. [Textual research on the edition of WU Kun's books].

    Science.gov (United States)

    Wang, Xu-Guang; Lu, Xiang

    2013-03-01

    WU Kun is a famous physician of the Xin'an School. He wrote a 6-volume Yi fang kao (Textual Research on Recipes) in 1584, a 24-volume Wu zhu su wen (Wu's Annotation of Plain Questions) in 1594, a 6-volume Zhen fang liu ji (Six Collections of Acupuncture and Prescription) in 1618 block-printed and funded by CHENG Biao, and a 2-volume Mai yu (Language of Pulse) with writing date unknown. For Yi fang kao, there were 4 editions, including Ming-dynasty block-printed edition, Japanese and Korean block-printed edition, old hand-copied edition and modern printing edition. For Wu zhu su wen, there were 5 editions, Ming-Qing-dynasty block-printed edition, Japanese block-printed edition, old hand-copied edition, photocopy and modern edition. For Zhen fang liu ji, there were Ming-dynasty block-printed edition, hand-copied edition, photocopy and modern edition. Mai yu always was printed together with Yi fang kao, and its editions were same as those of Yi fang kao, except some modern printing edition with Mai yu excluded. PMID:24135480

  16. Predicting RNA-RNA Interactions Using RNAstructure.

    Science.gov (United States)

    DiChiacchio, Laura; Mathews, David H

    2016-01-01

    RNA-RNA binding is a required step for many regulatory and catalytic processes in the cell. Identifying RNA-RNA hybridization sites is challenging because of the competition between intramolecular and intermolecular structure formation. A complete picture of RNA-RNA binding includes an understanding of single-stranded folding and binding site accessibility, and is strongly concentration-dependent. This chapter provides guidance for using RNAstructure to predict RNA-RNA binding sites and RNA-RNA structures, utilizing free energy minimization and partition function calculations. RNAstructure is freely available at http://rna.urmc.rochester.edu/RNAstructure.html . PMID:27665592

  17. 血清游离DNA甲基化检测对胶质瘤的意义%Detection of serum Alu element hypomethylation for the diagnosis and prognosis of glioma

    Institute of Scientific and Technical Information of China (English)

    龚铭杰; 陈建; 戚菁; 施金龙; 夏亮; 施炜

    2013-01-01

    Objective To investige the roles of measuring hypomethylation of serum Alu elements (Alu) in glima.Methods Tumor tissues and matched serum specimens from 65 glioma patients and serum samples from 30 healthy controls were examined for Alu hypomethylation by bisulfite sequencing.Results The median serum Alu methylation level was 47.30% in patients [interquartile range (IQR),(35.40 ± 54.25) %] and 57.90% in the controls [IQR,(55.25 ± 61.45) %].The median Alu methylation level in tumor samples was 40.30% [IQR,(36.80 ± 54.20) %],which showed the correlation of Alu hypomethylation between tumor and serum samples (r =0.882) in the study group.The methylation level was higher in the low-grade glioma group than in the high-grade group in tumor and serum samples.A correlation between high methylation level and longer survival time was detected in tumor and serum samples.Receiver operating characteristic (ROC) curve analysis revealed that the area-under-the-curve (AUC) for diagnosis was 0.861 (95% confidence interval:0.789 ± 0.933),suggesting that Alu hypomethylation in serum may be of diagnostic value.Conclusion Our results indicate that the detection of Alu hypomethylation in serum may be clinically useful for the diagnosis and prognosis of glioma.%目的 探讨血清游离DNA甲基化水平检测对胶质瘤的意义.方法 采用亚硫酸氢盐测序(BSP)法检测65例胶质瘤患者血清、组织和30例正常血清Alu甲基化水平并进行分析.结果 患者血清中Alu平均甲基化水平为47.30%[(35.40±54.25)%],正常血清是57.90%[(55.25±61.45)%,P<0.01];肿瘤组织Alu平均甲基化水平为40.30%[(36.80±54.20)%],与血清中一致(r=0.882);另外,高级别组的甲基化水平都低于低级别组(P<0.01);Alu甲基化水平越高预示更高的生存率(P<0.01);受试者工作特征(ROC)曲线下面积(AUC)为0.861(0.789 ~ 0.933,P<0.01).结论 血清游离DNA中Alu低甲基化的检测有助于胶质瘤的诊断及预后判断.

  18. Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system.

    Science.gov (United States)

    Zhou, Yan; Liu, Yong; Hussmann, Dianna; Brøgger, Peter; Al-Saaidi, Rasha Abdelkadhem; Tan, Shuang; Lin, Lin; Petersen, Trine Skov; Zhou, Guang Qian; Bross, Peter; Aagaard, Lars; Klein, Tino; Rønn, Sif Groth; Pedersen, Henrik Duelund; Bolund, Lars; Nielsen, Anders Lade; Sørensen, Charlotte Brandt; Luo, Yonglun

    2016-07-01

    Programmable DNA nucleases such as TALENs and CRISPR/Cas9 are emerging as powerful tools for genome editing. Dual-fluorescent surrogate systems have been demonstrated by several studies to recapitulate DNA nuclease activity and enrich for genetically edited cells. In this study, we created a single-strand annealing-directed, dual-fluorescent surrogate reporter system, referred to as C-Check. We opted for the Golden Gate Cloning strategy to simplify C-Check construction. To demonstrate the utility of the C-Check system, we used the C-Check in combination with TALENs or CRISPR/Cas9 in different scenarios of gene editing experiments. First, we disrupted the endogenous pIAPP gene (3.0 % efficiency) by C-Check-validated TALENs in primary porcine fibroblasts (PPFs). Next, we achieved gene-editing efficiencies of 9.0-20.3 and 4.9 % when performing single- and double-gene targeting (MAPT and SORL1), respectively, in PPFs using C-Check-validated CRISPR/Cas9 vectors. Third, fluorescent tagging of endogenous genes (MYH6 and COL2A1, up to 10.0 % frequency) was achieved in human fibroblasts with C-Check-validated CRISPR/Cas9 vectors. We further demonstrated that the C-Check system could be applied to enrich for IGF1R null HEK293T cells and CBX5 null MCF-7 cells with frequencies of nearly 100.0 and 86.9 %, respectively. Most importantly, we further showed that the C-Check system is compatible with multiplexing and for studying CRISPR/Cas9 sgRNA specificity. The C-Check system may serve as an alternative dual-fluorescent surrogate tool for measuring DNA nuclease activity and enrichment of gene-edited cells, and may thereby aid in streamlining programmable DNA nuclease-mediated genome editing and biological research. PMID:26755436

  19. Book Review: New Perspectives on Technical Editing

    Science.gov (United States)

    Murphy, A. J. (Ed.); Sterken, Christiaan

    2012-08-01

    New Perspectives on Technical Editing by Avon J. Murphy (ed.) ISBN : 978-0895033949 (2010) Baywood Publishing Company Inc, Hardcover, 210 pages, 35.5 GBP This book presents a collection of 10 chapters dealing with diverse aspects of technical editing (ie, editorial planning, and analysis and structural changes made to other people's technological documents): research in technical editing, trends and teaching of technical editing, copyediting, and technical journal editing. The role and function of the modern journal and book editor is also dealt with in detail. Each chapter is written by an expert in the field: senior editors, university professors in technical communication, technical writers and linguists. The ever-evolving role of the editor is clearly elucidated in several historical reviews, and in the descriptions of the expectations for the future. A very striking aspect of this book is its extensive collection of bibliographic resources: every chapter lists dozens of very useful references, and the closing chapter, and annotated bibliography, contain many not so well known references, and are most useful. All in all, the book is a treasure trove listing more than 400 references, in addition to numerous webpage URLs embedded in the texts. The book is designed to help the reader to understand current practices and norms in technical editing, and to help to take action in editing as well as in teaching and educating would-be editors. The audience for this book thus includes editors and teachers, but also writers, researchers and students. A deep reading of this book will result in a better understanding of the difference between full technical editing and its much narrower component so well known as copyediting, and will convince any prospective editor that editing should not be undertaken if the people involved do not master the art of precision and accuracy in technical (as well as in human) communication, do not possess the technical know how and computer

  20. A Parallel Error Detection Structure Design of SPARC V8 Architecture Compatible ALU%一种SPARC V8结构ALU的并行错误检测结构设计

    Institute of Scientific and Technical Information of China (English)

    张洵颖

    2011-01-01

    针对嵌入式处理器在线错误检测的需求,文中基于编码预测的并行错误检测策略,选择Berger编码作为编码预测错误检测编码,设计了SPARC V8结构的加/减、逻辑和移位运算的B0编码预测结构.该结构可以直接集成为带有并行错误检测结构的SPARC V8结构ALU,相比于电路复制这样的结构实现了硬件资源的节省.与对偶复执这样两倍的执行时间并且附带结果比较的策略相比,具有明显的性能优势.%According to the on-line error detection requirement of embedded processors, this paper presents a parallel error detection structure of SPARC V8 architecture compatible ALU, which takes the B0 encoding of Berger code prediction as the parallel on-line error detection strategy. This structure can be integrated into the SPARC V8 architecture compatible ALU directly, and forms a parallel error detection ALU. Comparing with the two-rail logic method, this structure gets the hardware cost decrease. Comparing with pseudoduplication method, in which the same circuit successively processes data twice but along different data path, this structure has an obviously performance advantage.

  1. The Express-Lane Edit: Making Editing Useful for Young Adolescents

    Science.gov (United States)

    Anderson, Jeff

    2008-01-01

    Editing is a powerful tool for writers, but are our methods of teaching it really demonstrating that power for young adolescents? The author, frustrated with students' inability to edit, blames his own approach and, beginning with a grocery store epiphany, works to develop a more effective system. Elements of his successful approach include time…

  2. Improving Student Revising and Editing Skills through the Use of Peer Editing and Writing Conferencing.

    Science.gov (United States)

    Kolling, Ann

    This report describes instructional strategies that will improve the revising and editing skills of sixth grade students during the writing process. The literature review suggested improved instruction and evaluation through a writer's workshop approach, which would include a positive environment, mini-lessons, teacher modeling, peer editing, and…

  3. Ca2+ permeability of unedited and edited versions of the kainate selective glutamate receptor GluR6.

    OpenAIRE

    Egebjerg, J; Heinemann, S F

    1993-01-01

    The Ca2+ permeability of the kainate selective glutamate receptor GluR6 depends on the editing of the RNA (or DNA). The unedited version of GluR6, GluR6Q, encodes a glutamine at position 621 (Q/R site) and exhibits a Ca2+/monovalent ion permeability ratio of 1.2, while the edited version of GluR6, GluR6R, encodes an arginine at position 621 and exhibits a permeability ratio of 0.47. Kainate activation of the GluR6 receptor results in currents that are modulated by extracellular calcium ions. ...

  4. Research on Tissue-type Plasminogen Acivator Gene Alu-repeat Polymorphiam in a Shangdong Chinese Population%组织型纤维酶原激活因子Alu重复序列基因多态性检测

    Institute of Scientific and Technical Information of China (English)

    魏然; 叶文静; 徐涛; 韩继举; 任道凌; 陈彬; 夏作理

    2003-01-01

    研究山东地区汉族人组织型纤维酶原激活因子(TPA)-Alu重复序列基因多态性特点.提取外周血DNA,采用聚合酶链反应扩增t-PA基因h(8th)内含子的Alu重复序列,记录Alu插入/缺失(I/D)结果.166例山东人中TPA-Alu-I/I纯合子基因型50个;TPA-Alu-D/D型46个;TPA-Alu-I/D杂合型70个.Alu序列的插入纯合(I/I)频率为30.12%,缺失纯合(D/D)频率为27.71%:(I/D)杂合型频率为42.17;经x2检验男女之间无显著性差异(P>0.05).受试者等位基因的频率符合Hardy-Wein-berg原则,结果有代表性.

  5. Detailed phenotypic and molecular analyses of genetically modified mice generated by CRISPR-Cas9-mediated editing.

    Directory of Open Access Journals (Sweden)

    Bijal A Parikh

    Full Text Available The bacterial CRISPR-Cas9 system has been adapted for use as a genome editing tool. While several recent reports have indicated that successful genome editing of mice can be achieved, detailed phenotypic and molecular analyses of the mutant animals are limited. Following pronuclear micro-injection of fertilized eggs with either wild-type Cas9 or the nickase mutant (D10A and single or paired guide RNA (sgRNA for targeting of the tyrosinase (Tyr gene, we assessed genome editing in mice using rapid phenotypic readouts (eye and coat color. Mutant mice with insertions or deletions (indels in Tyr were efficiently generated without detectable off-target cleavage events. Gene correction of a single nucleotide by homologous recombination (HR could only occur when the sgRNA recognition sites in the donor DNA were modified. Gene repair did not occur if the donor DNA was not modified because Cas9 catalytic activity was completely inhibited. Our results indicate that allelic mosaicism can occur following -Cas9-mediated editing in mice and appears to correlate with sgRNA cleavage efficiency at the single-cell stage. We also show that larger than expected deletions may be overlooked based on the screening strategy employed. An unbiased analysis of all the deleted nucleotides in our experiments revealed that the highest frequencies of nucleotide deletions were clustered around the predicted Cas9 cleavage sites, with slightly broader distributions than expected. Finally, additional analysis of founder mice and their offspring indicate that their general health, fertility, and the transmission of genetic changes were not compromised. These results provide the foundation to interpret and predict the diverse outcomes following CRISPR-Cas9-mediated genome editing experiments in mice.

  6. CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii.

    Science.gov (United States)

    Li, Qi; Chen, Jun; Minton, Nigel P; Zhang, Ying; Wen, Zhiqiang; Liu, Jinle; Yang, Haifeng; Zeng, Zhe; Ren, Xiaodan; Yang, Junjie; Gu, Yang; Jiang, Weihong; Jiang, Yu; Yang, Sheng

    2016-07-01

    Solventogenic clostridia are important industrial microorganisms that produce various chemicals and fuels. Effective genetic tools would facilitate physiological studies aimed both at improving our understanding of metabolism and optimizing solvent productivity through metabolic engineering. Here we have developed an all-in-one, CRISPR-based genome editing plasmid, pNICKclos, that can be used to achieve successive rounds of gene editing in Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB 8052 with efficiencies varying from 6.7% to 100% and 18.8% to 100%, respectively. The plasmid specifies the requisite target-specific guide RNA, the gene encoding the Streptococcus pyogenes Cas9 nickase and the genome editing template encompassing the gene-specific homology arms. It can be used to create single target mutants within three days, with a further two days required for the curing of the pNICKclos plasmid ready for a second round of mutagenesis. A S. pyogenes dCas9-mediated gene regulation control system, pdCASclos, was also developed and used in a CRISPRi strategy to successfully repress the expression of spo0A in C. acetobutylicum and C. beijerinckii. The combined application of the established high efficiency CRISPR-Cas9 based genome editing and regulation control systems will greatly accelerate future progress in the understanding and manipulation of metabolism in solventogenic clostridia. PMID:27213844

  7. The genome editing revolution: A CRISPR-Cas TALE off-target story.

    Science.gov (United States)

    Stella, Stefano; Montoya, Guillermo

    2016-07-01

    In the last 10 years, we have witnessed a blooming of targeted genome editing systems and applications. The area was revolutionized by the discovery and characterization of the transcription activator-like effector proteins, which are easier to engineer to target new DNA sequences than the previously available DNA binding templates, zinc fingers and meganucleases. Recently, the area experimented a quantum leap because of the introduction of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (clustered regularly interspaced short palindromic sequence). This ribonucleoprotein complex protects bacteria from invading DNAs, and it was adapted to be used in genome editing. The CRISPR ribonucleic acid (RNA) molecule guides to the specific DNA site the Cas9 nuclease to cleave the DNA target. Two years and more than 1000 publications later, the CRISPR-Cas system has become the main tool for genome editing in many laboratories. Currently the targeted genome editing technology has been used in many fields and may be a possible approach for human gene therapy. Furthermore, it can also be used to modifying the genomes of model organisms for studying human pathways or to improve key organisms for biotechnological applications, such as plants, livestock genome as well as yeasts and bacterial strains. PMID:27417121

  8. The genome editing revolution: A CRISPR-Cas TALE off-target story.

    Science.gov (United States)

    Stella, Stefano; Montoya, Guillermo

    2016-07-01

    In the last 10 years, we have witnessed a blooming of targeted genome editing systems and applications. The area was revolutionized by the discovery and characterization of the transcription activator-like effector proteins, which are easier to engineer to target new DNA sequences than the previously available DNA binding templates, zinc fingers and meganucleases. Recently, the area experimented a quantum leap because of the introduction of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system (clustered regularly interspaced short palindromic sequence). This ribonucleoprotein complex protects bacteria from invading DNAs, and it was adapted to be used in genome editing. The CRISPR ribonucleic acid (RNA) molecule guides to the specific DNA site the Cas9 nuclease to cleave the DNA target. Two years and more than 1000 publications later, the CRISPR-Cas system has become the main tool for genome editing in many laboratories. Currently the targeted genome editing technology has been used in many fields and may be a possible approach for human gene therapy. Furthermore, it can also be used to modifying the genomes of model organisms for studying human pathways or to improve key organisms for biotechnological applications, such as plants, livestock genome as well as yeasts and bacterial strains.

  9. Triplex-mediated genome targeting and editing.

    Science.gov (United States)

    Reza, Faisal; Glazer, Peter M

    2014-01-01

    Genome targeting and editing in vitro and in vivo can be achieved through an interplay of exogenously introduced molecules and the induction of endogenous recombination machinery. The former includes a repertoire of sequence-specific binding molecules for targeted induction and appropriation of this machinery, such as by triplex-forming oligonucleotides (TFOs) or triplex-forming peptide nucleic acids (PNAs) and recombinagenic donor DNA, respectively. This versatile targeting and editing via recombination approach facilitates high-fidelity and low-off-target genome mutagenesis, repair, expression, and regulation. Herein, we describe the current state-of-the-art in triplex-mediated genome targeting and editing with a perspective towards potential translational and therapeutic applications. We detail several materials and methods for the design, delivery, and use of triplex-forming and recombinagenic molecules for mediating and introducing specific, heritable, and safe genomic modifications. Furthermore we denote some guidelines for endogenous genome targeting and editing site identification and techniques to test targeting and editing efficiency. PMID:24557900

  10. Association between Alu insertion polymorphisms and HLA class T alleles in Chinese Lisu and Nu ethnic populations%中国傈僳族和怒族群体人类白细胞抗原Ⅰ类基因区Alu插入多态性研究

    Institute of Scientific and Technical Information of China (English)

    董兆梅; 姚宇峰; 史磊; 陶玉芬; 林克勤; 黄小琴; 杨昭庆; 褚嘉祐; 史荔

    2012-01-01

    Objective To investigate the frequencies of HLA-Alu repeat polymorphisms (AluMICB,AluTF,AluHJ,AluHG and AluHF) in Chinese Lisu and Nu ethnic populations.Methods The frequencies of HLA-Alu repeat polymorphisms in above populations were determined with polymerase chain reaction (PCR).The associations between HLA-Alu repeat polymorphisms and HLA-A,HLA-B and HLA-C alleles were also analyzed.Phylogenetic trees were constructed with genetic distance calculated from the frequencies of HLA-Alu repeat polymorphisms.Results Frequencies of AluTF * 2 and AluHF * 2 were different between the two populations (P<0.05),while those of other three insertions were similar.The strength of association between HLA-Alus and HLA alleles were different (P<0.05) in the two populations.Although AluMICB * 2 were associated with HLA-B* 56:01 in both populations,the association was stronger in Lisu population (74.0%) but moderate in Nu population (30.7%).HLA-Alus were associated with particular HLA subtypes,e.g.,AluHG * 2 with certain HLA-A * 02 subtypes.By phylogenetic analysis,Lisu and Nu were clustered together with southern Chinese and Thai populations.Conclusion The distribution of HLA-Alus and the strength of associations between HLA-Alus and HLA class I alleles have varied between the two populations.Study of this association may facilitate identification of origins,evolution,progenitor haplotypes and recombination within the HLA class I region.%目的 研究中国两个隔离群体(傈僳族和怒族)人类白细胞抗原(human leukocyte antigen,HLA)Ⅰ类基因区域内5个HLA-Alu插入多态性(AluMICB、AluTF、AluHJ、AluHG和AluHF)的分布特征.方法 应用聚合酶链反应技术对中国两个隔离群体傈僳族(107人)和怒族(104人)进行HLA-Alu多态性分型.结合HLA基因分型数据,分析这两个群体中HLA-Alu插入与HLA-A、HLA-B和HLA-C基因的关系.根据HLA-Alu频率计算各群体间遗传距离,构建系统进化树.结果 AluTF和AluHF插入

  11. Efficient Genome Editing in Apple Using a CRISPR/Cas9 system.

    Science.gov (United States)

    Nishitani, Chikako; Hirai, Narumi; Komori, Sadao; Wada, Masato; Okada, Kazuma; Osakabe, Keishi; Yamamoto, Toshiya; Osakabe, Yuriko

    2016-01-01

    Genome editing is a powerful technique for genome modification in molecular research and crop breeding, and has the great advantage of imparting novel desired traits to genetic resources. However, the genome editing of fruit tree plantlets remains to be established. In this study, we describe induction of a targeted gene mutation in the endogenous apple phytoene desaturase (PDS) gene using the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed and stably transformed with Cas9 separately in apple. Clear and partial albino phenotypes were observed in 31.8% of regenerated plantlets for one gRNA, and bi-allelic mutations in apple PDS were confirmed by DNA sequencing. In addition, an 18-bp gRNA also induced a targeted mutation. These CRIPSR/Cas9 induced-mutations in the apple genome suggest activation of the NHEJ pathway, but with some involvement also of the HR pathway. Our results demonstrate that genome editing can be practically applied to modify the apple genome. PMID:27530958

  12. CRISPR/Cas9: A powerful tool for crop genome editing

    Directory of Open Access Journals (Sweden)

    Gaoyuan Song

    2016-04-01

    Full Text Available The CRISPR/Cas9 technology is evolved from a type II bacterial immune system and represents a new generation of targeted genome editing technology that can be applied to nearly all organisms. Site-specific modification is achieved by a single guide RNA (usually about 20 nucleotides that is complementary to a target gene or locus and is anchored by a protospacer-adjacent motif. Cas9 nuclease then cleaves the targeted DNA to generate double-strand breaks (DSBs, which are subsequently repaired by non-homologous end joining (NHEJ or homology-directed repair (HDR mechanisms. NHEJ may introduce indels that cause frame shift mutations and hence the disruption of gene functions. When combined with double or multiplex guide RNA design, NHEJ may also introduce targeted chromosome deletions, whereas HDR can be engineered for target gene correction, gene replacement, and gene knock-in. In this review, we briefly survey the history of the CRISPR/Cas9 system invention and its genome-editing mechanism. We also describe the most recent innovation of the CRISPR/Cas9 technology, particularly the broad applications of modified Cas9 variants, and discuss the potential of this system for targeted genome editing and modification for crop improvement.

  13. CRISPR/Cas9:A powerful tool for crop genome editing

    Institute of Scientific and Technical Information of China (English)

    Gaoyuan Song; Meiling Jia; Kai Chen; Xingchen Kong; Bushra Khattak; Chuanxiao Xie; Aili Li; Long Mao

    2016-01-01

    The CRISPR/Cas9 technology is evolved from a type II bacterial immune system and represents a new generation of targeted genome editing technology that can be applied to nearly all organisms. Site-specific modification is achieved by a single guide RNA (usually about 20 nucleotides) that is complementary to a target gene or locus and is anchored by a protospacer-adjacent motif. Cas9 nuclease then cleaves the targeted DNA to generate double-strand breaks (DSBs), which are subsequently repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms. NHEJ may introduce indels that cause frame shift mutations and hence the disruption of gene functions. When combined with double or multiplex guide RNA design, NHEJ may also introduce targeted chromosome deletions, whereas HDR can be engineered for target gene correction, gene replacement, and gene knock-in. In this review, we briefly survey the history of the CRISPR/Cas9 system invention and its genome-editing mechanism. We also describe the most recent innovation of the CRISPR/Cas9 technology, particularly the broad applications of modified Cas9 variants, and discuss the potential of this system for targeted genome editing and modification for crop improvement.

  14. Introduction to nuclear science, second edition

    CERN Document Server

    Bryan, Jeff C.

    2013-01-01

    This book was written to provide students who have limited backgrounds in the physical sciences and math with an accessible textbook on nuclear science. Expanding on the foundation of the bestselling first edition, Introduction to Nuclear Science, Second Edition provides a clear and complete introduction to nuclear chemistry and physics, from basic concepts to nuclear power and medical applications. Incorporating suggestions from professors using this book for their courses, the author has created a new text that is approximately 60 percent larger and more comprehensive and flexible than the first.New to This Edition: Thorough review of nuclear forensics, radiology, gamma cameras, and decay through proton or neutron emission More detailed explanations of the necessary mathematics A chapter on dosimetry of radiation fields Expanded discussion of applications, introduced earlier in the text More in-depth coverage of nuclear reactors, including a new chapter examining more reactor types, their safety systems,...

  15. Louse (Insecta : Phthiraptera) mitochondrial 12S rRNA secondary structure is highly variable

    OpenAIRE

    Page, R.D.M.; Cruickshank, R.; Johnson, K P

    2002-01-01

    Lice are ectoparasitic insects hosted by birds and mammals. Mitochondrial 12S rRNA sequences obtained from lice show considerable length variation and are very difficult to align. We show that the louse 12S rRNA domain III secondary structure displays considerable variation compared to other insects, in both the shape and number of stems and loops. Phylogenetic trees constructed from tree edit distances between louse 12S rRNA structures do not closely resemble trees constructed from sequence ...

  16. 恒河猴基因组SINEs和Alu元件的数量和分布%The Numbers and Distribution of SINEs and Alu Elements in Rhesus Macaque Genome

    Institute of Scientific and Technical Information of China (English)

    夏珊; 范振鑫

    2014-01-01

    哺乳动物的基因组中富含各种类型的重复序列,其中的微卫星作为分子标记在相关研究中得到了广泛的应用.而重复序列中的SINEs元件,在各类群物种的分子系统发育、遗传多样性等方面的研究也得到了使用.其中,灵长类物种特有的SINEs元件、Alu元件,也在灵长类物种的进化研究中得到了成功的使用.本研究对于重要的医学模式动物恒河猴的基因组中SINEs和Alu元件进行了搜索,并进一步统计分析其分布规律、长度等信息.在恒河猴基因组的20条常染色体上共发现了Alu元件1 093 185个,在性染色体X上发现了45 215个.长度为200 bp至300 bp区间的Alu元件分布最多;Alu元件中75%的分化值至少都为10,而只有6.2%左右的元件分化值能达到至少20,这一结果表明绝大部分的Alu元件都比较年轻.本研究的统计结果为后续应用SINEs和Alu元件作为分子标记的研究提供了重要的信息.%There are many different types of repeat elements in mammal genomes,and microsatellite has been widely used as molecular marker in researches.Additionally,another type of repeat element,SINEs,has also been used as molecular markers in molecular phylogeny and genetic diversity.Alu element,which was the primate-specific SINEs element,has been widely used in the evolutionary study of primate species.In this study,SINEs and Alu elements were screened in the genome of rhesus macaque Macaca mulatta,which was an important model animal in biomedical study.In total,there were 1 093 185 Alu elements in the 20 autosomes,and 45 215 Alu elements in the chromosome X.Most of the Alu elements were ranged in length from 200 bp to 300 bp,and 75% of which had a minimal divergence value of 10,whereas only 6.2% could reach 20,indicating that most of the Alu elements were relatively young.These results may provide important information for the future studies on the application of SINEs and Alu elements as molecular

  17. 血管紧张素转化酶基因Alu I/D的多态性与心房颤动的关联研究%Association of Angiotensin-convertion enzyme (ACE) gene Alu I/D with Atrial Fibrillation

    Institute of Scientific and Technical Information of China (English)

    张复贵; 闵新文; 曾秋棠; 毛晓波; 易桂文; 吉庆伟

    2011-01-01

    目的:探讨血管紧张素转化酶(ACE)基因Alu I/D的多态性与心房颤动(Af)的关系.方法:研究对象均来自湖北地区汉族人群,120例Af患者(Af组),120例非Af者(对照组).采用成组配比研究,取静脉血,提取基因组DNA,采用聚合酶链反应-限制性酶切片段长度多态性(PCR-RFLP)分析技术对2组人群ACE基因Alu I/D的多态性进行分析.结果:ACE DD基因型频率在Af组与对照组之间存在显著差异(32.5%∶18.3%,P=0.008),等位基因在2组间亦存在同样的趋势(D/I=52.9%∶37.5%,P=0.001).在对混杂因素进行校正后,携带DD基因的人群患Af的危险性较高(OR=3.34,95%CI=1.58~7.04,P=0.002);携带ID基因型的人群患Af的危险性也升高,但差异无统计学意义(OR=1.95,95%CI=0.95~3.99,P=0.069);排除混杂因素后,左房内径与人群患Af的风险呈显著相关(OR=8.92,95%CI=3.72~21.40,P=0.000).结论:在湖北地区汉族人群中,ACE基因Alu I/D的多态性与Af的发病呈明显相关性,可能是Af的遗传危险因素,左房内径的增加与Af的发病呈显著相关.%Objective:To investigate the relationship between the polymorphism of the Angiotensin-convertion enzyme (ACE) gene Alu I/D and atrial fibrillation (Af). Method:All cases came from the Han population in Hubei Province, 120 cases were Af patients, 120 were non Af. Unitizing match investigate was used, vein blood were acquired to extracte genomic DNA, Polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) was used to analyze the polymorphism of the ACE gene Alu I/D analysis in both populations. Result:There was significant difference between the case group and control group in DD frequencies of genotype ACE I/D polymorphism (32.5%: 18.3%, P= 0.008), there was also the same trend between alleles (D/I= 52.9%: 37.5%,P= 0.001). After adjustment of confounding factor, there were more risk for the crowd carrying DD gene suffering from Af (OR= 3.34, 95% CI=1.58- 7.04, P= 0. 002

  18. 精子发生中Alu序列的返座作用%Retrotransposons Role of Alu Sequences in Human Spermatogenesis

    Institute of Scientific and Technical Information of China (English)

    王黎熔; 李建民; 单玉喜

    2001-01-01

    目的: 克隆人类精原细胞和初级精母细胞特异表达的基因. 方法: 应用改良的mRNA表达图谱分析技术,对正常男性、唯支持细胞综合征和生精阻滞在初级精母细胞病人的睾丸进行mRNA表达图谱分析的差异显示,选择唯支持细胞综合征缺失的表达基因并克隆测序. 结果: 正常男性、唯支持细胞综合征和生精阻滞在初级精母细胞症病人的睾丸组织表达图谱存在显著的表达差异,目前已获得唯支持细胞综合征缺失的片段88个.在已分析的6个片段中,1个与人类的Alu序列家族(Alu family)98%同源. 结论: Alu序列在生精细胞中通过返座作用形成新Alu序列,使其不断扩散.

  19. Partial protoporphyrinogen oxidase (PPOX gene deletions, due to different Alu-mediated mechanisms, identified by MLPA analysis in patients with variegate porphyria

    Directory of Open Access Journals (Sweden)

    Barbaro Michela

    2013-01-01

    Full Text Available Abstract Variegate porphyria (VP is an autosomal dominantly inherited hepatic porphyria. The genetic defect in the PPOX gene leads to a partial defect of protoporphyrinogen oxidase, the penultimate enzyme of heme biosynthesis. Affected individuals can develop cutaneous symptoms in sun-exposed areas of the skin and/or neuropsychiatric acute attacks. The identification of the genetic defect in VP families is of crucial importance to detect the carrier status which allows counseling to prevent potentially life threatening neurovisceral attacks, usually triggered by factors such as certain drugs, alcohol or fasting. In a total of 31 Swedish VP families sequence analysis had identified a genetic defect in 26. In the remaining five families an extended genetic investigation was necessary. After the development of a synthetic probe set, MLPA analysis to screen for single exon deletions/duplications was performed. We describe here, for the first time, two partial deletions within the PPOX gene detected by MLPA analysis. One deletion affects exon 5 and 6 (c.339-197_616+320del1099 and has been identified in four families, most probably after a founder effect. The other extends from exon 5 to exon 9 (c.339-350_987+229del2609 and was found in one family. We show that both deletions are mediated by Alu repeats. Our findings emphasize the usefulness of MLPA analysis as a complement to PPOX gene sequencing analysis for comprehensive genetic diagnostics in patients with VP.

  20. Establishment of Sequence-Tagged Sites on 15q11-q13 by Alu-Vector PCR Cloning of Yac-Generated Fragments

    Directory of Open Access Journals (Sweden)

    W. S. Kim

    1996-01-01

    Full Text Available Angelman syndrome (AS is caused by the loss of function of undefined gene(s on human chromosome 15. The majority of subjects have deletions involving maternally-derived chromosome 15q II-q 13, and the shortest region of deletion overlap (SRO has been localized to the region between D15S10 and D15S113. In this study, yeast artificial chromosomes (YACs, 6G-D4, 9H-D2 and 37D-F9, mapping within the AS SRO, were isolated from the ICI Y AC library. Alu-vector PCR products were amplified from the YACs and from YACs A229A2 and A33FI 0 which had been obtained from the St. Louis Y AC library. The PCR products were cloned and sequenced, and three new sequence-tagged sites were generated within the AS SRO, facilitating the characterization of gene(s involved in the Angelman syndrome.

  1. Transportation Energy Data Book, Edition 19

    Energy Technology Data Exchange (ETDEWEB)

    Davis, S.C.

    1999-09-01

    The Transportation Energy Data Book: Edition 19 is a statistical compendium prepared and published by Oak Ridge National Laboratory (ORNL) under contract with the Office of Transportation Technologies in the Department of Energy (DOE). Designed for use as a desk-top reference, the data book represents an assembly and display of statistics and information that characterize transportation activity, and presents data on other factors that influence transportation energy use. The purpose of this document is to present relevant statistical data in the form of tables and graphs. The latest editions of the Data Book are available to a larger audience via the Internet (http://www-cta.ornl.gov/data/tedb.htm).

  2. Prince2 2009 edition a pocket guide

    CERN Document Server

    Hedeman, Bert

    2010-01-01

    This Pocket Guide supplies a summary of the PRINCE2 method, to provide a quick introduction as well as a structured overview of the method;Main target Group for this pocket guide is anyone who wants to get to know the method PRINCE2 or a methodical approach for project management. The book is also very useful for members of a project management team on a project using the PRINCE2 method. Furthermore this pocket guide can be used as literature for the preparation of the PRINCE2 2009 Edition Foundation exam;This pocket guide is based on PRINCE2 2009 Edition;This pocket book deals with processes,

  3. [On two editions of Yunyu Xuanji and their source].

    Science.gov (United States)

    Deng, Yue-E

    2013-01-01

    Yunyu Xuanji is considered as one of the 400 key ancient books in the 'TCM Ancient Books Protection and Use Project' by the State Bureau of TCM. Two editions of this book are hand-written copies. The Qianyao edition was finished in the 52(nd) year of the Kangxi Period (1713). The other edition (Taoshi Xianyishukoucongshu) was probably done in the Late Qing Dynasty or the period of the Republic of China. By careful comparison, the contents of the two editions are basically the same, and only the preface of the former edition has been given some changes, which were probably made by later generations. The second manuscript was written in a more standard mode. Therefore the two editions were written according to the same literature and it is possible that the latter one is the copied edition of the Qianyao edition. PMID:23879984

  4. Experimental Stochatics (2nd edition)

    International Nuclear Information System (INIS)

    for teachers of computational stochastic methods, is the main contribution of this electronic monograph. However, both the book and software suffer from several severe problems. Firstly, I feel that the structure of the text is weak. Probably this is partly the result of the text from the CD-ROM being put into a book format, but the short paragraphs and poorly structured sentences destroy the reading experience. Secondly, although the software is functional, I believe that, like me, many users will be disappointed by the quality of the user interface and the visualizations. The opportunities to interact with the simulations are limited. Thirdly, the presentation is slightly old fashioned and lacking in pedagogical structure. For example, flow charts and Pascal programs are used to present algorithms. To conclude, I am surprised that this electronic monograph warranted a second edition in this form. Teachers may find the examples useful as a starting point, but students and researchers are advised to look elsewhere. (book review)

  5. Delivery of Genome Editing Reagents to Hematopoietic Stem/Progenitor Cells.

    Science.gov (United States)

    Hoban, Megan D; Romero, Zulema; Cost, Gregory J; Mendel, Matthew; Holmes, Michael; Kohn, Donald B

    2016-01-01

    This unit describes the protocol for the delivery of reagents for targeted genome editing to CD34(+) hematopoietic stem/progenitor cells (HSPCs). Specifically, this unit focuses on the process of thawing and pre-stimulating CD34(+) HSPCs, as well as the details of their electroporation with in vitro-transcribed mRNA-encoding site-specific nucleases [in this case zinc-finger nucleases (ZFNs)]. In addition, discussed is delivery of a gene editing donor template in the form of an oligonucleotide or integrase-defective lentiviral vector (IDLV). Finally, an analysis of cell survival following treatment and downstream culture conditions are presented. While optimization steps might be needed for each specific application with respect to nuclease and donor template amount, adherence to this protocol will serve as an excellent starting point for this further work. PMID:26840227

  6. Generation of an Oocyte-Specific Cas9 Transgenic Mouse for Genome Editing.

    Directory of Open Access Journals (Sweden)

    Linlin Zhang

    Full Text Available The CRISPR/Cas9 system has been developed as an easy-handle and multiplexable approach for engineering eukaryotic genomes by zygote microinjection of Cas9 and sgRNA, while preparing Cas9 for microinjection is laborious and introducing inconsistency into the experiment. Here, we describe a modified strategy for gene targeting through using oocyte-specific Cas9 transgenic mouse. With this mouse line, we successfully achieve precise gene targeting by injection of sgRNAs only into one-cell-stage embryos. Through comprehensive analysis, we also show allele complexity and off-target mutagenesis induced by this strategy is obviously lower than Cas9 mRNA/sgRNA injection. Thus, injection of sgRNAs into oocyte-specific Cas9 transgenic mouse embryo provides a convenient, efficient and reliable approach for mouse genome editing.

  7. FeynEdit - a tool for drawing Feynman diagrams

    OpenAIRE

    Hahn, T.; Lang, P.

    2007-01-01

    We describe the FeynEdit tool for drawing Feynman diagrams. Input and output is done using the LaTeX macros of FeynArts, which also implies that diagrams drawn by FeynArts can be edited with FeynEdit. The LaTeX code can be conveniently transferred using copy-and-paste.

  8. 45 CFR 73.735-705 - Writing and editing.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 1 2010-10-01 2010-10-01 false Writing and editing. 73.735-705 Section 73.735-705... Outside Activities § 73.735-705 Writing and editing. (a) Employees are encouraged to engage in outside writing and editing whether or not done for compensation, when such activity is not otherwise...

  9. Human Resources Administration: A School-Based Perspective. Fourth Edition

    Science.gov (United States)

    Smith, Richard

    2009-01-01

    Enhanced and updated, this Fourth Edition of Richard E. Smith's highly successful text examines the growing role of the principal in planning, hiring, staff development, supervision, and other human resource functions. The Fourth Edition includes new sections on ethics, induction, and the role of the mentor teacher. This edition also introduces…

  10. Soil Carbon Sequestration and the Greenhouse Effect (2nd Edition)

    Science.gov (United States)

    This volume is a second edition of the book “Soil Carbon Sequestration and The Greenhouse Effect”. The first edition was published in 2001 as SSSA Special Publ. #57. The present edition is an update of the concepts, processes, properties, practices and the supporting data. All chapters are new co...

  11. Editing modifies the GABA(A) receptor subunit alpha3

    DEFF Research Database (Denmark)

    Ohlson, Johan; Pedersen, Jakob Skou; Haussler, David;

    2007-01-01

    to find selectively edited sites and combined it with bioinformatic techniques that find stem-loop structures suitable for editing. We present here the first verified editing candidate detected by this screening procedure. We show that Gabra-3, which codes for the alpha3 subunit of the GABA...

  12. The staging system: Display and edit module

    Science.gov (United States)

    Edwards, E.; Bernier, L.

    1976-01-01

    The Display and Edit (D and E) Module described is one of six major modules being developed for the STAGING (STructural Analysis through Generalized INteractive Graphics) System. Several remarks are included concerning the computer environment and the architecture of the data base. The utility of this module is emphasized.

  13. Thermodynamics of Fluids Under Flow Second Edition

    CERN Document Server

    Jou, David; Criado-Sancho, Manuel

    2011-01-01

    This is the second edition of the book “Thermodynamics of Fluids under Flow,” which was published in 2000 and has now been corrected, expanded and updated. This is a companion book to our other title Extended irreversible thermodynamics (D. Jou, J. Casas-Vázquez and G. Lebon, Springer, 4th edition 2010), and of the textbook Understanding non-equilibrium thermodynamics (G. Lebon, D. Jou and J. Casas-Vázquez, Springer, 2008. The present book is more specialized than its counterpart, as it focuses its attention on the non-equilibrium thermodynamics of flowing fluids, incorporating non-trivial thermodynamic contributions of the flow, going beyond local equilibrium theories, i.e., including the effects of internal variables and of external forcing due to the flow. Whereas the book's first edition was much more focused on polymer solutions, with brief glimpses into ideal and real gases, the present edition covers a much wider variety of systems, such as: diluted and concentrated polymer solutions, polymer ble...

  14. The Landscape of Qualitative Research. Third Edition

    Science.gov (United States)

    Denzin, Norman K., Ed.; Lincoln, Yvonna, Ed.

    2007-01-01

    This book, the first volume of the paperback versions of the "The SAGE Handbook of Qualitative Research, Third Edition," takes a look at the field from a broadly theoretical perspective, and is composed of the Handbook's Parts I ("Locating the Field"), II ("Major Paradigms and Perspectives"), and VI ("The Future of Qualitative Research"). "The…

  15. Collecting and Interpreting Qualitative Materials. Third Edition

    Science.gov (United States)

    Denzin, Norman K., Ed.; Lincoln, Yvonna, Ed.

    2007-01-01

    This book is the third volume of the paperback versions of "The SAGE Handbook of Qualitative Research, Third Edition." This portion of the handbook considers the tasks of collecting, analyzing, and interpreting empirical materials, and comprises the Handbook's Parts IV ("Methods of Collecting and Analyzing Empirical Materials") and V ("The Art and…

  16. Science Editing in America: An Overview

    Institute of Scientific and Technical Information of China (English)

    Barbara Gastel

    2003-01-01

    @@ In America, science editing appears to be an increasingly recognized field. In what settings do American science editors work, and what kinds of work do they do ? What is their educational background? What style manuals and other resources do they use? What organizations serve them? What topics and issues do they find of professional interest?

  17. Handbook of Qualitative Research. Second Edition.

    Science.gov (United States)

    Denzin, Norman K., Ed.; Lincoln, Yvonna S., Ed.

    This handbook's second edition represents the state of the art for the theory and practice of qualitative inquiry. It features eight new topics, including autoethnography, critical race theory, applied ethnography, queer theory, and "testimonio"every chapter in the handbook has been thoroughly revised and updated. The book contains:"Preface" (1…

  18. Prescott’s Microbiology, Eighth Edition

    Directory of Open Access Journals (Sweden)

    Joanne J. Dobbins

    2010-04-01

    Full Text Available Review of: Prescott’s Microbiology, Eighth Edition. Joanne M. Willey, Linda M. Sherwood, and Christopher J. Woolverton. 2011. McGraw-Hill Higher Education, NewYork, NY. 1070 pages. ISBN- 978-0-07-337526-7.

  19. A Model for Flexibly Editing CSCL Scripts

    Science.gov (United States)

    Sobreira, Pericles; Tchounikine, Pierre

    2012-01-01

    This article presents a model whose primary concern and design rationale is to offer users (teachers) with basic ICT skills an intuitive, easy, and flexible way of editing scripts. The proposal is based on relating an end-user representation as a table and a machine model as a tree. The table-tree model introduces structural expressiveness and…

  20. The NMC Horizon Report: 2014 Library Edition

    NARCIS (Netherlands)

    Johnson, L; Adams Becker, S.; Estrada, V.; Freeman, A.; van den Brekel, Guus

    2014-01-01

    The NMC Horizon Report: 2014 Library Edition, examines key trends, significant challenges, and emerging technologies for their potential impact on academic and research libraries worldwide. While there are many local factors affecting libraries, there are also issues that transcend regional boundari

  1. Critical Social Theories. 2nd Edition

    Science.gov (United States)

    Agger, Ben

    2006-01-01

    Praised for its clarity and accessibility, this fully updated edition of "Critical Social Theories" presents a comprehensive analysis of leading social and cultural theories today. Diverse perspectives are addressed from feminism and cultural studies to postmodernism and critical theory. Written accessibly for students and faculty, the second…

  2. Edit propagation using geometric relationship functions

    KAUST Repository

    Guerrero, Paul

    2014-03-01

    We propose a method for propagating edit operations in 2D vector graphics, based on geometric relationship functions. These functions quantify the geometric relationship of a point to a polygon, such as the distance to the boundary or the direction to the closest corner vertex. The level sets of the relationship functions describe points with the same relationship to a polygon. For a given query point, we first determine a set of relationships to local features, construct all level sets for these relationships, and accumulate them. The maxima of the resulting distribution are points with similar geometric relationships. We show extensions to handle mirror symmetries, and discuss the use of relationship functions as local coordinate systems. Our method can be applied, for example, to interactive floorplan editing, and it is especially useful for large layouts, where individual edits would be cumbersome. We demonstrate populating 2D layouts with tens to hundreds of objects by propagating relatively few edit operations. © 2014 ACM 0730-0301/2014/03- ART15 $15.00.

  3. The NMC Horizon Report: 2013 Museum Edition

    Science.gov (United States)

    Johnson, L.; Adams Becker, S.; Freeman, A.

    2013-01-01

    The "NMC Horizon Report: 2013 Museum Edition," is a co-production with the Marcus Institute for Digital Education in the Arts (MIDEA), and examines six emerging technologies for their potential impact on and use in education and interpretation within the museum environment: BYOD (Bring Your Own Device), crowdsourcing, electronic…

  4. Does Money Matter in Education? Second Edition

    Science.gov (United States)

    Baker, Bruce D.

    2016-01-01

    This second edition policy brief revisits the long and storied literature on whether money matters in providing a quality education. It includes research released since the original brief in 2012 and covers a handful of additional topics. Increasingly, political rhetoric adheres to the unfounded certainty that money does not make a difference in…

  5. Nanoparticles for Site Specific Genome Editing

    Science.gov (United States)

    McNeer, Nicole Ali

    Triplex-forming peptide nucleic acids (PNAs) can be used to coordinate the recombination of short 50-60 by "donor DNA" fragments into genomic DNA, resulting in site-specific correction of genetic mutations or the introduction of advantageous genetic modifications. Site-specific gene editing in hematopoietic stem and progenitor cells (HSPCs) could result in treatment or cure of inherited disorders of the blood such as beta-thalassemia. Gene editing in HSPCs and differentiated T cells could help combat HIV/AIDs by modifying receptors, such as CCR5, necessary for R5-tropic HIV entry. However, translation of genome modification technologies to clinical practice is limited by challenges in intracellular delivery, especially in difficult-to-transfect hematolymphoid cells. In vivo gene editing could also provide novel treatment for systemic monogenic disorders such as cystic fibrosis, an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane receptor. Here, we have engineered biodegradable nanoparticles to deliver oligonucleotides for site-specific genome editing of disease-relevant genes in human cells, with high efficiency, low toxicity, and editing of clinically relevant cell types. We designed nanoparticles to edit the human beta-globin and CCR5 genes in hematopoietic cells. We show that poly(lactic-co-glycolic acid) (PLGA) nanoparticles can delivery PNA and donor DNA for site-specific gene modification in human hematopoietic cells in vitro and in vivo in NOD-scid IL2rgammanull mice. Nanoparticles delivered by tail vein localized to hematopoietic compartments in the spleen and bone marrow of humanized mice, resulting in modification of the beta-globin and CCR5 genes. Modification frequencies ranged from 0.005 to 20% of cells depending on the organ and cell type, without detectable toxicity. This project developed highly versatile methods for delivery of therapeutics to hematolymphoid cells and hematopoietic stem cells, and will help to

  6. 基于多能干细胞的Alport综合征的microRNA差异性表达与碱基编辑功能分析%Identification of different expression microRNA and base edit function in Alport syndrome base on iPSCs

    Institute of Scientific and Technical Information of China (English)

    陈文标; 彭武建; 林小聪; 戴勇

    2014-01-01

    目的 分析Alport综合征(AS)的诱导多能干细胞(iPSCs)全基因微小核糖核酸(microRNA)的表达谱,筛选出具有差异性表达的microRNA与发生碱基突变的microRNA.方法 收集1例AS患者与1例健康人的尿液,从尿液中分离尿肾脏管细胞,尿肾脏管细胞分化诱导成iPSCs.运用高通量测序方法对iPSCs的microRNA进行测序与表达量测定,比较分析两人microRNA的表达差异,寻找具有特异性的microRNA靶点.同时,将mi-croRNA序列与miRBase数据库(http://www.mirbase.org/)中已知成熟microRNA序列进行比对,比较两标本mi-croRNA碱基编辑数量,找出发生了碱基突变的microRNA.结果 在两人标本中发现30个microRNA具有显著的差异性表达,其中19个microRNAs表达上调,11个microRNA表达下调.has-miR-3117-3p的倍比值(log2 Ratio)为7.79,在表达上调的microRNA最具有特异性;has-miR-544b的倍比值为-9.09,在表达下调的microRNA最具有特异性.同时在两标本中共有208个microRNA发生了碱基突变,其中104个microRNA,AS患者发生碱基编辑概率比健康人大;77个microRNA,健康人发生碱基编辑概率比AS患者大.结论 AS患者与健康人肾脏组织中的microRNA存在差异性,AS发生碱基编辑引起基因突变的microRNA比健康人更常见.这些差异性表达的microRNAs与microRNA碱基突变的概率可能在AS发病机制中起重要作用.

  7. Nuclear-physical data editing and analysis

    International Nuclear Information System (INIS)

    Full text: At the modern stage of high energy physics development, investigation of multiple production processes of particles and nucleus-fragments and their automatical editing becomes one of the central problems of high energy physics (elementary-particles). Enormous experimental information accumulated from various registering installation put global physical problems before physicists-experimenters. One of these problems -automatic editing and receiving of correct physical data and thereby to make scientific conclusions [1]. In the article, some questions of automatic editing of experimental data are presented. Modern automatic systems of mathematical editing of data are complex programme, consisting of various components, which are called in definite sequence [2]. The editing of experimental results on ECM is a complex process, connected with using of tens thousands of information storage devices (discs and cartridges) and other external memory equipment. For processing of experimental data the programme system complexes are prepared and created. This system is used for study of hypernuclear and multiparticle processes in different scientific research institutions of Commonwealth of Independent Countries and other countries abroad [3]. At the present in Andijan State University some of processors and methodic systems are used by students of Physics Department in preparing graduation papers. In particular, the dependence of average number of fragments from energy of particles bombarding nucleus-target was investigated. The investigations made in the framework of different models of nuclei, show slow increase of the number of nuclei-fragments, depending of the energy of bombarding particle [4]. In the further the similar systems for investigation and automation of rapid and superrapid reactions, as well as processes in solid and fluid crystals will be created [5]. (author)

  8. Cloud Properties of CERES-MODIS Edition 4 and CERES-VIIRS Edition 1

    Science.gov (United States)

    Sun-Mack, Sunny; Minnis, Patrick; Chang, Fu-Lung; Hong, Gang; Arduini, Robert; Chen, Yan; Trepte, Qing; Yost, Chris; Smith, Rita; Brown, Ricky; Chu, Churngwei; Heckert, Elizabeth; Gibson, Sharon; Heck, Patrick W.

    2015-01-01

    The Clouds and Earth's Radiant Energy System (CERES) analyzes MODerate-resolution Imaging Spectroradiometer (MODIS) data and Visible Infrared Imaging Radiometer Suite (VIIRS) to derive cloud properties that are combine with aerosol and CERES broadband flux data to create a multi-parameter data set for climate study. CERES has produced over 15 years of data from Terra and over 13 years of data from Aqua using the CERES-MODIS Edition-2 cloud retrieval algorithm. A recently revised algorithm, CERESMODIS Edition 4, has been developed and is now generating enhanced cloud data for climate research (over 10 years for Terra and 8 years for Aqua). New multispectral retrievals of properties are included along with a multilayer cloud retrieval system. Cloud microphysical properties are reported at 3 wavelengths, 0.65, 1.24, and 2.1 microns to enable better estimates of the vertical profiles of cloud water contents. Cloud properties over snow are retrieved using the 1.24-micron channel. A new CERES-VIIRS cloud retrieval package was developed for the VIIRS spectral complement and is currently producing the CERES-VIIRS Edition 1 cloud dataset. The results from CERES-MODIS Edition 4 and CERES-VIIRS Edition 1 are presented and compared with each other and other datasets, including CALIPSO, CloudSat and the CERES-MODIS Edition-2 results.

  9. A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation

    OpenAIRE

    Farasat, Iman; Salis, Howard M.

    2016-01-01

    The ability to precisely modify genomes and regulate specific genes will greatly accelerate several medical and engineering applications. The CRISPR/Cas9 (Type II) system binds and cuts DNA using guide RNAs, though the variables that control its on-target and off-target activity remain poorly characterized. Here, we develop and parameterize a system-wide biophysical model of Cas9-based genome editing and gene regulation to predict how changing guide RNA sequences, DNA superhelical densities, ...

  10. A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation.

    OpenAIRE

    Iman Farasat; Salis, Howard M.

    2016-01-01

    The ability to precisely modify genomes and regulate specific genes will greatly accelerate several medical and engineering applications. The CRISPR/Cas9 (Type II) system binds and cuts DNA using guide RNAs, though the variables that control its on-target and off-target activity remain poorly characterized. Here, we develop and parameterize a system-wide biophysical model of Cas9-based genome editing and gene regulation to predict how changing guide RNA sequences, DNA superhelical densities, ...

  11. Application of SLIC Am79R70 in SPC ALU%用户线接口芯片Am79R70在交换机ALU中的应用

    Institute of Scientific and Technical Information of China (English)

    龚军; 汪小燕

    2003-01-01

    Am79R70是Legerity公司生产的模拟用户线接口电路(SLIC)芯片,它能提供连续可调的电流馈电,可在其控制端C1、C2和C3输入不同的逻辑电平以控制芯片的工作方式.文章介绍了Am79R70的性能特点及其在用户接口单元(ALU)中的应用.

  12. Efficient CRISPR-Cas9-mediated genome editing in Plasmodium falciparum.

    Science.gov (United States)

    Wagner, Jeffrey C; Platt, Randall J; Goldfless, Stephen J; Zhang, Feng; Niles, Jacquin C

    2014-09-01

    Malaria is a major cause of global morbidity and mortality, and new strategies for treating and preventing this disease are needed. Here we show that the Streptococcus pyogenes Cas9 DNA endonuclease and single guide RNAs (sgRNAs) produced using T7 RNA polymerase (T7 RNAP) efficiently edit the Plasmodium falciparum genome. Targeting the genes encoding native knob-associated histidine-rich protein (kahrp) and erythrocyte binding antigen 175 (eba-175), we achieved high (≥ 50-100%) gene disruption frequencies within the usual time frame for generating transgenic parasites.

  13. Generating Mouse Models Using CRISPR-Cas9-Mediated Genome Editing.

    Science.gov (United States)

    Qin, Wenning; Kutny, Peter M; Maser, Richard S; Dion, Stephanie L; Lamont, Jeffrey D; Zhang, Yingfan; Perry, Greggory A; Wang, Haoyi

    2016-01-01

    The CRISPR-Cas9 system in bacteria and archaea has recently been exploited for genome editing in various model organisms, including mice. The CRISPR-Cas9 reagents can be delivered directly into the mouse zygote to derive a mutant animal carrying targeted genetic modifications. The major components of the system include the guide RNA, which provides target specificity, the Cas9 nuclease that creates the DNA double-strand break, and the donor oligonucleotide or plasmid carrying the intended mutation flanked by sequences homologous to the target site. Here we describe the general considerations and experimental protocols for creating genetically modified mice using the CRISPR-Cas9 system. PMID:26928663

  14. The CRISPR/Cas9 system for gene editing and its potential application in pain research

    Science.gov (United States)

    Sun, Linlin; Lutz, Brianna Marie; Tao, Yuan-Xiang

    2016-01-01

    The CRISPR/Cas9 system is a research hotspot in genome editing and regulation. Currently, it is used in genomic silencing and knock-in experiments as well as transcriptional activation and repression. This versatile system consists of two components: a guide RNA (gRNA) and a Cas9 nuclease. Recognition of a genomic DNA target is mediated through base pairing with a 20-base gRNA. The latter further recruits the Cas9 endonuclease protein to the target site and creates double-stranded breaks in the target DNA. Compared with traditional genome editing directed by DNA-binding protein domains, this short RNA-directed Cas9 endonuclease system is simple and easily programmable. Although this system may have off-target effects and in vivo delivery and immune challenges, researchers have employed this system in vivo to establish disease models, study specific gene functions under certain disease conditions, and correct genomic information for disease treatment. In regards to pain research, the CRISPR/Cas9 system may act as a novel tool in gene correction therapy for pain-associated hereditary diseases and may be a new approach for RNA-guided transcriptional activation or repression of pain-related genes. In addition, this system is also applied to loss-of-function mutations in pain-related genes and knockin of reporter genes or loxP tags at pain-related genomic loci. The CRISPR/Cas9 system will likely be carried out widely in both bench work and clinical settings in the pain field. PMID:27500183

  15. Building java programs (3rd edition)

    CERN Document Server

    Reges, Stuart

    2013-01-01

    Building Java Programs: A Back to Basics Approach, Third Edition, introduces novice programmers to basic constructs and common pitfalls by emphasizing the essentials of procedural programming, problem solving, and algorithmic reasoning. By using objects early to solve interesting problems and defining objects later in the course, Building Java Programs develops programming knowledge for a broad audience. NEW! This edition is available with MyProgrammingLab, an innovative online homework and assessment tool. Through the power of practice and immediate personalized feedback, MyProgrammingLab helps students fully grasp the logic, semantics, and syntax of programming. Note: If you are purchasing the standalone text or electronic version, MyProgrammingLab does not come automatically packaged with the text. To purchase MyProgrammingLab, please visit: myprogramminglab.com or you can purchase a package of the physical text + MyProgrammingLab by searching the Pearson Higher Education web site. MyProgrammi...

  16. Transportation Energy Data Book, Edition 18

    Energy Technology Data Exchange (ETDEWEB)

    Davis, Stacy C.

    1998-09-01

    The Transportation Energy Data Book: Edition 18 is a statistical compendium prepared and published by Oak Ridge National Laboratory (ORNL) under contract with the Office of Transportation Technologies in the Department of Energy (DOE). Designed for use as a desk-top reference, the data book represents an assembly and display of statistics and information that characterize transportation activity, and presents data on other factors that influence transportation energy use. The purpose of this document is to present relevant statistical data in the form of tables and graphs. This edition of the Data Book has 11 chapters which focus on various aspects of the transportation industry. Chapter 1 focuses on petroleum; Chapter 2 - energy Chapter 3 - emissions; Chapter 4 - transportation and the economy; Chapter 5 - highway vehicles; Chapter 6 - Light vehicles; Chapter 7 - heavy vehicles; Chapter 8 - alternative fuel vehicles; Chapter 9 - fleet vehicles; Chapter 10 - household vehicles; and Chapter 11 - nonhighway modes. The sources used represent the latest available data.

  17. Motion Editing for Time-Varying Mesh

    Science.gov (United States)

    Xu, Jianfeng; Yamasaki, Toshihiko; Aizawa, Kiyoharu

    2008-12-01

    Recently, time-varying mesh (TVM), which is composed of a sequence of mesh models, has received considerable interest due to its new and attractive functions such as free viewpoint and interactivity. TVM captures the dynamic scene of the real world from multiple synchronized cameras. However, it is expensive and time consuming to generate a TVM sequence. In this paper, an editing system is presented to reuse the original data, which reorganizes the motions to obtain a new sequence based on the user requirements. Hierarchical motion structure is observed and parsed in TVM sequences. Then, the representative motions are chosen into a motion database, where a motion graph is constructed to connect those motions with smooth transitions. After the user selects some desired motions from the motion database, the best paths are searched by a modified Dijkstra algorithm to achieve a new sequence. Our experimental results demonstrate that the edited sequences are natural and smooth.

  18. Motion Editing for Time-Varying Mesh

    Directory of Open Access Journals (Sweden)

    Kiyoharu Aizawa

    2008-08-01

    Full Text Available Recently, time-varying mesh (TVM, which is composed of a sequence of mesh models, has received considerable interest due to its new and attractive functions such as free viewpoint and interactivity. TVM captures the dynamic scene of the real world from multiple synchronized cameras. However, it is expensive and time consuming to generate a TVM sequence. In this paper, an editing system is presented to reuse the original data, which reorganizes the motions to obtain a new sequence based on the user requirements. Hierarchical motion structure is observed and parsed in TVM sequences. Then, the representative motions are chosen into a motion database, where a motion graph is constructed to connect those motions with smooth transitions. After the user selects some desired motions from the motion database, the best paths are searched by a modified Dijkstra algorithm to achieve a new sequence. Our experimental results demonstrate that the edited sequences are natural and smooth.

  19. Engineering Delivery Vehicles for Genome Editing.

    Science.gov (United States)

    Nelson, Christopher E; Gersbach, Charles A

    2016-06-01

    The field of genome engineering has created new possibilities for gene therapy, including improved animal models of disease, engineered cell therapies, and in vivo gene repair. The most significant challenge for the clinical translation of genome engineering is the development of safe and effective delivery vehicles. A large body of work has applied genome engineering to genetic modification in vitro, and clinical trials have begun using cells modified by genome editing. Now, promising preclinical work is beginning to apply these tools in vivo. This article summarizes the development of genome engineering platforms, including meganucleases, zinc finger nucleases, TALENs, and CRISPR/Cas9, and their flexibility for precise genetic modifications. The prospects for the development of safe and effective viral and nonviral delivery vehicles for genome editing are reviewed, and promising advances in particular therapeutic applications are discussed.

  20. Video summarization and semantics editing tools

    Science.gov (United States)

    Xu, Li-Qun; Zhu, Jian; Stentiford, Fred

    2001-01-01

    This paper describes a video summarization and semantics editing tool that is suited for content-based video indexing and retrieval with appropriate human operator assistance. The whole system has been designed with a clear focus on the extraction and exploitation of motion information inherent in the dynamic video scene. The dominant motion information has ben used explicitly for shot boundary detection, camera motion characterization, visual content variations description, and for key frame extraction. Various contributions have been made to ensure that the system works robustly with complex scenes and across different media types. A window-based graphical user interface has been designed to make the task very easy for interactive analysis and editing of semantic events and episode where appropriate.