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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

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  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. DHX9 suppresses RNA processing defects originating from the Alu invasion of the human genome.

    Science.gov (United States)

    Aktaş, Tuğçe; Avşar Ilık, İbrahim; Maticzka, Daniel; Bhardwaj, Vivek; Pessoa Rodrigues, Cecilia; Mittler, Gerhard; Manke, Thomas; Backofen, Rolf; Akhtar, Asifa

    2017-04-06

    Transposable elements are viewed as 'selfish genetic elements', yet they contribute to gene regulation and genome evolution in diverse ways. More than half of the human genome consists of transposable elements. Alu elements belong to the short interspersed nuclear element (SINE) family of repetitive elements, and with over 1 million insertions they make up more than 10% of the human genome. Despite their abundance and the potential evolutionary advantages they confer, Alu elements can be mutagenic to the host as they can act as splice acceptors, inhibit translation of mRNAs and cause genomic instability. Alu elements are the main targets of the RNA-editing enzyme ADAR and the formation of Alu exons is suppressed by the nuclear ribonucleoprotein HNRNPC, but the broad effect of massive secondary structures formed by inverted-repeat Alu elements on RNA processing in the nucleus remains unknown. Here we show that DHX9, an abundant nuclear RNA helicase, binds specifically to inverted-repeat Alu elements that are transcribed as parts of genes. Loss of DHX9 leads to an increase in the number of circular-RNA-producing genes and amount of circular RNAs, translational repression of reporters containing inverted-repeat Alu elements, and transcriptional rewiring (the creation of mostly nonsensical novel connections between exons) of susceptible loci. Biochemical purifications of DHX9 identify the interferon-inducible isoform of ADAR (p150), but not the constitutively expressed ADAR isoform (p110), as an RNA-independent interaction partner. Co-depletion of ADAR and DHX9 augments the double-stranded RNA accumulation defects, leading to increased circular RNA production, revealing a functional link between these two enzymes. Our work uncovers an evolutionarily conserved function of DHX9. We propose that it acts as a nuclear RNA resolvase that neutralizes the immediate threat posed by transposon insertions and allows these elements to evolve as tools for the post

  6. Identification of RNA polymerase III-transcribed Alu loci by computational screening of RNA-Seq data.

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    Conti, Anastasia; Carnevali, Davide; Bollati, Valentina; Fustinoni, Silvia; Pellegrini, Matteo; Dieci, Giorgio

    2015-01-01

    Of the ∼ 1.3 million Alu elements in the human genome, only a tiny number are estimated to be active in transcription by RNA polymerase (Pol) III. Tracing the individual loci from which Alu transcripts originate is complicated by their highly repetitive nature. By exploiting RNA-Seq data sets and unique Alu DNA sequences, we devised a bioinformatic pipeline allowing us to identify Pol III-dependent transcripts of individual Alu elements. When applied to ENCODE transcriptomes of seven human cell lines, this search strategy identified ∼ 1300 Alu loci corresponding to detectable transcripts, with ∼ 120 of them expressed in at least three cell lines. In vitro transcription of selected Alus did not reflect their in vivo expression properties, and required the native 5'-flanking region in addition to internal promoter. We also identified a cluster of expressed AluYa5-derived transcription units, juxtaposed to snaR genes on chromosome 19, formed by a promoter-containing left monomer fused to an Alu-unrelated downstream moiety. Autonomous Pol III transcription was also revealed for Alus nested within Pol II-transcribed genes. The ability to investigate Alu transcriptomes at single-locus resolution will facilitate both the identification of novel biologically relevant Alu RNAs and the assessment of Alu expression alteration under pathological conditions.

  7. REDIdb: the RNA editing database.

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    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.

  8. Caractérisation de l'expression des éléments Alu et du phénomène d'édition de l'ARN chez l'humain et la souris

    OpenAIRE

    2012-01-01

    The Alu repeats comprise more than 10% of the human genome. They spread in the genome by retrotransposition. As a response to this invasion, organisms developed mechanisms to preserve the integrity of their genome, such as RNA editing. The most abundant type of editing in mammals is A-to-I editing where the ADAR proteins transform adenosine into inosine and targets mainly Alu elements in human. Editing of the Alu elements leads to their sequestration in the nucleus and mutates their internal ...

  9. Investigating RNA editing factors from trypanosome mitochondria

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    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

  10. RNA Editing in Plant Mitochondria

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    Hiesel, Rudolf; Wissinger, Bernd; Schuster, Wolfgang; Brennicke, Axel

    1989-12-01

    Comparative sequence analysis of genomic and complementary DNA clones from several mitochondrial genes in the higher plant Oenothera revealed nucleotide sequence divergences between the genomic and the messenger RNA-derived sequences. These sequence alterations could be most easily explained by specific post-transcriptional nucleotide modifications. Most of the nucleotide exchanges in coding regions lead to altered codons in the mRNA that specify amino acids better conserved in evolution than those encoded by the genomic DNA. Several instances show that the genomic arginine codon CGG is edited in the mRNA to the tryptophan codon TGG in amino acid positions that are highly conserved as tryptophan in the homologous proteins of other species. This editing suggests that the standard genetic code is used in plant mitochondria and resolves the frequent coincidence of CGG codons and tryptophan in different plant species. The apparently frequent and non-species-specific equivalency of CGG and TGG codons in particular suggests that RNA editing is a common feature of all higher plant mitochondria.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. RNA Editing and Its Molecular Mechanism in Plant Organelles

    OpenAIRE

    2016-01-01

    RNA editing by cytidine (C) to uridine (U) conversions is widespread in plant mitochondria and chloroplasts. In some plant taxa, “reverse” U-to-C editing also occurs. However, to date, no instance of RNA editing has yet been reported in green algae and the complex thalloid liverworts. RNA editing may have evolved in early land plants 450 million years ago. However, in some plant species, including the liverwort, Marchantia polymorpha, editing may have been lost during evolution. Most RNA edit...

  16. The art of editing RNA structural alignments

    DEFF Research Database (Denmark)

    Andersen, Ebbe Sloth

    2014-01-01

    Manual editing of RNA structural alignments may be considered more art than science, since it still requires an expert biologist to take multiple levels of information into account and be slightly creative when constructing high-quality alignments. Even though the task is rather tedious, it is re......Manual editing of RNA structural alignments may be considered more art than science, since it still requires an expert biologist to take multiple levels of information into account and be slightly creative when constructing high-quality alignments. Even though the task is rather tedious...

  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. Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition.

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    Madina, Bhaskara R; Kumar, Vikas; Metz, Richard; Mooers, Blaine H M; Bundschuh, Ralf; Cruz-Reyes, Jorge

    2014-07-01

    Mitochondrial mRNAs in kinetoplastids require extensive U-insertion/deletion editing that progresses 3'-to-5' in small blocks, each directed by a guide RNA (gRNA), and exhibits substrate and developmental stage-specificity by unsolved mechanisms. Here, we address compositionally related factors, collectively known as the mitochondrial RNA-binding complex 1 (MRB1) or gRNA-binding complex (GRBC), that contain gRNA, have a dynamic protein composition, and transiently associate with several mitochondrial factors including RNA editing core complexes (RECC) and ribosomes. MRB1 controls editing by still unknown mechanisms. We performed the first next-generation sequencing study of native subcomplexes of MRB1, immunoselected via either RNA helicase 2 (REH2), that binds RNA and associates with unwinding activity, or MRB3010, that affects an early editing step. The particles contain either REH2 or MRB3010 but share the core GAP1 and other proteins detected by RNA photo-crosslinking. Analyses of the first editing blocks indicate an enrichment of several initiating gRNAs in the MRB3010-purified complex. Our data also indicate fast evolution of mRNA 3' ends and strain-specific alternative 3' editing within 3' UTR or C-terminal protein-coding sequence that could impact mitochondrial physiology. Moreover, we found robust specific copurification of edited and pre-edited mRNAs, suggesting that these particles may bind both mRNA and gRNA editing substrates. We propose that multiple subcomplexes of MRB1 with different RNA/protein composition serve as a scaffold for specific assembly of editing substrates and RECC, thereby forming the editing holoenzyme. The MRB3010-subcomplex may promote early editing through its preferential recruitment of initiating gRNAs.

  2. 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.

  3. 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/.

  4. Ebola virus RNA editing depends on the primary editing site sequence and an upstream secondary structure.

    Directory of Open Access Journals (Sweden)

    Masfique Mehedi

    Full Text Available Ebolavirus (EBOV, the causative agent of a severe hemorrhagic fever and a biosafety level 4 pathogen, increases its genome coding capacity by producing multiple transcripts encoding for structural and nonstructural glycoproteins from a single gene. This is achieved through RNA editing, during which non-template adenosine residues are incorporated into the EBOV mRNAs at an editing site encoding for 7 adenosine residues. However, the mechanism of EBOV RNA editing is currently not understood. In this study, we report for the first time that minigenomes containing the glycoprotein gene editing site can undergo RNA editing, thereby eliminating the requirement for a biosafety level 4 laboratory to study EBOV RNA editing. Using a newly developed dual-reporter minigenome, we have characterized the mechanism of EBOV RNA editing, and have identified cis-acting sequences that are required for editing, located between 9 nt upstream and 9 nt downstream of the editing site. Moreover, we show that a secondary structure in the upstream cis-acting sequence plays an important role in RNA editing. EBOV RNA editing is glycoprotein gene-specific, as a stretch encoding for 7 adenosine residues located in the viral polymerase gene did not serve as an editing site, most likely due to an absence of the necessary cis-acting sequences. Finally, the EBOV protein VP30 was identified as a trans-acting factor for RNA editing, constituting a novel function for this protein. Overall, our results provide novel insights into the RNA editing mechanism of EBOV, further understanding of which might result in novel intervention strategies against this viral pathogen.

  5. A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing.

    Science.gov (United States)

    Fukuda, Masatora; Kurihara, Kei; Tanaka, Yasuyoshi; Deshimaru, Masanobu

    2012-09-01

    Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo.

  6. The emerging role of RNA editing in plasticity.

    Science.gov (United States)

    Rosenthal, Joshua J C

    2015-06-01

    All true metazoans modify their RNAs by converting specific adenosine residues to inosine. Because inosine binds to cytosine, it is a biological mimic for guanosine. This subtle change, termed RNA editing, can have diverse effects on various RNA-mediated cellular pathways, including RNA interference, innate immunity, retrotransposon defense and messenger RNA recoding. Because RNA editing can be regulated, it is an ideal tool for increasing genetic diversity, adaptation and environmental acclimation. This review will cover the following themes related to RNA editing: (1) how it is used to modify different cellular RNAs, (2) how frequently it is used by different organisms to recode mRNA, (3) how specific recoding events regulate protein function, (4) how it is used in adaptation and (5) emerging evidence that it can be used for acclimation. Organismal biologists with an interest in adaptation and acclimation, but with little knowledge of RNA editing, are the intended audience.

  7. Putative impact of RNA editing on drug discovery.

    Science.gov (United States)

    Decher, Niels; Netter, Michael F; Streit, Anne K

    2013-01-01

    Virtually all organisms use RNA editing as a powerful post-transcriptional mechanism to recode genomic information and to increase functional protein diversity. The enzymatic editing of pre-mRNA by ADARs and CDARs is known to change the functional properties of neuronal receptors and ion channels regulating cellular excitability. However, RNA editing is also an important mechanism for genes expressed outside the brain. The fact that RNA editing breaks the 'one gene encodes one protein' hypothesis is daunting for scientists and a probable drawback for drug development, as scientists might search for drugs targeting the 'wrong' protein. This possible difficulty for drug discovery and development became more evident from recent publications, describing that RNA editing events have profound impact on the pharmacology of some common drug targets. These recent studies highlight that RNA editing can cause massive discrepancies between the in vitro and in vivo pharmacology. Here, we review the putative impact of RNA editing on drug discovery, as RNA editing has to be considered before using high-throughput screens, rational drug design or choosing the right model organism for target validation.

  8. Comparative RNA editing in autistic and neurotypical cerebella.

    Science.gov (United States)

    Eran, A; Li, J B; Vatalaro, K; McCarthy, J; Rahimov, F; Collins, C; Markianos, K; Margulies, D M; Brown, E N; Calvo, S E; Kohane, I S; Kunkel, L M

    2013-09-01

    Adenosine-to-inosine (A-to-I) RNA editing is a neurodevelopmentally regulated epigenetic modification shown to modulate complex behavior in animals. Little is known about human A-to-I editing, but it is thought to constitute one of many molecular mechanisms connecting environmental stimuli and behavioral outputs. Thus, comprehensive exploration of A-to-I RNA editing in human brains may shed light on gene-environment interactions underlying complex behavior in health and disease. Synaptic function is a main target of A-to-I editing, which can selectively recode key amino acids in synaptic genes, directly altering synaptic strength and duration in response to environmental signals. Here, we performed a high-resolution survey of synaptic A-to-I RNA editing in a human population, and examined how it varies in autism, a neurodevelopmental disorder in which synaptic abnormalities are a common finding. Using ultra-deep (>1000 × ) sequencing, we quantified the levels of A-to-I editing of 10 synaptic genes in postmortem cerebella from 14 neurotypical and 11 autistic individuals. A high dynamic range of editing levels was detected across individuals and editing sites, from 99.6% to below detection limits. In most sites, the extreme ends of the population editing distributions were individuals with autism. Editing was correlated with isoform usage, clusters of correlated sites were identified, and differential editing patterns examined. Finally, a dysfunctional form of the editing enzyme adenosine deaminase acting on RNA B1 was found more commonly in postmortem cerebella from individuals with autism. These results provide a population-level, high-resolution view of A-to-I RNA editing in human cerebella and suggest that A-to-I editing of synaptic genes may be informative for assessing the epigenetic risk for autism.

  9. Selectively Constrained RNA Editing Regulation Crosstalks with piRNA Biogenesis in Primates.

    Science.gov (United States)

    Yang, Xin-Zhuang; Chen, Jia-Yu; Liu, Chu-Jun; Peng, Jiguang; Wee, Yin Rei; Han, Xiaorui; Wang, Chenqu; Zhong, Xiaoming; Shen, Qing Sunny; Liu, Hsuan; Cao, Huiqing; Chen, Xiao-Wei; Tan, Bertrand Chin-Ming; Li, Chuan-Yun

    2015-12-01

    Although millions of RNA editing events have been reported to modify hereditary information across the primate transcriptome, evidence for their functional significance remains largely elusive, particularly for the vast majority of editing sites in noncoding regions. Here, we report a new mechanism for the functionality of RNA editing-a crosstalk with PIWI-interacting RNA (piRNA) biogenesis. Exploiting rhesus macaque as an emerging model organism closely related to human, in combination with extensive genome and transcriptome sequencing in seven tissues of the same animal, we deciphered accurate RNA editome across both long transcripts and the piRNA species. Superimposing and comparing these two distinct RNA editome profiles revealed 4,170 editing-bearing piRNA variants, or epiRNAs, that primarily derived from edited long transcripts. These epiRNAs represent distinct entities that evidence an intersection between RNA editing regulations and piRNA biogenesis. Population genetics analyses in a macaque population of 31 independent animals further demonstrated that the epiRNA-associated RNA editing is maintained by purifying selection, lending support to the functional significance of this crosstalk in rhesus macaque. Correspondingly, these findings are consistent in human, supporting the conservation of this mechanism during the primate evolution. Overall, our study reports the earliest lines of evidence for a crosstalk between selectively constrained RNA editing regulation and piRNA biogenesis, and further illustrates that such an interaction may contribute substantially to the diversification of the piRNA repertoire in primates.

  10. Uncovering RNA editing sites in long non-coding RNAs

    Directory of Open Access Journals (Sweden)

    Ernesto ePicardi

    2014-12-01

    Full Text Available RNA editing is an important co/post-transcriptional molecular process able to modify RNAs by nucleotide insertions/deletions or substitutions. In human, the most common RNA editing event involves the deamination of adenosine (A into inosine (I through the adenosine deaminase acting on RNA (ADAR proteins. Although A-to-I editing can occur in both coding and non coding RNAs, recent findings, based on RNA-seq experiments, have clearly demonstrated that a large fraction of RNA editing events alter non-coding RNAs sequences including untranslated regions of mRNAs (UTRs, introns, long non-coding RNAs (lncRNA and low molecular weight RNAs (tRNA, miRNAs and others. An accurate detection of A-to-I events occurring in non-coding RNAs is of utmost importance to clarify yet unknown functional roles of RNA editing in the context of gene expression regulation and maintenance of cell homeostasis. In the last few years, massive transcriptome sequencing has been employed to identify putative RNA editing changes at genome scale. Despite several efforts, the computational prediction of A-to-I sites in complete eukaryotic genomes is yet a challenging task. We have recently developed a software package, called REDItools, in order to simplify the detection of RNA editing events from deep sequencing data. In the present work, we show the potential of our tools in recovering A-to-I candidates from RNA-Seq experiments as well as guidelines to improve the RNA editing detection in non-coding RNAs, with specific attention to the lncRNAs.

  11. PREPACT 2.0: Predicting C-to-U and U-to-C RNA Editing in Organelle Genome Sequences with Multiple References and Curated RNA Editing Annotation

    OpenAIRE

    2013-01-01

    RNA editing is vast in some genetic systems, with up to thousands of targeted C-to-U and U-to-C substitutions in mitochondria and chloroplasts of certain plants. Efficient prognoses of RNA editing in organelle genomes will help to reveal overlooked cases of editing. We present PREPACT 2.0 (http://www.prepact.de) with numerous enhancements of our previously developed Plant RNA Editing Prediction & Analysis Computer Tool. Reference organelle transcriptomes for editing prediction have been exten...

  12. 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......RNA editing is a post-transcriptional event that recodes hereditary information. Here we describe a comprehensive profile of the RNA editome of a male Han Chinese individual based on analysis of ∼767 million sequencing reads from poly(A)(+), poly(A)(-) and small RNA samples. We developed...... 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...

  13. 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.

  14. Selectively Constrained RNA Editing Regulation Crosstalks with piRNA Biogenesis in Primates

    OpenAIRE

    2015-01-01

    Although millions of RNA editing events have been reported to modify hereditary information across the primate transcriptome, evidence for their functional significance remains largely elusive, particularly for the vast majority of editing sites in noncoding regions. Here, we report a new mechanism for the functionality of RNA editing—a crosstalk with PIWI-interacting RNA (piRNA) biogenesis. Exploiting rhesus macaque as an emerging model organism closely related to human, in combination with ...

  15. Construction of a guide-RNA for site-directed RNA mutagenesis utilising intracellular A-to-I RNA editing

    Science.gov (United States)

    Fukuda, Masatora; Umeno, Hiromitsu; Nose, Kanako; Nishitarumizu, Azusa; Noguchi, Ryoma; Nakagawa, Hiroyuki

    2017-01-01

    As an alternative to DNA mutagenesis, RNA mutagenesis can potentially become a powerful gene-regulation method for fundamental research and applied life sciences. Adenosine-to-inosine (A-to-I) RNA editing alters genetic information at the transcript level and is an important biological process that is commonly conserved in metazoans. Therefore, a versatile RNA-mutagenesis method can be achieved by utilising the intracellular RNA-editing mechanism. Here, we report novel guide RNAs capable of inducing A-to-I mutations by guiding the editing enzyme, human adenosine deaminase acting on RNA (ADAR). These guide RNAs successfully introduced A-to-I mutations into the target-site, which was determined by the reprogrammable antisense region. In ADAR2-over expressing cells, site-directed RNA editing could also be performed by simply introducing the guide RNA. Our guide RNA framework provides basic insights into establishing a generally applicable RNA-mutagenesis method. PMID:28148949

  16. A single alteration 20 nt 5′ to an editing target inhibits chloroplast RNA editing in vivo

    OpenAIRE

    2001-01-01

    Transcripts of typical dicot plant plastid genes undergo C→U RNA editing at approximately 30 locations, but there is no consensus sequence surrounding the C targets of editing. The cis-acting elements required for editing of the C located at tobacco rpoB editing site II were investigated by introducing translatable chimeric minigenes containing sequence –20 to +6 surrounding the C target of editing. When the –20 to +6 sequence specified by the homologous region pre...

  17. 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.

  18. Trans and cis factors affecting A-to-I RNA editing efficiency of a noncoding editing target in C. elegans.

    Science.gov (United States)

    Washburn, Michael C; Hundley, Heather A

    2016-05-01

    Adenosine-to-inosine RNA editing by ADARs affects thousands of adenosines in an organism's transcriptome. However, adenosines are not edited at equal levels nor do these editing levels correlate well with ADAR expression levels. Therefore, additional mechanisms are utilized by the cell to dictate the editing efficiency at a given adenosine. To examine cis-and trans-acting factors that regulate A-to-I editing levels specifically in neural cells, we utilized the model organism Caenorhabditis elegans We demonstrate that a double-stranded RNA (dsRNA) binding protein, ADR-1, inhibits editing in neurons, which is largely masked when examining editing levels from whole animals. Furthermore, expression of ADR-1 and mRNA expression of the editing target can act synergistically to regulate editing efficiency. In addition, we identify a dsRNA region within the Y75B8A.83' UTR that acts as acis-regulatory element by enhancing ADR-2 editing efficiency. Together, this work identifies mechanisms that regulate editing efficiency of noncoding A-to-I editing sites, which comprise the largest class of ADAR targets.

  19. A survey of genomic traces reveals a common sequencing error, RNA editing, and DNA editing.

    Directory of Open Access Journals (Sweden)

    Alexander Wait Zaranek

    2010-05-01

    Full Text Available While it is widely held that an organism's genomic information should remain constant, several protein families are known to modify it. Members of the AID/APOBEC protein family can deaminate DNA. Similarly, members of the ADAR family can deaminate RNA. Characterizing the scope of these events is challenging. Here we use large genomic data sets, such as the two billion sequences in the NCBI Trace Archive, to look for clusters of mismatches of the same type, which are a hallmark of editing events caused by APOBEC3 and ADAR. We align 603,249,815 traces from the NCBI trace archive to their reference genomes. In clusters of mismatches of increasing size, at least one systematic sequencing error dominates the results (G-to-A. It is still present in mismatches with 99% accuracy and only vanishes in mismatches at 99.99% accuracy or higher. The error appears to have entered into about 1% of the HapMap, possibly affecting other users that rely on this resource. Further investigation, using stringent quality thresholds, uncovers thousands of mismatch clusters with no apparent defects in their chromatograms. These traces provide the first reported candidates of endogenous DNA editing in human, further elucidating RNA editing in human and mouse and also revealing, for the first time, extensive RNA editing in Xenopus tropicalis. We show that the NCBI Trace Archive provides a valuable resource for the investigation of the phenomena of DNA and RNA editing, as well as setting the stage for a comprehensive mapping of editing events in large-scale genomic datasets.

  20. Group II intron RNA catalysis of progressive nucleotide insertion: a model for RNA editing.

    Science.gov (United States)

    Mueller, M W; Hetzer, M; Schweyen, R J

    1993-08-20

    The self-splicing bl1 intron lariat from mitochondria of Saccharomyces cerevisiae catalyzed the insertion of nucleotidyl monomers derived from the 3' end of a donor RNA into an acceptor RNA in a 3' to 5' direction in vitro. In this catalyzed reaction, the site specificity provided by intermolecular base pair interactions, the formation of chimeric intermediates, the polarity of the nucleotidyl insertion, and its reversibility all resemble such properties in previously proposed models of RNA editing in kinetoplastid mitochondria. These results suggest that RNA editing occurs by way of a concerted, two-step transesterification mechanism and that RNA splicing and RNA editing might be prebiotically related mechanisms; possibly, both evolved from a primordial demand for self-replication.

  1. 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.

  2. 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.

  3. 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 ...

  4. 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.

  5. A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing

    OpenAIRE

    Fukuda, Masatora; Kurihara, Kei; Tanaka, Yasuyoshi; Deshimaru, Masanobu

    2012-01-01

    Engineered site-specific RNA cleavage is widely used for gene regulation, RNA mapping, and synthetic RNA production. Here the authors extend the range of engineered recognition selectivity to include cleavage of sequence motifs containing naturally occurring base modifications. They describe and implement a designer hammerhead ribozyme that cleaves a target sequence 1 nt from a site of adenosine to inosine (A-to-I) or cytosine to uracil (C-to-U) editing in synthetic or physiological mRNA cont...

  6. Cas9-Guide RNA Directed Genome Editing in Soybean.

    Science.gov (United States)

    Li, Zhongsen; Liu, Zhan-Bin; Xing, Aiqiu; Moon, Bryan P; Koellhoffer, Jessica P; Huang, Lingxia; Ward, R Timothy; Clifton, Elizabeth; Falco, S Carl; Cigan, A Mark

    2015-10-01

    Recently discovered bacteria and archaea adaptive immune system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) endonuclease has been explored in targeted genome editing in different species. Streptococcus pyogenes Cas9-guide RNA (gRNA) was successfully applied to generate targeted mutagenesis, gene integration, and gene editing in soybean (Glycine max). Two genomic sites, DD20 and DD43 on chromosome 4, were mutagenized with frequencies of 59% and 76%, respectively. Sequencing randomly selected transgenic events confirmed that the genome modifications were specific to the Cas9-gRNA cleavage sites and consisted of small deletions or insertions. Targeted gene integrations through homology-directed recombination were detected by border-specific polymerase chain reaction analysis for both sites at callus stage, and one DD43 homology-directed recombination event was transmitted to T1 generation. T1 progenies of the integration event segregated according to Mendelian laws and clean homozygous T1 plants with the donor gene precisely inserted at the DD43 target site were obtained. The Cas9-gRNA system was also successfully applied to make a directed P178S mutation of acetolactate synthase1 gene through in planta gene editing.

  7. 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.

  8. 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.

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

    OpenAIRE

    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 t...

  10. CURE-Chloroplast: A chloroplast C-to-U RNA editing predictor for seed plants

    OpenAIRE

    2009-01-01

    Abstract Background RNA editing is a type of post-transcriptional modification of RNA and belongs to the class of mechanisms that contribute to the complexity of transcriptomes. C-to-U RNA editing is commonly observed in plant mitochondria and chloroplasts. The in vivo mechanism of recognizing C-to-U RNA editing sites is still unknown. In recent years, many efforts have been made to computationally predict C-to-U RNA editing sites in the mitochondria of seed plants, but there is still no algo...

  11. 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.

  12. Regulation of Gene Expression by DNA Methylation and RNA Editing in Animals

    DEFF Research Database (Denmark)

    Li, Qiye

    , there has been growing interest in exploring the modifications occurring at the RNA level, which can impact the fate and function of mRNA. One fascinating type of such modifications is RNA editing, which alters specific nucleotides in transcribed RNA and thus can produce transcripts that are not encoded...... (Heterocephalus glaber), a eusocial mammal living in cooperative colonies. Finally, I introduce a software package that I developed that is specifically designed for the genome-wide identification of RNA-editing sites in animals, with the ultimate aim of promoting the evolutionary and functional study of RNA...... editing in different species....

  13. 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.

  14. How do ADARs bind RNA? New protein-RNA structures illuminate substrate recognition by the RNA editing ADARs.

    Science.gov (United States)

    Thomas, Justin M; Beal, Peter A

    2017-02-20

    Deamination of adenosine in RNA to form inosine has wide ranging consequences on RNA function including amino acid substitution to give proteins not encoded in the genome. What determines which adenosines in an mRNA are subject to this modification reaction? The answer lies in an understanding of the mechanism and substrate recognition properties of adenosine deaminases that act on RNA (ADARs). Our recent publication of X-ray crystal structures of the human ADAR2 deaminase domain bound to RNA editing substrates shed considerable light on how the catalytic domains of these enzymes bind RNA and promote adenosine deamination. Here we review in detail the deaminase domain-RNA contact surfaces and present models of how full length ADARs, bearing double stranded RNA-binding domains (dsRBDs) and deaminase domains, could process naturally occurring substrate RNAs.

  15. 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.

  16. 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.

    2015-01-01

    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

  17. 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.

  18. Identification of an RNA element for specific coordination of A-to-I RNA editing on HTR2C pre-mRNA.

    Science.gov (United States)

    Fukuda, Masatora; Oyama, Yui; Nishitarumizu, Azusa; Omura, Miki; Nose, Kanako; Deshimaru, Masanobu

    2015-10-01

    Adenosine-to-Inosine (A-to-I) RNA editing is an intracellular mechanism in which inosine is specifically substituted against adenosine by the action of adenosine deaminases acting on RNA (ADARs). Serotonin 2C receptor (HTR2C) is encoded through combinatorial A-to-I RNA editing at recoding sites (A - E site) on its pre-mRNA. Although the efficiency of RNA editing at particular sites is known to be critical for modulating the serotonin signaling, the mechanistic details of site-specific editing on HTR2C pre-mRNA are not fully understood. Toward complete understanding of this mechanism, we discovered an RNA element, which coordinates site-specific RNA editing on HTR2C pre-mRNA by an in vitro editing assay and secondary structural analysis of mutant HTR2C RNA fragments. Our results showed that HTR2C pre-mRNA forms a characteristic structure, which was restricted by the internal loop and Watson-Crick base-pair interaction on site E, for intrinsic editing. We suggest that the internal loop would contribute toward adjusting the relative distance and/or geometry between the editing sites and the scaffold for ADAR.

  19. Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array

    NARCIS (Netherlands)

    Zetsche, Bernd; Heidenreich, Matthias; Mohanraju, Prarthana; Fedorova, Iana; Kneppers, Jeroen; Degennaro, Ellen M.; Winblad, Nerges; Choudhury, Sourav R.; Abudayyeh, Omar O.; Wu, Wen Y.; Oost, van der John

    2017-01-01

    Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to fo

  20. Deciphering the functions and regulation of brain-enriched A-to-I RNA editing.

    Science.gov (United States)

    Li, Jin Billy; Church, George M

    2013-11-01

    Adenosine-to-inosine (A-to-I) RNA editing, in which genomically encoded adenosine is changed to inosine in RNA, is catalyzed by adenosine deaminase acting on RNA (ADAR). This fine-tuning mechanism is critical during normal development and diseases, particularly in relation to brain functions. A-to-I RNA editing has also been hypothesized to be a driving force in human brain evolution. A large number of RNA editing sites have recently been identified, mostly as a result of the development of deep sequencing and bioinformatic analyses. Deciphering the functional consequences of RNA editing events is challenging, but emerging genome engineering approaches may expedite new discoveries. To understand how RNA editing is dynamically regulated, it is imperative to construct a spatiotemporal atlas at the species, tissue and cell levels. Future studies will need to identify the cis and trans regulatory factors that drive the selectivity and frequency of RNA editing. We anticipate that recent technological advancements will aid researchers in acquiring a much deeper understanding of the functions and regulation of RNA editing.

  1. 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

  2. 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.

  3. The RNA-Editing Enzyme ADAR1 Controls Innate Immune Responses to RNA

    Directory of Open Access Journals (Sweden)

    Niamh M. Mannion

    2014-11-01

    Full Text Available The ADAR RNA-editing enzymes deaminate adenosine bases to inosines in cellular RNAs. Aberrant interferon expression occurs in patients in whom ADAR1 mutations cause Aicardi-Goutières syndrome (AGS or dystonia arising from striatal neurodegeneration. Adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day E12.5. We demonstrate that Adar1 embryonic lethality is rescued to live birth in Adar1; Mavs double mutants in which the antiviral interferon induction response to cytoplasmic double-stranded RNA (dsRNA is prevented. Aberrant immune responses in Adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing-active cytoplasmic ADARs. We propose that inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions. Transfecting dsRNA oligonucleotides containing inosine-uracil base pairs into Adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response. ADAR1 mutations causing AGS affect the activity of the interferon-inducible cytoplasmic isoform more severely than the nuclear isoform.

  4. Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing

    OpenAIRE

    Fukuda, Masatora; Kurihara, Kei; Yamaguchi, Shota; Oyama, Yui; Deshimaru, Masanobu

    2014-01-01

    Adenosine-to-inosine RNA editing is an endogenous mechanism for regulating various biological processes. A method for site-specific and editing-state–dependent degradation of target RNA may be a powerful tool both for analyzing the mechanism of RNA editing and for regulating biological processes. In this paper, we describe a strategy for constructing a trans-acting hammerhead ribozyme that specifically cleaves target RNA dependent on the editing state at the specific site.

  5. 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.

  6. Condition-specific RNA editing in the coral symbiont Symbiodinium microadriaticum

    KAUST Repository

    Liew, Yi Jin

    2017-03-01

    RNA editing is a rare post-transcriptional event that provides cells with an additional level of gene expression regulation. It has been implicated in various processes including adaptation, viral defence and RNA interference; however, its potential role as a mechanism in acclimatization has just recently been recognised. Here, we show that RNA editing occurs in 1.6% of all nuclear-encoded genes of Symbiodinium microadriaticum, a dinoflagellate symbiont of reef-building corals. All base-substitution edit types were present, and statistically significant motifs were associated with three edit types. Strikingly, a subset of genes exhibited condition-specific editing patterns in response to different stressors that resulted in significant increases of non-synonymous changes. We posit that this previously unrecognised mechanism extends this organism’s capability to respond to stress beyond what is encoded by the genome. This in turn may provide further acclimatization capacity to these organisms, and by extension, their coral hosts.

  7. 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.

  8. In vitro RNA editing in plant mitochondria does not require added energy.

    Science.gov (United States)

    Takenaka, Mizuki; Verbitskiy, Daniil; van der Merwe, Johannes A; Zehrmann, Anja; Plessmann, Uwe; Urlaub, Henning; Brennicke, Axel

    2007-06-12

    RNA editing in flowering plant mitochondria is investigated by in vitro assays. These cauliflower mitochondrial lysates require added NTP or dNTP. We have now resolved the reason for this requirement to be the inhibition of the RNA binding activity of the glutamate dehydrogenases (GDH). Both GDH1 and GDH2 were identified in RNA-protein cross-links. The inhibition of in vitro RNA editing by GDH is confirmed by the ability of the GDH-specific herbicide phosphinothricin to substitute for NTP. NADH and NADPH, but not NAD or NADP, can also replace NTP, suggesting that the NAD(P)H-binding-pocket configuration of the GDH contacts the RNA. RNA editing in plant mitochondria is thus intrinsically independent of added energy in the form of NTP.

  9. Loss of matK RNA editing in seed plant chloroplasts

    Directory of Open Access Journals (Sweden)

    Maier Uwe G

    2009-08-01

    Full Text Available Abstract Background RNA editing in chloroplasts of angiosperms proceeds by C-to-U conversions at specific sites. Nuclear-encoded factors are required for the recognition of cis-elements located immediately upstream of editing sites. The ensemble of editing sites in a chloroplast genome differs widely between species, and editing sites are thought to evolve rapidly. However, large-scale analyses of the evolution of individual editing sites have not yet been undertaken. Results Here, we analyzed the evolution of two chloroplast editing sites, matK-2 and matK-3, for which DNA sequences from thousands of angiosperm species are available. Both sites are found in most major taxa, including deep-branching families such as the nymphaeaceae. However, 36 isolated taxa scattered across the entire tree lack a C at one of the two matK editing sites. Tests of several exemplary species from this in silico analysis of matK processing unexpectedly revealed that one of the two sites remain unedited in almost half of all species examined. A comparison of sequences between editors and non-editors showed that specific nucleotides co-evolve with the C at the matK editing sites, suggesting that these nucleotides are critical for editing-site recognition. Conclusion (i Both matK editing sites were present in the common ancestor of all angiosperms and have been independently lost multiple times during angiosperm evolution. (ii The editing activities corresponding to matK-2 and matK-3 are unstable. (iii A small number of third-codon positions in the vicinity of editing sites are selectively constrained independent of the presence of the editing site, most likely because of interacting RNA-binding proteins.

  10. RNA editing of the GABAA receptor α3 subunit alters the functional properties of recombinant receptors

    OpenAIRE

    Nimmich, Mitchell L.; Heidelberg, Laura S.; Fisher, Janet L.

    2009-01-01

    RNA editing provides a post-transcriptional mechanism to increase structural heterogeneity of gene products. Recently, the α3 subunit of the GABAA receptors has been shown to undergo RNA editing. As a result, a highly conserved isoleucine residue in the third transmembrane domain is replaced with a methionine. To determine the effect of this structural change on receptor function, we compared the GABA sensitivity, pharmacological properties and macroscopic kinetics of recombinant receptors co...

  11. 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...... in detailed data of both of these fine-tuning mechanisms in the embryonic development of the pig. Editing levels of transcripts examined are generally seen to increase through development, in agreement with editing of specific microRNA also examined in the Solexa sequencing study. Three studies examining...

  12. 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

  13. Extensive Mitochondrial mRNA Editing and Unusual Mitochondrial Genome Organization in Calcaronean Sponges.

    Science.gov (United States)

    Lavrov, Dennis V; Adamski, Marcin; Chevaldonné, Pierre; Adamska, Maja

    2016-01-11

    One of the unusual features of DNA-containing organelles in general and mitochondria in particular is the frequent occurrence of RNA editing [1]. The term "RNA editing" refers to a variety of mechanistically unrelated biochemical processes that alter RNA sequence during or after transcription [2]. The editing can be insertional, deletional, or substitutional and has been found in all major types of RNAs [3, 4]. Although mitochondrial mRNA editing is widespread in some eukaryotic lineages [5-7], it is rare in animals, with reported cases limited both in their scope and in phylogenetic distribution [8-11] (see also [12]). While analyzing genomic data from calcaronean sponges Sycon ciliatum and Leucosolenia complicata, we were perplexed by the lack of recognizable mitochondrial coding sequences. Comparison of genomic and transcriptomic data from these species revealed the presence of mitochondrial cryptogenes whose transcripts undergo extensive editing. This editing consisted of single or double uridylate (U) insertions in pre-existing short poly(U) tracts. Subsequent analysis revealed the presence of similar editing in Sycon coactum and the loss of editing in Petrobiona massiliana, a hypercalcified calcaronean sponge. In addition, mitochondrial genomes of at least some calcaronean sponges were found to have a highly unusual architecture, with nearly all genes located on individual and likely linear chromosomes. Phylogenetic analysis of mitochondrial coding sequences revealed accelerated rates of sequence evolution in this group. The latter observation presents a challenge for the mutational-hazard hypothesis [13], which posits that mRNA editing should not occur in lineages with an elevated mutation rate.

  14. 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.

  15. Abnormalities in A-to-I RNA editing patterns in CNS injuries correlate with dynamic changes in cell type composition

    Science.gov (United States)

    Gal-Mark, Nurit; Shallev, Lea; Sweetat, Sahar; Barak, Michal; Billy Li, Jin; Levanon, Erez Y.; Eisenberg, Eli; Behar, Oded

    2017-01-01

    Adenosine to Inosine (A-to-I) RNA editing is a co- or post-transcriptional mechanism that modifies genomically encoded nucleotides at the RNA level. A-to-I RNA editing is abundant in the brain, and altered editing levels have been reported in various neurological pathologies and following spinal cord injury (SCI). The prevailing concept is that the RNA editing process itself is dysregulated by brain pathologies. Here we analyzed recent RNA-seq data, and found that, except for few mammalian conserved editing sites, editing is significantly higher in neurons than in other cell populations of the brain. We studied A-to-I RNA editing in stab wound injury (SWI) and SCI models and showed that the apparent under-editing observed after injury correlates with an approximately 20% reduction in the relative density of neurons, due to cell death and immune cell infiltration that may account for the observed under-editing. Studies of neuronal and astrocyte cultures and a computational analysis of SCI RNA-seq data further supported the possibility that a reduction in neuronal density is responsible for alterations in the tissue-wide editing patterns upon injury. Thus, our data suggest that the case for a mechanistic linkage between A-to-I RNA editing and brain pathologies should be revisited. PMID:28266523

  16. Computational detection and functional analysis of human tissue-specific A-to-I RNA editing.

    Directory of Open Access Journals (Sweden)

    Tao He

    Full Text Available A-to-I RNA editing is a widespread post-transcriptional modification event in vertebrates. It could increase transcriptome and proteome diversity through recoding the genomic information and cross-linking other regulatory events, such as those mediated by alternative splicing, RNAi and microRNA (miRNA. Previous studies indicated that RNA editing can occur in a tissue-specific manner in response to the requirements of the local environment. We set out to systematically detect tissue-specific A-to-I RNA editing sites in 43 human tissues using bioinformatics approaches based on the Fisher's exact test and the Benjamini & Hochberg false discovery rate (FDR multiple testing correction. Twenty-three sites in total were identified to be tissue-specific. One of them resulted in an altered amino acid residue which may prevent the phosphorylation of PARP-10 and affect its activity. Eight and two tissue-specific A-to-I RNA editing sites were predicted to destroy putative exonic splicing enhancers (ESEs and exonic splicing silencers (ESSs, respectively. Brain-specific and ovary-specific A-to-I RNA editing sites were further verified by comparing the cDNA sequences with their corresponding genomic templates in multiple cell lines from brain, colon, breast, bone marrow, lymph, liver, ovary and kidney tissue. Our findings help to elucidate the role of A-to-I RNA editing in the regulation of tissue-specific development and function, and the approach utilized here can be broadened to study other types of tissue-specific substitution editing.

  17. 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

    2016-06-02

    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.

  18. A computational screen for site selective A-to-I editing detects novel sites in neuron specific Hu proteins

    Directory of Open Access Journals (Sweden)

    Furey Terrence S

    2010-01-01

    Full Text Available Abstract Background Several bioinformatic approaches have previously been used to find novel sites of ADAR mediated A-to-I RNA editing in human. These studies have discovered thousands of genes that are hyper-edited in their non-coding intronic regions, especially in alu retrotransposable elements, but very few substrates that are site-selectively edited in coding regions. Known RNA edited substrates suggest, however, that site selective A-to-I editing is particularly important for normal brain development in mammals. Results We have compiled a screen that enables the identification of new sites of site-selective editing, primarily in coding sequences. To avoid hyper-edited repeat regions, we applied our screen to the alu-free mouse genome. Focusing on the mouse also facilitated better experimental verification. To identify candidate sites of RNA editing, we first performed an explorative screen based on RNA structure and genomic sequence conservation. We further evaluated the results of the explorative screen by determining which transcripts were enriched for A-G mismatches between the genomic template and the expressed sequence since the editing product, inosine (I, is read as guanosine (G by the translational machinery. For expressed sequences, we only considered coding regions to focus entirely on re-coding events. Lastly, we refined the results from the explorative screen using a novel scoring scheme based on characteristics for known A-to-I edited sites. The extent of editing in the final candidate genes was verified using total RNA from mouse brain and 454 sequencing. Conclusions Using this method, we identified and confirmed efficient editing at one site in the Gabra3 gene. Editing was also verified at several other novel sites within candidates predicted to be edited. Five of these sites are situated in genes coding for the neuron-specific RNA binding proteins HuB and HuD.

  19. 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...... 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...... 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...

  20. 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.

  1. Comparative analysis of A-to-I editing in human and non-human primate brains reveals conserved patterns and context-dependent regulation of RNA editing.

    Science.gov (United States)

    O'Neil, Richard T; Wang, Xiaojing; Morabito, Michael V; Emeson, Ronald B

    2017-04-06

    A-to-I RNA editing is an important process for generating molecular diversity in the brain through modification of transcripts encoding several proteins important for neuronal signaling. We investigated the relationships between the extent of editing at multiple substrate transcripts (5HT2C, MGLUR4, CADPS, GLUR2, GLUR4, and GABRA3) in brain tissue obtained from adult humans and rhesus macaques. Several patterns emerged from these studies revealing conservation of editing across primate species. Additionally, variability in the human population allows us to make novel inferences about the co-regulation of editing at different editing sites and even across different brain regions.

  2. 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.

  3. RNA-guided genome editing in plants using a CRISPR-Cas system.

    Science.gov (United States)

    Xie, Kabin; Yang, Yinong

    2013-11-01

    Precise and straightforward methods to edit the plant genome are much needed for functional genomics and crop improvement. Recently, RNA-guided genome editing using bacterial Type II cluster regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) is emerging as an efficient tool for genome editing in microbial and animal systems. Here, we report the genome editing and targeted gene mutation in plants via the CRISPR-Cas9 system. Three guide RNAs (gRNAs) with a 20-22-nt seed region were designed to pair with distinct rice genomic sites which are followed by the protospacer-adjacent motif (PAM). The engineered gRNAs were shown to direct the Cas9 nuclease for precise cleavage at the desired sites and introduce mutation (insertion or deletion) by error-prone non-homologous end joining DNA repairing. By analyzing the RNA-guided genome-editing events, the mutation efficiency at these target sites was estimated to be 3-8%. In addition, the off-target effect of an engineered gRNA-Cas9 was found on an imperfectly paired genomic site, but it had lower genome-editing efficiency than the perfectly matched site. Further analysis suggests that mismatch position between gRNA seed and target DNA is an important determinant of the gRNA-Cas9 targeting specificity, and specific gRNAs could be designed to target more than 90% of rice genes. Our results demonstrate that the CRISPR-Cas system can be exploited as a powerful tool for gene targeting and precise genome editing in plants.

  4. A-to-I RNA editing: the "ADAR" side of human cancer.

    Science.gov (United States)

    Galeano, Federica; Tomaselli, Sara; Locatelli, Franco; Gallo, Angela

    2012-05-01

    Carcinogenesis is a complex, multi-stage process depending on both endogenous and exogenous factors. In the past years, DNA mutations provided important clues to the comprehension of the molecular pathways involved in numerous cancers. Recently, post-transcriptional modification events, such as RNA editing, are emerging as new players in several human diseases, including tumours. A-to-I RNA editing changes the nucleotide sequence of target RNAs, introducing A-to-I/G "mutations". Since ADAR enzymes catalyse this nucleotide conversion, their expression/activity is essential and finely regulated in normal cells. This review summarizes the available knowledge on A-to-I RNA editing in the cancer field, giving a new view on how ADARs may play a role in carcinogenesis.

  5. Inherited variants affecting RNA editing may contribute to ovarian cancer susceptibility

    DEFF Research Database (Denmark)

    Permuth, Jennifer B; Reid, Brett; Earp, Madalene

    2016-01-01

    RNA editing in mammals is a form of post-transcriptional modification in which adenosine is converted to inosine by the adenosine deaminases acting on RNA (ADAR) family of enzymes. Based on evidence of altered ADAR expression in epithelial ovarian cancers (EOC), we hypothesized that single nucleo......, including rs1127313 (G/A), a SNP in the 3' untranslated region. In summary, germline variation involving RNA editing genes may influence EOC susceptibility, warranting further investigation of inherited and acquired alterations affecting RNA editing.......RNA editing in mammals is a form of post-transcriptional modification in which adenosine is converted to inosine by the adenosine deaminases acting on RNA (ADAR) family of enzymes. Based on evidence of altered ADAR expression in epithelial ovarian cancers (EOC), we hypothesized that single...... nucleotide polymorphisms (SNPs) in ADAR genes modify EOC susceptibility, potentially by altering ovarian tissue gene expression. Using directly genotyped and imputed data from 10,891 invasive EOC cases and 21,693 controls, we evaluated the associations of 5,303 SNPs in ADAD1, ADAR, ADAR2, ADAR3, and SND1...

  6. Site-specific factor involved in the editing of the psbL mRNA in tobacco plastids.

    OpenAIRE

    1995-01-01

    In tobacco plastids, functional psbL mRNA is created by editing an ACG codon to an AUG translation initiation codon. To determine if editing may occur in a chimeric mRNA, the N-terminal part of psbL containing the editing site was translationally fused with the aadA and kan bacterial genes. The chimeric constructs were introduced into the tobacco plastid genome by targeted gene insertion. Editing of the chimeric mRNAs indicated that the 98 nt fragment spanning the psbL editing site contains a...

  7. 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.

  8. Integrative analyses of RNA editing, alternative splicing, and expression of young genes in human brain transcriptome by deep RNA sequencing.

    Science.gov (United States)

    Wu, Dong-Dong; Ye, Ling-Qun; Li, Yan; Sun, Yan-Bo; Shao, Yi; Chen, Chunyan; Zhu, Zhu; Zhong, Li; Wang, Lu; Irwin, David M; Zhang, Yong E; Zhang, Ya-Ping

    2015-08-01

    Next-generation RNA sequencing has been successfully used for identification of transcript assembly, evaluation of gene expression levels, and detection of post-transcriptional modifications. Despite these large-scale studies, additional comprehensive RNA-seq data from different subregions of the human brain are required to fully evaluate the evolutionary patterns experienced by the human brain transcriptome. Here, we provide a total of 6.5 billion RNA-seq reads from different subregions of the human brain. A significant correlation was observed between the levels of alternative splicing and RNA editing, which might be explained by a competition between the molecular machineries responsible for the splicing and editing of RNA. Young human protein-coding genes demonstrate biased expression to the neocortical and non-neocortical regions during evolution on the lineage leading to humans. We also found that a significantly greater number of young human protein-coding genes are expressed in the putamen, a tissue that was also observed to have the highest level of RNA-editing activity. The putamen, which previously received little attention, plays an important role in cognitive ability, and our data suggest a potential contribution of the putamen to human evolution.

  9. 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.

  10. 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.

  11. 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.

  12. RNA编辑研究进展%The Advances of RNA Editing

    Institute of Scientific and Technical Information of China (English)

    王兰

    2011-01-01

    RNA editing is a post-transcriptional process changing the identity of individual nucleotides in a transcript, and made the mature RNA was not completely alike the coding sequence of its template DNA.With further researching of molecular biology, some new RNA-editing types were found, and some of which showed RNA editing had some memory function.In this study, RNA editing was simply introduced including its types,and its mechanism, and biological function.%PNA编辑是近年来发现的一种转录后水平的修饰方式,通过改变单个核苷酸使得转录的成熟RNA与模板DNA的编码序列不完全一致.随着分子生物学的深入研究,一些新的RNA编辑类型不断被发现,有些编辑类型揭示RNA编辑具有一定的记忆功能.笔者AkRNA编辑的类型,RNA编辑的机制和生物学意义对RNA编辑进行简单的介绍.

  13. 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.

  14. Extensive and evolutionarily persistent mitochondrial tRNA editing in Velvet Worms (phylum Onychophora).

    Science.gov (United States)

    Segovia, Romulo; Pett, Walker; Trewick, Steve; Lavrov, Dennis V

    2011-10-01

    Mitochondrial genomes of onychophorans (velvet worms) present an interesting problem: Some previous studies reported them lacking several transfer RNA (tRNA) genes, whereas others found that all their tRNA genes were present but severely reduced. To resolve this discrepancy, we determined complete mitochondrial DNA (mtDNA) sequences of the onychophorans Oroperipatus sp. and Peripatoides sympatrica as well as cDNA sequences from 14 and 10 of their tRNAs, respectively. We show that tRNA genes in these genomes are indeed highly reduced and encode truncated molecules, which are restored to more conventional structures by extensive tRNA editing. During this editing process, up to 34 nucleotides are added to the tRNA sequences encoded in Oroperipatus sp. mtDNA, rebuilding the aminoacyl acceptor stem, the TΨC arm, and in some extreme cases, the variable arm and even a part of the anticodon stem. The editing is less extreme in P. sympatrica in which at least a part of the TΨC arm is always encoded in mtDNA. When the entire TΨC arm is added de novo in Oroperipatus sp., the sequence of this arm is either identical or similar among different tRNA species, yet the sequences show substantial variation for each tRNA. These observations suggest that the arm is rebuilt, at least in part, by a template-independent mechanism and argue against the alternative possibility that tRNA genes or their parts are imported from the nucleus. By contrast, the 3' end of the aminoacyl acceptor stem is likely restored by a template-dependent mechanism. The extreme tRNA editing reported here has been preserved for >140 My as it was found in both extant families of onychophorans. Furthermore, a similar type of tRNA editing may be present in several other groups of arthropods, which show a high degree of tRNA gene reduction in their mtDNA.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. Electroporation of DNA into Physarum polycephalum Mitochondria: Effects on Transcription and RNA Editing in Isolated Organelles

    Directory of Open Access Journals (Sweden)

    Jonatha M. Gott

    2016-12-01

    Full Text Available Mitochondrial RNAs in the acellular slime mold Physarum polycephalum contain nucleotides that are not encoded in the mitochondrial genes from which they are transcribed. These site-specific changes are quite extensive, comprising ~4% of the residues within mRNAs and ~2% of rRNAs and tRNAs. These “extra” nucleotides are added co-transcriptionally, but the means by which this is accomplished have not been elucidated. The cox1 mRNA also contains four sites of C to U changes, which occur post-transcriptionally, most likely via targeted deamination. The currently available in vitro systems for studying P. polycephalum editing are limited in that the template is the entire ~63,000 bp mitochondrial genome. This presents a significant challenge when trying to define the signals that specify editing sites. In an attempt to overcome this issue, a method for introducing DNA into isolated P. polycephalum mitochondria via electroporation has been developed. Exogenous DNA is expressed, but the transcripts synthesized from these templates are not edited under the conditions tested. However, transcripts derived from the mitochondrial genome are accurately edited after electroporation, indicating that the editing machinery is still functional. These findings suggest that this method may ultimately provide a feasible approach to elucidating editing signals.

  20. Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing.

    Science.gov (United States)

    Fukuda, Masatora; Kurihara, Kei; Yamaguchi, Shota; Oyama, Yui; Deshimaru, Masanobu

    2014-03-01

    Adenosine-to-inosine (A-to-I) RNA editing is an endogenous regulatory mechanism involved in various biological processes. Site-specific, editing-state-dependent degradation of target RNA may be a powerful tool both for analyzing the mechanism of RNA editing and for regulating biological processes. Previously, we designed an artificial hammerhead ribozyme (HHR) for selective, site-specific RNA cleavage dependent on the A-to-I RNA editing state. In the present work, we developed an improved strategy for constructing a trans-acting HHR that specifically cleaves target editing sites in the adenosine but not the inosine state. Specificity for unedited sites was achieved by utilizing a sequence encoding the intrinsic cleavage specificity of a natural HHR. We used in vitro selection methods in an HHR library to select for an extended HHR containing a tertiary stabilization motif that facilitates HHR folding into an active conformation. By using this method, we successfully constructed highly active HHRs with unedited-specific cleavage. Moreover, using HHR cleavage followed by direct sequencing, we demonstrated that this ribozyme could cleave serotonin 2C receptor (HTR2C) mRNA extracted from mouse brain, depending on the site-specific editing state. This unedited-specific cleavage also enabled us to analyze the effect of editing state at the E and C sites on editing at other sites by using direct sequencing for the simultaneous quantification of the editing ratio at multiple sites. Our approach has the potential to elucidate the mechanism underlying the interdependencies of different editing states in substrate RNA with multiple editing sites.

  1. 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.

  2. 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

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

    OpenAIRE

    Farboud, B; Meyer,BJ

    2015-01-01

    © 2015 by the Genetics Society of America.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 w...

  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. An Organelle RNA Recognition Motif Protein Is Required for Photosystem II Subunit psbF Transcript Editing1[OPEN

    Science.gov (United States)

    Lucas, Meriah K.

    2017-01-01

    Loss-of-function mutations in ORGANELLE RNA RECOGNITION MOTIF PROTEIN6 (ORRM6) result in the near absence of RNA editing of psbF-C77 and the reduction in accD-C794 editing in Arabidopsis (Arabidopsis thaliana). The orrm6 mutants have decreased levels of photosystem II (PSII) proteins, especially PsbF, lower PSII activity, pale green pigmentation, smaller leaf and plant sizes, and retarded growth. Stable expression of ORRM6 rescues the orrm6 editing defects and mutant phenotype. Unlike ORRM1, the other known ORRM plastid editing factor, ORRM6, does not contain RNA editing interacting protein/multiple organellar RNA editing factor (RIP/MORF) boxes, which are required for ORRM1 to interact with site-specific pentatricopeptide repeat protein editing factors. ORRM6 interacts with RIP1/MORF8, RIP2/MORF2, and RIP9/MORF9, known components of RNA editosomes. While some plastid RRM proteins are involved in other forms of RNA processing and translation, the primary function of ORRM6 is evidently to mediate psbF-C77 editing, like the essential site-specific pentatricopeptide repeat protein LOW PSII ACCUMULATION66. Stable expression in the orrm6 mutants of a nucleus-encoded, plastid-targeted PsbF protein from a psbF gene carrying a T at nucleotide 77 significantly increases leaf and plant sizes, chlorophyll content, and PSII activity. These transformants demonstrate that plastid RNA editing can be bypassed through the expression of nucleus-encoded, edited forms of plastid genes. PMID:28213559

  6. An Organelle RNA Recognition Motif Protein Is Required for Photosystem II Subunit psbF Transcript Editing.

    Science.gov (United States)

    Hackett, Justin B; Shi, Xiaowen; Kobylarz, Amy T; Lucas, Meriah K; Wessendorf, Ryan L; Hines, Kevin M; Bentolila, Stephane; Hanson, Maureen R; Lu, Yan

    2017-04-01

    Loss-of-function mutations in ORGANELLE RNA RECOGNITION MOTIF PROTEIN6 (ORRM6) result in the near absence of RNA editing of psbF-C77 and the reduction in accD-C794 editing in Arabidopsis (Arabidopsis thaliana). The orrm6 mutants have decreased levels of photosystem II (PSII) proteins, especially PsbF, lower PSII activity, pale green pigmentation, smaller leaf and plant sizes, and retarded growth. Stable expression of ORRM6 rescues the orrm6 editing defects and mutant phenotype. Unlike ORRM1, the other known ORRM plastid editing factor, ORRM6, does not contain RNA editing interacting protein/multiple organellar RNA editing factor (RIP/MORF) boxes, which are required for ORRM1 to interact with site-specific pentatricopeptide repeat protein editing factors. ORRM6 interacts with RIP1/MORF8, RIP2/MORF2, and RIP9/MORF9, known components of RNA editosomes. While some plastid RRM proteins are involved in other forms of RNA processing and translation, the primary function of ORRM6 is evidently to mediate psbF-C77 editing, like the essential site-specific pentatricopeptide repeat protein LOW PSII ACCUMULATION66. Stable expression in the orrm6 mutants of a nucleus-encoded, plastid-targeted PsbF protein from a psbF gene carrying a T at nucleotide 77 significantly increases leaf and plant sizes, chlorophyll content, and PSII activity. These transformants demonstrate that plastid RNA editing can be bypassed through the expression of nucleus-encoded, edited forms of plastid genes.

  7. 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

  8. Mutational analysis of Trypanosoma brucei RNA editing ligase reveals regions critical for interaction with KREPA2.

    Directory of Open Access Journals (Sweden)

    Vaibhav Mehta

    Full Text Available The Trypanosoma brucei parasite causes the vector-borne disease African sleeping sickness. Mitochondrial mRNAs of T. brucei undergo posttranscriptional RNA editing to make mature, functional mRNAs. The final step of this process is catalyzed by the essential ligase, T. brucei RNA Editing Ligase 1 (TbREL1 and the closely related T. brucei RNA Editing Ligase 2 (TbREL2. While other ligases such as T7 DNA ligase have both a catalytic and an oligonucleotide/oligosaccharide-binding (OB-fold domain, T. brucei RNA editing ligases contain only the catalytic domain. The OB-fold domain, which is required for interaction with the substrate RNA, is provided in trans by KREPA2 (for TbREL1 and KREPA1 (for TbREL2. KREPA2 enhancement of TbREL1 ligase activity is presumed to occur via an OB-fold-mediated increase in substrate specificity and catalysis. We characterized the interaction between TbREL1 and KREPA2 in vitro using full-length, truncated, and point-mutated ligases. As previously shown, our data indicate strong, specific stimulation of TbREL1 catalytic activity by KREPA2. We narrowed the region of contact to the final 59 C-terminal residues of TbREL1. Specifically, the TbREL1 C-terminal KWKE (441-444 sequence appear to coordinate the KREPA2-mediated enhancement of TbREL1 activities. N-terminal residues F206, T264 and Y275 are crucial for the overall activity of TbREL1, particularly for F206, a mutation of this residue also disrupts KREPA2 interaction. Thus, we have identified the critical TbREL1 regions and amino acids that mediate the KREPA2 interaction.

  9. 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.

  10. RNA editing differently affects protein-coding genes in D. melanogaster and H. sapiens.

    Science.gov (United States)

    Grassi, Luigi; Leoni, Guido; Tramontano, Anna

    2015-07-14

    When an RNA editing event occurs within a coding sequence it can lead to a different encoded amino acid. The biological significance of these events remains an open question: they can modulate protein functionality, increase the complexity of transcriptomes or arise from a loose specificity of the involved enzymes. We analysed the editing events in coding regions that produce or not a change in the encoded amino acid (nonsynonymous and synonymous events, respectively) in D. melanogaster and in H. sapiens and compared them with the appropriate random models. Interestingly, our results show that the phenomenon has rather different characteristics in the two organisms. For example, we confirm the observation that editing events occur more frequently in non-coding than in coding regions, and report that this effect is much more evident in H. sapiens. Additionally, in this latter organism, editing events tend to affect less conserved residues. The less frequently occurring editing events in Drosophila tend to avoid drastic amino acid changes. Interestingly, we find that, in Drosophila, changes from less frequently used codons to more frequently used ones are favoured, while this is not the case in H. sapiens.

  11. Alternative splicing and extensive RNA editing of human TPH2 transcripts.

    Directory of Open Access Journals (Sweden)

    Maik Grohmann

    Full Text Available Brain serotonin (5-HT neurotransmission plays a key role in the regulation of mood and has been implicated in a variety of neuropsychiatric conditions. Tryptophan hydroxylase (TPH is the rate-limiting enzyme in the biosynthesis of 5-HT. Recently, we discovered a second TPH isoform (TPH2 in vertebrates, including man, which is predominantly expressed in brain, while the previously known TPH isoform (TPH1 is primarly a non-neuronal enzyme. Overwhelming evidence now points to TPH2 as a candidate gene for 5-HT-related psychiatric disorders. To assess the role of TPH2 gene variability in the etiology of psychiatric diseases we performed cDNA sequence analysis of TPH2 transcripts from human post mortem amygdala samples obtained from individuals with psychiatric disorders (drug abuse, schizophrenia, suicide and controls. Here we show that TPH2 exists in two alternatively spliced variants in the coding region, denoted TPH2a and TPH2b. Moreover, we found evidence that the pre-mRNAs of both splice variants are dynamically RNA-edited in a mutually exclusive manner. Kinetic studies with cell lines expressing recombinant TPH2 variants revealed a higher activity of the novel TPH2B protein compared with the previously known TPH2A, whereas RNA editing was shown to inhibit the enzymatic activity of both TPH2 splice variants. Therefore, our results strongly suggest a complex fine-tuning of central nervous system 5-HT biosynthesis by TPH2 alternative splicing and RNA editing. Finally, we present molecular and large-scale linkage data evidencing that deregulated alternative splicing and RNA editing is involved in the etiology of psychiatric diseases, such as suicidal behaviour.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. RNA-guided genome editing for target gene mutations in wheat.

    Science.gov (United States)

    Upadhyay, Santosh Kumar; Kumar, Jitesh; Alok, Anshu; Tuli, Rakesh

    2013-12-09

    The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system has been used as an efficient tool for genome editing. We report the application of CRISPR-Cas-mediated genome editing to wheat (Triticum aestivum), the most important food crop plant with a very large and complex genome. The mutations were targeted in the inositol oxygenase (inox) and phytoene desaturase (pds) genes using cell suspension culture of wheat and in the pds gene in leaves of Nicotiana benthamiana. The expression of chimeric guide RNAs (cgRNA) targeting single and multiple sites resulted in indel mutations in all the tested samples. The expression of Cas9 or sgRNA alone did not cause any mutation. The expression of duplex cgRNA with Cas9 targeting two sites in the same gene resulted in deletion of DNA fragment between the targeted sequences. Multiplexing the cgRNA could target two genes at one time. Target specificity analysis of cgRNA showed that mismatches at the 3' end of the target site abolished the cleavage activity completely. The mismatches at the 5' end reduced cleavage, suggesting that the off target effects can be abolished in vivo by selecting target sites with unique sequences at 3' end. This approach provides a powerful method for genome engineering in plants.

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

    Science.gov (United States)

    Jia, Hongge; Wang, Nian

    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 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.

  20. 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.

  1. APOBEC1 complementation factor (A1CF) is dispensable for C-to-U RNA editing in vivo

    Science.gov (United States)

    Snyder, Elizabeth M.; McCarty, Christopher; Mehalow, Adrienne; Svenson, Karen L.; Murray, Stephen A.; Korstanje, Ron; Braun, Robert E.

    2017-01-01

    Editing of the human and murine ApoB mRNA by APOBEC1, the catalytic enzyme of the protein complex that catalyzes C-to-U RNA editing, creates an internal stop codon within the APOB coding sequence, generating two protein isoforms. It has been long held that APOBEC1-mediated editing activity is dependent on the RNA binding protein A1CF. The function of A1CF in adult tissues has not been reported because a previously reported null allele displays embryonic lethality. This work aimed to address the function of A1CF in adult mouse tissues using a conditional A1cf allele. Unexpectedly, A1cf-null mice were viable and fertile with modest defects in hematopoietic, immune, and metabolic parameters. C-to-U RNA editing was quantified for multiple targets, including ApoB, in the small intestine and liver. In all cases, no changes in RNA editing efficiency were observed. Blood plasma analysis demonstrated a male-specific increase in solute concentration and increased cellularity in the glomeruli of male A1cf-null mice. Urine analysis showed a reduction in solute concentration, suggesting abnormal water homeostasis and possible kidney abnormalities exclusive to the male. Computational identification of kidney C-to-U editing sites from polyadenylated RNA-sequencing identified a number of editing sites exclusive to the kidney. However, molecular analysis of kidney C-to-U editing showed no changes in editing efficiency with A1CF loss. Taken together, these observations demonstrate that A1CF does not act as the APOBEC1 complementation factor in vivo under normal physiological conditions and suggests new roles for A1CF, specifically within the male adult kidney. PMID:28069890

  2. APOBEC1 complementation factor (A1CF) is dispensable for C-to-U RNA editing in vivo.

    Science.gov (United States)

    Snyder, Elizabeth M; McCarty, Christopher; Mehalow, Adrienne; Svenson, Karen L; Murray, Stephen A; Korstanje, Ron; Braun, Robert E

    2017-04-01

    Editing of the human and murine ApoB mRNA by APOBEC1, the catalytic enzyme of the protein complex that catalyzes C-to-U RNA editing, creates an internal stop codon within the APOB coding sequence, generating two protein isoforms. It has been long held that APOBEC1-mediated editing activity is dependent on the RNA binding protein A1CF. The function of A1CF in adult tissues has not been reported because a previously reported null allele displays embryonic lethality. This work aimed to address the function of A1CF in adult mouse tissues using a conditional A1cf allele. Unexpectedly, A1cf-null mice were viable and fertile with modest defects in hematopoietic, immune, and metabolic parameters. C-to-U RNA editing was quantified for multiple targets, including ApoB, in the small intestine and liver. In all cases, no changes in RNA editing efficiency were observed. Blood plasma analysis demonstrated a male-specific increase in solute concentration and increased cellularity in the glomeruli of male A1cf-null mice. Urine analysis showed a reduction in solute concentration, suggesting abnormal water homeostasis and possible kidney abnormalities exclusive to the male. Computational identification of kidney C-to-U editing sites from polyadenylated RNA-sequencing identified a number of editing sites exclusive to the kidney. However, molecular analysis of kidney C-to-U editing showed no changes in editing efficiency with A1CF loss. Taken together, these observations demonstrate that A1CF does not act as the APOBEC1 complementation factor in vivo under normal physiological conditions and suggests new roles for A1CF, specifically within the male adult kidney.

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

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    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 geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing

    OpenAIRE

    2015-01-01

    CRISPR/Cas has emerged as potent genome editing technology and has successfully been applied in many organisms, including several plant species. However, delivery of genome editing reagents remains a challenge in plants. Here, we report a virus-based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant genome editing (VIGE) that can be used to precisely target genome locations and cause mutations. VIGE is performed by using a modified Cabbage Leaf Curl virus (CaLCuV) vector to expr...

  5. RNA editing in gymnosperms and its impact on the evolution of the mitochondrial coxI gene.

    Science.gov (United States)

    Lu, M Z; Szmidt, A E; Wang, X R

    1998-05-01

    Sequence analysis of the mitochondrial coxI gene in eight gymnosperm species revealed a high rate of nonsynonymous nucleotide substitutions with a strong (98%) predominance of C-T substitutions. Further analysis of the corresponding coxI cDNA sequences showed that all the non-synonymous C-T changes in the coxI genomic DNA sequences were eliminated by RNA editing resulting in nearly identical mRNA (amino acid) sequences among the species. Pronounced variation in the number and location of edited sites was found among species. Most species had a relatively large number of edited sites (from 25 to 34). However, no RNA editing of the coxI sequence was found in Gingko biloba or Larix sibirica. The sequence composition of the investigated coxI fragment suggests that the coxI gene in G. biloba and L. sibirica originated from edited mitochondrial coxI transcripts by reverse transcription followed by insertion into the nuclear genome or back into the mitochondrial genome. Our results also demonstrate that where there are a large number of edited sites, RNA editing can accelerate the divergence of nucleotide sequences among species.

  6. Noncoding RNAs and RNA editing in brain development, functional diversification, and neurological disease.

    Science.gov (United States)

    Mehler, Mark F; Mattick, John S

    2007-07-01

    The progressive maturation and functional plasticity of the nervous system in health and disease involve a dynamic interplay between the transcriptome and the environment. There is a growing awareness that the previously unexplored molecular and functional interface mediating these complex gene-environmental interactions, particularly in brain, may encompass a sophisticated RNA regulatory network involving the twin processes of RNA editing and multifaceted actions of numerous subclasses of non-protein-coding RNAs. The mature nervous system encompasses a wide range of cell types and interconnections. Long-term changes in the strength of synaptic connections are thought to underlie memory retrieval, formation, stabilization, and effector functions. The evolving nervous system involves numerous developmental transitions, such as neurulation, neural tube patterning, neural stem cell expansion and maintenance, lineage elaboration, differentiation, axonal path finding, and synaptogenesis. Although the molecular bases for these processes are largely unknown, RNA-based epigenetic mechanisms appear to be essential for orchestrating these precise and versatile biological phenomena and in defining the etiology of a spectrum of neurological diseases. The concerted modulation of RNA editing and the selective expression of non-protein-coding RNAs during seminal as well as continuous state transitions may comprise the plastic molecular code needed to couple the intrinsic malleability of neural network connections to evolving environmental influences to establish diverse forms of short- and long-term memory, context-specific behavioral responses, and sophisticated cognitive capacities.

  7. 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.

  8. 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.

  9. Alu-directed transcriptional regulation of some novel miRNAs

    Directory of Open Access Journals (Sweden)

    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.

  10. 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.

  11. 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

  12. 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

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

    Science.gov (United States)

    Doetschman, Thomas; Georgieva, Teodora

    2017-03-03

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

  14. Identification of Alternative Variants and Insertion of the Novel Polymorphic AluYl17 in TSEN54 Gene during Primate Evolution

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    Ja-Rang Lee

    2016-01-01

    Full Text Available TSEN54 encodes a subunit of the tRNA-splicing endonuclease complex, which catalyzes the identification and cleavage of introns from precursor tRNAs. Previously, we identified an AluSx-derived alternative transcript in TSEN54 of cynomolgus monkey. Reverse transcription-polymerase chain reaction (RT-PCR amplification and TSEN54 sequence analysis of primate and human samples identified five novel alternative transcripts, including the AluSx exonized transcript. Additionally, we performed comparative expression analysis via RT-qPCR in various cynomolgus, rhesus monkey, and human tissues. RT-qPCR amplification revealed differential expression patterns. Furthermore, genomic PCR amplification and sequencing of primate and human DNA samples revealed that AluSx elements were integrated in human and all of the primate samples tested. Intriguingly, in langur genomic DNA, an additional AluY element was inserted into AluSx of intron eight of TSEN54. The new AluY element showed polymorphic insertion. Using standardized nomenclature for Alu repeats, the polymorphic AluY of the langur TSEN54 was designated as being of the AluYl17 subfamily. Our results suggest that integration of the AluSx element in TSEN54 contributed to diversity in transcripts and induced lineage- or species-specific evolutionary events such as alternative splicing and polymorphic insertion during primate evolution.

  15. 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.

  16. [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.

  17. Whole-Transcriptome RNA-seq, Gene Set Enrichment Pathway Analysis, and Exon Coverage Analysis of Two Plastid RNA Editing Mutants.

    Science.gov (United States)

    Hackett, Justin B; Lu, Yan

    2017-04-07

    In land plants, plastid and mitochondrial RNAs are subject to post-transcriptional C-to-U RNA editing. T-DNA insertions in the ORGANELLE RNA RECOGNITION MOTIF PROTEIN6 gene resulted in reduced photosystem II (PSII) activity and smaller plant and leaf sizes. Exon coverage analysis of the ORRM6 gene showed that orrm6-1 and orrm6-2 are loss-of-function mutants. Compared to other ORRM proteins, ORRM6 affects a relative small number of RNA editing sites. Sanger sequencing of reverse transcription-PCR products of plastid transcripts revealed two plastid RNA editing sites that are substantially affected in the orrm6 mutants: psbF-C77 and accD-C794. The psbF gene encodes the beta subunit of cytochrome b559, an essential component of PSII. The accD gene encodes the beta subunit of acetyl-CoA carboxylase, a protein required in plastid fatty acid biosynthesis. Whole-transcriptome RNA-seq demonstrated that editing at psbF-C77 is nearly absent and the editing extent at accD-C794 was significantly reduced. Gene set enrichment pathway analysis showed that expression of multiple gene sets involved in photosynthesis, especially photosynthetic electron transport, is significantly up-regulated in both orrm6 mutants. The up-regulation could be a mechanism to compensate for the reduced PSII electron transport rate in the orrm6 mutants. These results further demonstrated that Organelle RNA Recognition Motif protein ORRM6 is required in editing of specific RNAs in the Arabidopsis (Arabidopsis thaliana) plastid.

  18. 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

  19. 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

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

    Science.gov (United States)

    Sun, Yongmei; Li, Xing; Wu, Di; Pan, Qi; Ji, Yuefeng; Ren, Hong; Ding, Keyue

    2016-01-01

    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.

  1. 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

    2016-08-25

    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.

  2. High-efficiency targeted editing of large viral genomes by RNA-guided nucleases.

    Directory of Open Access Journals (Sweden)

    Yanwei Bi

    2014-05-01

    Full Text Available A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR-associated (Cas RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ and homology-directed repair (HDR pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses.

  3. 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...

  4. 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.

  5. 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.

  6. 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

  7. RNA Editing Genes Associated with Extreme Old Age in Humans and with Lifespan in C. elegans

    Science.gov (United States)

    Puca, Annibale; Solovieff, Nadia; Kojima, Toshio; Wang, Meng C.; Melista, Efthymia; Meltzer, Micah; Fischer, Sylvia E. J.; Andersen, Stacy; Hartley, Stephen H.; Sedgewick, Amanda; Arai, Yasumichi; Bergman, Aviv; Barzilai, Nir; Terry, Dellara F.; Riva, Alberto; Anselmi, Chiara Viviani; Malovini, Alberto; Kitamoto, Aya; Sawabe, Motoji; Arai, Tomio; Gondo, Yasuyuki; Steinberg, Martin H.; Hirose, Nobuyoshi; Atzmon, Gil; Ruvkun, Gary; Baldwin, Clinton T.; Perls, Thomas T.

    2009-01-01

    Background The strong familiality of living to extreme ages suggests that human longevity is genetically regulated. The majority of genes found thus far to be associated with longevity primarily function in lipoprotein metabolism and insulin/IGF-1 signaling. There are likely many more genetic modifiers of human longevity that remain to be discovered. Methodology/Principal Findings Here, we first show that 18 single nucleotide polymorphisms (SNPs) in the RNA editing genes ADARB1 and ADARB2 are associated with extreme old age in a U.S. based study of centenarians, the New England Centenarian Study. We describe replications of these findings in three independently conducted centenarian studies with different genetic backgrounds (Italian, Ashkenazi Jewish and Japanese) that collectively support an association of ADARB1 and ADARB2 with longevity. Some SNPs in ADARB2 replicate consistently in the four populations and suggest a strong effect that is independent of the different genetic backgrounds and environments. To evaluate the functional association of these genes with lifespan, we demonstrate that inactivation of their orthologues adr-1 and adr-2 in C. elegans reduces median survival by 50%. We further demonstrate that inactivation of the argonaute gene, rde-1, a critical regulator of RNA interference, completely restores lifespan to normal levels in the context of adr-1 and adr-2 loss of function. Conclusions/Significance Our results suggest that RNA editors may be an important regulator of aging in humans and that, when evaluated in C. elegans, this pathway may interact with the RNA interference machinery to regulate lifespan. PMID:20011587

  8. A novel type of RNA editing occurs in the mitochondrial tRNAs of the centipede Lithobius forficatus

    Science.gov (United States)

    Lavrov, Dennis V.; Brown, Wesley M.; Boore, Jeffrey L.

    2000-01-01

    We determined the complete mtDNA sequence of the centipede Lithobius forficatus and found that only one of the 22 inferred tRNA genes encodes a fully paired aminoacyl acceptor stem. The other 21 genes encode tRNAs with up to five mismatches in these stems, and some of these overlap extensively with the downstream genes. Because a well-paired acceptor stem is required for proper tRNA functioning, RNA editing in the products of these genes was suspected. We investigated this hypothesis by studying cDNA sequences from eight tRNAs and found the editing of up to 5 nt at their 3′ ends. This editing appears to occur by a novel mechanism with the 5′ end of the acceptor stem being used as a template for the de novo synthesis of the 3′ end, presumably by an RNA-dependent RNA polymerase. In addition, unusual secondary structures for several tRNAs were found, including those lacking a TΨC (T) or a dihydrouridine (D) arm, and having an unusual number of base pairs in the acceptor or anticodon stems. PMID:11095730

  9. 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...

  10. 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 genes harboring Alus revealed significant enrichment for immune......-mediated processes (p-value genes harboring inverted 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...

  11. A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing.

    Science.gov (United States)

    Yin, Kangquan; Han, Ting; Liu, Guang; Chen, Tianyuan; Wang, Ying; Yu, Alice Yunzi L; Liu, Yule

    2015-10-09

    CRISPR/Cas has emerged as potent genome editing technology and has successfully been applied in many organisms, including several plant species. However, delivery of genome editing reagents remains a challenge in plants. Here, we report a virus-based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant genome editing (VIGE) that can be used to precisely target genome locations and cause mutations. VIGE is performed by using a modified Cabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stable transgenic plants expressing Cas9. DNA sequencing confirmed VIGE of endogenous NbPDS3 and NbIspH genes in non-inoculated leaves because CaLCuV can infect plants systemically. Moreover, VIGE of NbPDS3 and NbIspH in newly developed leaves caused photo-bleached phenotype. These results demonstrate that geminivirus-based VIGE could be a powerful tool in plant genome editing.

  12. Evolution of Alu elements toward enhancers.

    Science.gov (United States)

    Su, Ming; Han, Dali; Boyd-Kirkup, Jerome; Yu, Xiaoming; Han, Jing-Dong J

    2014-04-24

    The human genome contains approximately one million Alu repetitive elements comprising 10% of the genome, yet their functions are not well understood. Here, we show that Alu elements resemble enhancers. Alu elements are bound by two well-phased nucleosomes that contain histones bearing marks of active chromatin, and they show tissue-specific enrichment for the enhancer mark H3K4me1. A proportion of Alu elements were experimentally validated as bona fide active enhancers with an in vitro reporter assay. In addition, Hi-C data indicate that Alus show long-range interactions with gene promoters. We also find that Alus are generally more conserved when located in the proximal upstream region of genes. Their similarity to enhancers becomes more prominent with their age in the human genome, following a clear evolutionary continuum reminiscent of the evolutionary pattern of proto-genes. Therefore, we conclude that some Alu elements can function as enhancers and propose that many more may be proto-enhancers that serve as a repertoire for the de novo birth of enhancers.

  13. 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.

  14. Sendai virus, an RNA virus with no risk of genomic integration, delivers CRISPR/Cas9 for efficient gene editing.

    Science.gov (United States)

    Park, Arnold; Hong, Patrick; Won, Sohui T; Thibault, Patricia A; Vigant, Frederic; Oguntuyo, Kasopefoluwa Y; Taft, Justin D; Lee, Benhur

    2016-01-01

    The advent of RNA-guided endonuclease (RGEN)-mediated gene editing, specifically via CRISPR/Cas9, has spurred intensive efforts to improve the efficiency of both RGEN delivery and targeted mutagenesis. The major viral vectors in use for delivery of Cas9 and its associated guide RNA, lentiviral and adeno-associated viral systems, have the potential for undesired random integration into the host genome. Here, we repurpose Sendai virus, an RNA virus with no viral DNA phase and that replicates solely in the cytoplasm, as a delivery system for efficient Cas9-mediated gene editing. The high efficiency of Sendai virus infection resulted in high rates of on-target mutagenesis in cell lines (75-98% at various endogenous and transgenic loci) and primary human monocytes (88% at the ccr5 locus) in the absence of any selection. In conjunction with extensive former work on Sendai virus as a promising gene therapy vector that can infect a wide range of cell types including hematopoietic stem cells, this proof-of-concept study opens the door to using Sendai virus as well as other related paramyxoviruses as versatile and efficient tools for gene editing.

  15. Alu elements and DNA double-strand break repair

    OpenAIRE

    White, Travis B; Morales, Maria E.; Deininger, Prescott L.

    2015-01-01

    Alu elements represent one of the most common sources of homology and homeology in the human genome. Homeologous recombination between Alu elements represents a major form of genetic instability leading to deletions and duplications. Although these types of events have been studied extensively through genomic sequencing to assess the impact of Alu elements on disease mutations and genome evolution, the overall abundance of Alu elements in the genome often makes it difficult to assess the rele...

  16. 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

  17. 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.

  18. 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

    2016-07-05

    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.

  19. Kaitseliidu koolimõis Alu / Helju Koger

    Index Scriptorium Estoniae

    Koger, Helju, 1943-

    2003-01-01

    2002. a. sügisest on Alu mõisahoones Kaitseliidu kool. Mõisa ajaloost. 1875. a. valminud härrastemaja projekteeris Paul Friedrich Wilhelm Alisch. Mõisahoone restaureerimisest. 2000. a. restaureerimisprojekti autorid Anu Vaarpuu ja Urmas Sepp arhitektibüroost Hoerdel. 15 ill

  20. 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.

  1. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing

    Energy Technology Data Exchange (ETDEWEB)

    Church, George M.; Esvelt, Kevin; Mali, Prashant

    2017-03-07

    Methods of modulating expression of a target nucleic acid in a cell are provided including use of multiple orthogonal Cas9 proteins to simultaneously and independently regulate corresponding genes or simultaneously and independently edit corresponding genes.

  2. Mouse let-7 miRNA populations exhibit RNA editing that is constrained in the 5′-seed/ cleavage/anchor regions and stabilize predicted mmu-let-7a:mRNA duplexes

    Science.gov (United States)

    Reid, Jeffrey G.; Nagaraja, Ankur K.; Lynn, Francis C.; Drabek, Rafal B.; Muzny, Donna M.; Shaw, Chad A.; Weiss, Michelle K.; Naghavi, Arash O.; Khan, Mahjabeen; Zhu, Huifeng; Tennakoon, Jayantha; Gunaratne, Gemunu H.; Corry, David B.; Miller, Jonathan; McManus, Michael T.; German, Michael S.; Gibbs, Richard A.; Matzuk, Martin M.; Gunaratne, Preethi H.

    2008-01-01

    Massively parallel sequencing of millions of <30-nt RNAs expressed in mouse ovary, embryonic pancreas (E14.5), and insulin-secreting beta-cells (βTC-3) reveals that ∼50% of the mature miRNAs representing mostly the mmu-let-7 family display internal insertion/deletions and substitutions when compared to precursor miRNA and the mouse genome reference sequences. Approximately, 12%–20% of species associated with mmu-let-7 populations exhibit sequence discrepancies that are dramatically reduced in nucleotides 3–7 (5′-seed) and 10–15 (cleavage and anchor sites). This observation is inconsistent with sequencing error and leads us to propose that the changes arise predominantly from post-transcriptional RNA-editing activity operating on miRNA:target mRNA complexes. Internal nucleotide modifications are most enriched at the ninth nucleotide position. A common ninth base edit of U-to-G results in a significant increase in stability of down-regulated let-7a targets in inhibin-deficient mice (Inha−/−). An excess of U-insertions (14.8%) over U-deletions (1.5%) and the presence of cleaved intermediates suggest that a mammalian TUTase (terminal uridylyl transferase) mediated dUTP-dependent U-insertion/U-deletion cycle may be a possible mechanism. We speculate that mRNA target site-directed editing of mmu-let-7a duplex-bulges stabilizes “loose” miRNA:mRNA target associations and functions to expand the target repertoire and/or enhance mRNA decay over translational repression. Our results also demonstrate that the systematic study of sequence variation within specific RNA classes in a given cell type from millions of sequences generated by next-generation sequencing (NGS) technologies (“intranomics”) can be used broadly to infer functional constraints on specific parts of completely uncharacterized RNAs. PMID:18614752

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. Non-Viral CRISPR/Cas Gene Editing In Vitro and In Vivo Enabled by Synthetic Nanoparticle Co-Delivery of Cas9 mRNA and sgRNA.

    Science.gov (United States)

    Miller, Jason B; Zhang, Shuyuan; Kos, Petra; Xiong, Hu; Zhou, Kejin; Perelman, Sofya S; Zhu, Hao; Siegwart, Daniel J

    2017-01-19

    CRISPR/Cas is a revolutionary gene editing technology with wide-ranging utility. The safe, non-viral delivery of CRISPR/Cas components would greatly improve future therapeutic utility. We report the synthesis and development of zwitterionic amino lipids (ZALs) that are uniquely able to (co)deliver long RNAs including Cas9 mRNA and sgRNAs. ZAL nanoparticle (ZNP) delivery of low sgRNA doses (15 nm) reduces protein expression by >90 % in cells. In contrast to transient therapies (such as RNAi), we show that ZNP delivery of sgRNA enables permanent DNA editing with an indefinitely sustained 95 % decrease in protein expression. ZNP delivery of mRNA results in high protein expression at low doses in vitro (gene editing.

  8. 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.

  9. The dsRBP and Inactive Editor ADR-1 Utilizes dsRNA Binding to Regulate A-to-I RNA Editing across the C. elegans Transcriptome

    OpenAIRE

    Michael C. Washburn; Boyko Kakaradov; Balaji Sundararaman; Emily Wheeler; Shawn Hoon; Gene W. Yeo; Heather A. Hundley

    2014-01-01

    Inadequate adenosine-to-inosine editing of noncoding regions occurs in disease but is often uncorrelated with ADAR levels, underscoring the need to study deaminase-independent control of editing. C. elegans have two ADAR proteins, ADR-2 and the theoretically catalytically inactive ADR-1. Using high-throughput RNA sequencing of wild-type and adr mutant worms, we expand the repertoire of C. elegans edited transcripts over 5-fold and confirm that ADR-2 is the only active deaminase in vivo. Despi...

  10. The role of RNA editing of the serotonin 2C receptor in a rat model of oro-facial neuropathic pain.

    Science.gov (United States)

    Nakae, Aya; Nakai, Kunihiro; Tanaka, Tatsuya; Hagihira, Saotoshi; Shibata, Masahiko; Ueda, Koichi; Masimo, Takashi

    2008-05-01

    We examined whether infraorbital nerve injury affected the RNA editing efficiency of the serotonin (5HT) 2C receptor in the cervical spinal cord, in association with increased pain thresholds, and whether a 5HT reuptake inhibitor (fluvoxamine; Depromel, Meiji Seika, Tokyo, Japan) altered this editing. Accordingly, we injured rats with an infraorbital nerve loose ligation and examined the pain thresholds, mRNA and mRNA editing of the 5HT2C receptor. We evaluated changes in mRNA editing and 5HT2C mRNA expression using cloning along with sequence analysis and quantitative reverse transcription-polymerase chain reaction to compare samples taken at post-injury day 28 from spinal cord sites, including the trigeminal nucleus caudalis, in naive, sham and injured rats (groups of each type had also received fluvoxamine). 5HT2C receptor expression was maintained post-injury. The RNA editing efficiency was statistically significantly lower at molecular sites A and B in ipsilateral spinal cord samples from injured rats than in bilateral samples from naive and sham rats, and in contralateral samples from injured rats. After injury, the proportional presence of two receptor isoforms changed, i.e. statistically significantly less VNV and significantly more INV and ISV. The proportions reverted after fluvoxamine administration. The post-injury change might be evidence of a functional adaptation mechanism that increases the expression of 5HT2C mRNA isoforms that encode receptors that are more sensitive to 5HT. This would activate the brainstem-spinal descending 5HT systems and, in effect, suppress nociceptive signals from primary afferent neurons to the spinal trigeminal nucleus caudalis.

  11. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.

    Science.gov (United States)

    Kim, Sojung; Kim, Daesik; Cho, Seung Woo; Kim, Jungeun; Kim, Jin-Soo

    2014-06-01

    RNA-guided engineered nucleases (RGENs) derived from the prokaryotic adaptive immune system known as CRISPR (clustered, regularly interspaced, short palindromic repeat)/Cas (CRISPR-associated) enable genome editing in human cell lines, animals, and plants, but are limited by off-target effects and unwanted integration of DNA segments derived from plasmids encoding Cas9 and guide RNA at both on-target and off-target sites in the genome. Here, we deliver purified recombinant Cas9 protein and guide RNA into cultured human cells including hard-to-transfect fibroblasts and pluripotent stem cells. RGEN ribonucleoproteins (RNPs) induce site-specific mutations at frequencies of up to 79%, while reducing off-target mutations associated with plasmid transfection at off-target sites that differ by one or two nucleotides from on-target sites. RGEN RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects. Furthermore, RNP delivery is less stressful to human embryonic stem cells, producing at least twofold more colonies than does plasmid transfection.

  12. 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.

  13. 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.

  14. APOBEC3A is implicated in a novel class of G-to-A mRNA editing in WT1 transcripts.

    Directory of Open Access Journals (Sweden)

    Ahmadreza Niavarani

    Full Text Available Classic deamination mRNA changes, including cytidine to uridine (C-to-U and adenosine to inosine (A-to-I, are important exceptions to the central dogma and lead to significant alterations in gene transcripts and products. Although there are a few reports of non-classic mRNA alterations, as yet there is no molecular explanation for these alternative changes. Wilms Tumor 1 (WT1 mutations and variants are implicated in several diseases, including Wilms tumor and acute myeloid leukemia (AML. We observed two alternative G-to-A changes, namely c.1303G>A and c.1586G>A in cDNA clones and found them to be recurrent in a series of 21 umbilical cord blood mononuclear cell (CBMC samples studied. Two less conserved U-to-C changes were also observed. These alternative changes were found to be significantly higher in non-progenitor as compared to progenitor CBMCs, while they were found to be absent in a series of AML samples studied, indicating they are targeted, cell type-specific mRNA editing modifications. Since APOBEC/ADAR family members are implicated in RNA/DNA editing, we screened them by RNA-interference (RNAi for WT1-mRNA changes and observed near complete reversal of WT1 c.1303G>A alteration upon APOBEC3A (A3A knockdown. The role of A3A in mediating this change was confirmed by A3A overexpression in Fujioka cells, which led to a significant increase in WT1 c.1303G>A mRNA editing. Non-progenitor CBMCs showed correspondingly higher levels of A3A-mRNA and protein as compared to the progenitor ones. To our knowledge, this is the first report of mRNA modifying activity for an APOBEC3 protein and implicates A3A in a novel G-to-A form of editing. These findings open the way to further investigations into the mechanisms of other potential mRNA changes, which will help to redefine the RNA editing paradigm in both health and disease.

  15. 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

  16. 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...

  17. 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.

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

    Science.gov (United States)

    Barresi, Sabina; Tomaselli, Sara; Athanasiadis, Alekos; Galeano, Federica; Locatelli, Franco; Bertini, Enrico; Zanni, Ginevra; Gallo, Angela

    2014-01-01

    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.

  19. 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.

  20. 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…

  1. Polymorphic Alu insertions among Mayan populations.

    Science.gov (United States)

    Herrera, R J; Rojas, D P; Terreros, M C

    2007-01-01

    The Mayan homeland within Mesoamerica spans five countries: Belize, El Salvador, Guatemala, Honduras and Mexico. There are indications that the people we call the Maya migrated from the north to the highlands of Guatemala as early as 4000 B.C. Their existence was village-based and agricultural. The culture of these Preclassic Mayans owes much to the earlier Olmec civilization, which flourished in the southern portion of North America. In this study, four different Mayan groups were examined to assess their genetic variability. Ten polymorphic Alu insertion (PAI) loci were employed to ascertain the genetic affinities among these Mayan groups. North American, African, European and Asian populations were also examined as reference populations. Our results suggest that the Mayan groups examined in this study are not genetically homogeneous.

  2. 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.

  3. 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.

  4. DESIGN OF 16-BIT LOW POWER ALU - DBGPU

    Directory of Open Access Journals (Sweden)

    Dhanabal R

    2013-06-01

    Full Text Available Arithmetic and Logic Unit (ALU is one of the common and the most crucial components of an embedded system. Power consumption is a major design issue in the case of embedded systems. Usually ALU’s consists of a number of functional units for different arithmetic and logic operations which are realised using combinational circuits. Each of the functional unit performs a specific arithmetic or logic operation. In this paper the main concern is given for reducing the power of the adder and multiplier modules which are important functional units of ALU thereby reducing the overall power consumption without compromising the speed of the processor. The ALU circuit ensures the execution of either arithmetic or logic operation only at a time so that only one set of circuits is active at a time thus ensuring low power consumption. The entire ALU circuit isrealised using Verilog HDL and power analysis is obtained through same.

  5. 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.

  6. 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.

  7. ALU Using Area Optimized Vedic Multiplier

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    Anshul Khare

    2014-07-01

    Full Text Available —The load on general processor is increasing. For Fast Operations it is an extreme importance in Arithmetic Unit. The performance of Arithmetic Unit depends greatly on it multipliers. So, researchers are continuous searching for new approaches and hardware to implement arithmetic operation in huge efficient way in the terms of speed and area. Vedic Mathematics is the old system of mathematics which has a different technique of calculations based on total 16 Sutras. Proposed work has discussion of the quality of Urdhva Triyakbhyam Vedic approach for multiplication which uses different way than actual process of multiplication itself. It allows parallel generation of elements of products also eliminates undesired multiplication steps with zeros and mapped to higher level of bit using Karatsuba technique with processors, the compatibility to various data types. It is been observed that lot of delay is required by the conventional adders which are needed to have the partial products so in the work it is further optimized the Vedic multiplier type Urdhva Triyakbhyam by replacing the traditional adder with Carry save Adder to have more Delay Optimization. The proposed work shows improvement of speed as compare with the traditional designs. After the proposal discussion of the Vedic multiplier in the paper, It is been used for the implementation of Arithmetic unit using proposed efficient Vedic Multiplier it is not only useful for the improve efficiency the arithmetic module of ALU but also it is useful in the area of digital signal processing. The RTL entry of proposed Arithmetic unit done in VHDL it is synthesized and simulated with Xilinx ISE EDA tool. At the last the proposed Arithmetic Unit is validated on a FPGA device Vertex-IV.

  8. 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.

  9. 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.

  10. Modeling the amplification dynamics of human Alu retrotransposons.

    Directory of Open Access Journals (Sweden)

    Dale J Hedges

    2005-09-01

    Full Text Available Retrotransposons have had a considerable impact on the overall architecture of the human genome. Currently, there are three lineages of retrotransposons (Alu, L1, and SVA that are believed to be actively replicating in humans. While estimates of their copy number, sequence diversity, and levels of insertion polymorphism can readily be obtained from existing genomic sequence data and population sampling, a detailed understanding of the temporal pattern of retrotransposon amplification remains elusive. Here we pose the question of whether, using genomic sequence and population frequency data from extant taxa, one can adequately reconstruct historical amplification patterns. To this end, we developed a computer simulation that incorporates several known aspects of primate Alu retrotransposon biology and accommodates sampling effects resulting from the methods by which mobile elements are typically discovered and characterized. By modeling a number of amplification scenarios and comparing simulation-generated expectations to empirical data gathered from existing Alu subfamilies, we were able to statistically reject a number of amplification scenarios for individual subfamilies, including that of a rapid expansion or explosion of Alu amplification at the time of human-chimpanzee divergence.

  11. 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...

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

    Science.gov (United States)

    Peixoto, Ana; Santos, Catarina; Pinheiro, Manuela; Pinto, Pedro; Soares, Maria José; Rocha, Patrícia; Gusmão, Leonor; Amorim, António; van der Hout, Annemarie; Gerdes, Anne-Marie; Thomassen, Mads; Kruse, Torben A; Cruger, Dorthe; Sunde, Lone; Bignon, Yves-Jean; Uhrhammer, Nancy; Cornil, Lucie; Rouleau, Etienne; Lidereau, Rosette; Yannoukakos, Drakoulis; Pertesi, Maroulio; Narod, Steven; Royer, Robert; Costa, Maurício M; Lazaro, Conxi; Feliubadaló, Lidia; Graña, Begoña; Blanco, Ignacio; de la Hoya, Miguel; Caldés, Trinidad; Maillet, Philippe; Benais-Pont, Gaelle; Pardo, Bruno; Laitman, Yael; Friedman, Eitan; Velasco, Eladio A; Durán, Mercedes; Miramar, Maria-Dolores; Valle, Ana Rodriguez; Calvo, María-Teresa; Vega, Ana; Blanco, Ana; Diez, Orland; Gutiérrez-Enríquez, Sara; Balmaña, Judith; Ramon y Cajal, Teresa; Alonso, Carmen; Baiget, Montserrat; Foulkes, William; Tischkowitz, Marc; Kyle, Rachel; Sabbaghian, Nelly; Ashton-Prolla, Patricia; Ewald, Ingrid P; Rajkumar, Thangarajan; Mota-Vieira, Luisa; Giannini, Giuseppe; Gulino, Alberto; Achatz, Maria I; Carraro, Dirce M; de Paillerets, Brigitte Bressac; Remenieras, Audrey; Benson, Cindy; Casadei, Silvia; King, Mary-Claire; Teugels, Erik; Teixeira, Manuel R

    2011-06-01

    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 individuals requesting predictive testing living in France and in the USA, all being Portuguese immigrants. After performing an extensive haplotype study in carrier families, we estimate that this founder mutation occurred 558 ± 215 years ago. We further demonstrate significant quantitative differences 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 HBOC families from Portugal or with Portuguese ancestry are specifically tested for this rearrangement.

  13. 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%)

  14. 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.

  15. 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.

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

    Science.gov (United States)

    Wang, Mugui; Liu, Yujun; Zhang, Cuicui; Liu, Jianping; Liu, Xin; Wang, Liangchao; Wang, Wenyi; Chen, Hao; Wei, Chuchu; Ye, Xiufen; Li, Xinyuan; Tu, Jumin

    2015-01-01

    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.

  17. The CP2 domain of leucyl-tRNA synthetase is crucial for amino acid activation and post-transfer editing.

    Science.gov (United States)

    Zhou, Xiao-Long; Zhu, Bin; Wang, En-Duo

    2008-12-26

    Leucyl-tRNA synthetase (LeuRS) has an insertion domain, called connective peptide 2 (CP2), either directly preceding or following the editing domain (CP1 domain), depending on the species. The global structures of the CP2 domains from all LeuRSs are similar. Although the CP1 domain has been extensively explored to be responsible for hydrolysis of mischarged tRNALeu, the role of the CP2 domain remains undefined. In the present work, deletion of the CP2 domain of Giardia lamblia LeuRS (GlLeuRS) showed that the CP2 domain is indispensable for amino acid activation and post-transfer editing and that it contributes to LeuRS-tRNALeu binding affinity. In addition, its functions are conserved in both eukaryotic/archaeal and prokaryotic LeuRSs from G. lamblia, Pyrococcus horikoshii (PhLeuRS), and Escherichia coli (EcLeuRS). Alanine scanning and site-directed mutagenesis assays of the CP2 domain identified several residues that are crucial for its various functions. Data from the chimeric mutants, which replaced the CP2 domain of GlLeuRS with either PhLeuRS or EcLeuRS, showed that the CP2 domain of PhLeuRS but not that of EcLeuRS can partially restore amino acid activation and post-transfer editing functions, suggesting that the functions of the CP2 domain are dependent on its location in the primary sequence of LeuRS.

  18. 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.

  19. An Alu-Based Phylogeny of Lemurs (Infraorder: Lemuriformes)

    OpenAIRE

    2012-01-01

    LEMURS (INFRAORDER: Lemuriformes) are a radiation of strepsirrhine primates endemic to the island of Madagascar. As of 2012, 101 lemur species, divided among five families, have been described. Genetic and morphological evidence indicates all species are descended from a common ancestor that arrived in Madagascar ∼55-60 million years ago (mya). Phylogenetic relationships in this species-rich infraorder have been the subject of debate. Here we use Alu elements, a family of primate-specific Sho...

  20. An Alu-Based Phylogeny of Lemurs (Infraorder: Lemuriformes)

    Science.gov (United States)

    McLain, Adam T.; Meyer, Thomas J.; Faulk, Christopher; Herke, Scott W.; Oldenburg, J. Michael; Bourgeois, Matthew G.; Abshire, Camille F.

    2012-01-01

    Lemurs (infraorder: Lemuriformes) are a radiation of strepsirrhine primates endemic to the island of Madagascar. As of 2012, 101 lemur species, divided among five families, have been described. Genetic and morphological evidence indicates all species are descended from a common ancestor that arrived in Madagascar ∼55–60 million years ago (mya). Phylogenetic relationships in this species-rich infraorder have been the subject of debate. Here we use Alu elements, a family of primate-specific Short INterspersed Elements (SINEs), to construct a phylogeny of infraorder Lemuriformes. Alu elements are particularly useful SINEs for the purpose of phylogeny reconstruction because they are identical by descent and confounding events between loci are easily resolved by sequencing. The genome of the grey mouse lemur (Microcebus murinus) was computationally assayed for synapomorphic Alu elements. Those that were identified as Lemuriformes-specific were analyzed against other available primate genomes for orthologous sequence in which to design primers for PCR (polymerase chain reaction) verification. A primate phylogenetic panel of 24 species, including 22 lemur species from all five families, was examined for the presence/absence of 138 Alu elements via PCR to establish relationships among species. Of these, 111 were phylogenetically informative. A phylogenetic tree was generated based on the results of this analysis. We demonstrate strong support for the monophyly of Lemuriformes to the exclusion of other primates, with Daubentoniidae, the aye-aye, as the basal lineage within the infraorder. Our results also suggest Lepilemuridae as a sister lineage to Cheirogaleidae, and Indriidae as sister to Lemuridae. Among the Cheirogaleidae, we show strong support for Microcebus and Mirza as sister genera, with Cheirogaleus the sister lineage to both. Our results also support the monophyly of the Lemuridae. Within Lemuridae we place Lemur and Hapalemur together to the exclusion of

  1. An alu-based phylogeny of lemurs (infraorder: Lemuriformes.

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    Adam T McLain

    Full Text Available LEMURS (INFRAORDER: Lemuriformes are a radiation of strepsirrhine primates endemic to the island of Madagascar. As of 2012, 101 lemur species, divided among five families, have been described. Genetic and morphological evidence indicates all species are descended from a common ancestor that arrived in Madagascar ∼55-60 million years ago (mya. Phylogenetic relationships in this species-rich infraorder have been the subject of debate. Here we use Alu elements, a family of primate-specific Short INterspersed Elements (SINEs, to construct a phylogeny of infraorder Lemuriformes. Alu elements are particularly useful SINEs for the purpose of phylogeny reconstruction because they are identical by descent and confounding events between loci are easily resolved by sequencing. The genome of the grey mouse lemur (Microcebus murinus was computationally assayed for synapomorphic Alu elements. Those that were identified as Lemuriformes-specific were analyzed against other available primate genomes for orthologous sequence in which to design primers for PCR (polymerase chain reaction verification. A primate phylogenetic panel of 24 species, including 22 lemur species from all five families, was examined for the presence/absence of 138 Alu elements via PCR to establish relationships among species. Of these, 111 were phylogenetically informative. A phylogenetic tree was generated based on the results of this analysis. We demonstrate strong support for the monophyly of Lemuriformes to the exclusion of other primates, with Daubentoniidae, the aye-aye, as the basal lineage within the infraorder. Our results also suggest Lepilemuridae as a sister lineage to Cheirogaleidae, and Indriidae as sister to Lemuridae. Among the Cheirogaleidae, we show strong support for Microcebus and Mirza as sister genera, with Cheirogaleus the sister lineage to both. Our results also support the monophyly of the Lemuridae. Within Lemuridae we place Lemur and Hapalemur together to the

  2. Alu recombination-mediated structural deletions in the chimpanzee genome.

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    Kyudong Han

    2007-10-01

    Full Text Available With more than 1.2 million copies, Alu elements are one of the most important sources of structural variation in primate genomes. Here, we compare the chimpanzee and human genomes to determine the extent of Alu recombination-mediated deletion (ARMD in the chimpanzee genome since the divergence of the chimpanzee and human lineages ( approximately 6 million y ago. Combining computational data analysis and experimental verification, we have identified 663 chimpanzee lineage-specific deletions (involving a total of approximately 771 kb of genomic sequence attributable to this process. The ARMD events essentially counteract the genomic expansion caused by chimpanzee-specific Alu inserts. The RefSeq databases indicate that 13 exons in six genes, annotated as either demonstrably or putatively functional in the human genome, and 299 intronic regions have been deleted through ARMDs in the chimpanzee lineage. Therefore, our data suggest that this process may contribute to the genomic and phenotypic diversity between chimpanzees and humans. In addition, we found four independent ARMD events at orthologous loci in the gorilla or orangutan genomes. This suggests that human orthologs of loci at which ARMD events have already occurred in other nonhuman primate genomes may be "at-risk" motifs for future deletions, which may subsequently contribute to human lineage-specific genetic rearrangements and disorders.

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

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    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.

  4. 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

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

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    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:

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

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    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.

  7. Mitochondrial DNA of Clathrina clathrus (Calcarea, Calcinea): six linear chromosomes, fragmented rRNAs, tRNA editing, and a novel genetic code.

    Science.gov (United States)

    Lavrov, Dennis V; Pett, Walker; Voigt, Oliver; Wörheide, Gert; Forget, Lise; Lang, B Franz; Kayal, Ehsan

    2013-04-01

    Sponges (phylum Porifera) are a large and ancient group of morphologically simple but ecologically important aquatic animals. Although their body plan and lifestyle are relatively uniform, sponges show extensive molecular and genetic diversity. In particular, mitochondrial genomes from three of the four previously studied classes of Porifera (Demospongiae, Hexactinellida, and Homoscleromorpha) have distinct gene contents, genome organizations, and evolutionary rates. Here, we report the mitochondrial genome of Clathrina clathrus (Calcinea, Clathrinidae), a representative of the fourth poriferan class, the Calcarea, which proves to be the most unusual. Clathrina clathrus mitochondrial DNA (mtDNA) consists of six linear chromosomes 7.6-9.4 kb in size and encodes at least 37 genes: 13 protein codings, 2 ribosomal RNAs (rRNAs), and 24 transfer RNAs (tRNAs). Protein genes include atp9, which has now been found in all major sponge lineages, but no atp8. Our analyses further reveal the presence of a novel genetic code that involves unique reassignments of the UAG codons from termination to tyrosine and of the CGN codons from arginine to glycine. Clathrina clathrus mitochondrial rRNAs are encoded in three (srRNA) and ≥6 (lrRNA) fragments distributed out of order and on several chromosomes. The encoded tRNAs contain multiple mismatches in the aminoacyl acceptor stems that are repaired posttranscriptionally by 3'-end RNA editing. Although our analysis does not resolve the phylogenetic position of calcareous sponges, likely due to their high rates of mitochondrial sequence evolution, it confirms mtDNA as a promising marker for population studies in this group. The combination of unusual mitochondrial features in C. clathrus redefines the extremes of mtDNA evolution in animals and further argues against the idea of a "typical animal mtDNA."

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

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    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

  9. 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…

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

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    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.

  11. 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

  12. 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

    2016-10-26

    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.

  13. New phagotrophic euglenoid species (new genus Decastava; Scytomonas saepesedens; Entosiphon oblongum), Hsp90 introns, and putative euglenoid Hsp90 pre-mRNA insertional editing.

    Science.gov (United States)

    Cavalier-Smith, Thomas; Chao, Ema E; Vickerman, Keith

    2016-10-01

    We describe three new phagotrophic euglenoid species by light microscopy and 18S rDNA and Hsp90 sequencing: Scytomonas saepesedens; Decastava edaphica; Entosiphon oblongum. We studied Scytomonas and Decastava ultrastructure. Scytomonas saepesedens feeds when sessile with actively beating cilium, and has five pellicular strips with flush joints and Calycimonas-like microtubule-supported cytopharynx. Decastava, sister to Keelungia forming new clade Decastavida on 18S rDNA trees, has 10 broad strips with cusp-like joints, not bifurcate ridges like Ploeotia and Serpenomonas (phylogenetically and cytologically distinct genera), and Serpenomonas-like feeding apparatus (8-9 unreinforced microtubule pairs loop from dorsal jaw support to cytostome). Hsp90 and 18S rDNA trees group Scytomonas with Petalomonas and show Entosiphon as the earliest euglenoid branch. Basal euglenoids have rigid longitudinal strips; derived clade Spirocuta has spiral often slideable strips. Decastava Hsp90 genes have introns. Decastava/Entosiphon Hsp90 frameshifts imply insertional RNA editing. Petalomonas is too heterogeneous in pellicle structure for one genus; we retain Scytomonas (sometimes lumped with it) and segregate four former Petalomonas as new genus Biundula with pellicle cross section showing 2-8 smooth undulations and typified by Biundula (=Petalomonas) sphagnophila comb. n. Our taxon-rich site-heterogeneous rDNA trees confirm that Heteronema is excessively heterogeneous; therefore we establish new genus Teloprocta for Heteronema scaphurum.

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

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    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.

  15. 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.

  16. 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 -

  17. Alu polymorphic insertions reveal genetic structure of north Indian populations.

    Science.gov (United States)

    Tripathi, Manorama; Tripathi, Piyush; Chauhan, Ugam Kumari; Herrera, Rene J; Agrawal, Suraksha

    2008-10-01

    The Indian subcontinent is characterized by the ancestral and cultural diversity of its people. Genetic input from several unique source populations and from the unique social architecture provided by the caste system has shaped the current genetic landscape of India. In the present study 200 individuals each from three upper-caste and four middle-caste Hindu groups and from two Muslim populations in North India were examined for 10 polymorphic Alu insertions (PAIs). The investigated PAIs exhibit high levels of polymorphism and average heterozygosity. Limited interpopulation variance and genetic flow in the present study suggest admixture. The results of this study demonstrate that, contrary to common belief, the caste system has not provided an impermeable barrier to genetic exchange among Indian groups.

  18. The mitochondrial genome of the gymnosperm Cycas taitungensis contains a novel family of short interspersed elements, Bpu sequences, and abundant RNA editing sites.

    Science.gov (United States)

    Chaw, Shu-Miaw; Shih, Arthur Chun-Chieh; Wang, Daryi; Wu, Yu-Wei; Liu, Shu-Mei; Chou, The-Yuan

    2008-03-01

    The mtDNA of Cycas taitungensis is a circular molecule of 414,903 bp, making it 2- to 6-fold larger than the known mtDNAs of charophytes and bryophytes, but similar to the average of 7 elucidated angiosperm mtDNAs. It is characterized by abundant RNA editing sites (1,084), more than twice the number found in the angiosperm mtDNAs. The A + T content of Cycas mtDNA is 53.1%, the lowest among known land plants. About 5% of the Cycas mtDNA is composed of a novel family of mobile elements, which we designated as "Bpu sequences." They share a consensus sequence of 36 bp with 2 terminal direct repeats (AAGG) and a recognition site for the Bpu 10I restriction endonuclease (CCTGAAGC). Comparison of the Cycas mtDNA with other plant mtDNAs revealed many new insights into the biology and evolution of land plant mtDNAs. For example, the noncoding sequences in mtDNAs have drastically expanded as land plants have evolved, with abrupt increases appearing in the bryophytes, and then in the seed plants. As a result, the genomic organizations of seed plant mtDNAs are much less compact than in other plants. Also, the Cycas mtDNA appears to have been exempted from the frequent gene loss observed in angiosperm mtDNAs. Similar to the angiosperms, the 3 Cycas genes nad1, nad2, and nad5 are disrupted by 5 group II intron squences, which have brought the genes into trans-splicing arrangements. The evolutionary origin and invasion/duplication mechanism of the Bpu sequences in Cycas mtDNA are hypothesized and discussed.

  19. 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.

  20. 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.

  1. Molecular docking and molecular dynamics simulation studies on Thermus thermophilus leucyl-tRNA synthetase complexed with different amino acids and pre-transfer editing substrates

    Directory of Open Access Journals (Sweden)

    Rayevsky A. V.

    2016-02-01

    Full Text Available Aim. To investigate the structural bases for the amino acid selectivity of the Thermus thermophilus leucyl-tRNA synthetase (LeuRSTT aminoacylation site and to disclose the binding pattern of pre-transfer editing substrates. Methods. Eight amino acids proposed as semi-cognate substrates for aminoacylation and eight aminoacyl-adenylates (formed from AMP and eight amino acids were prepared in zwitterions form. The protein structure with a co-crystallized substrate in the aminoacylation site [PDBID: 1OBH] was taken from RCSB. Docking settings and evaluation of substrate efficiency were followed by twofold docking function analysis for each conformation with Gold CCDC. The molecular dynamics simulation was performed using Gromacs. The procedures of relaxation and binding study were separated in two different subsequent simulations for 50ns and 5ns. Results. The evaluation of substrate efficiency for 8 amino acids by twofold docking function analysis, based on score values,has shown that the ligands of LeuRSTT can be positioned in the following order: Leu>Nva>Hcy>Nle>Met>Cys>Ile >Val. MD simulation has revealed lower electrostatic interactions of isoleucine with the active site of the enzyme compared with those for norvaline and leucine. In the case of aminoacyl-adenylates no significant differences were found based on score values for both GoldScore and Asp functions. Molecular dynamics of leucyl-, isoleucyl- and norvalyl-adenylates showed that the most stable and conformationally favorable is leucine, then follow norvaline and isoleucine. It has been also found that the TYR43 of the active site covers carboxyl group of leucine and norvaline like a shield and deflected towards isoleucine, allowing water molecules to come closer. Conclusions. In this study we revealed some structural basis for screening unfavorable substrates by shape, size and flexibility of a radical. The results obtained for different amino acids by molecular docking and MD studies

  2. 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.

  3. Enrichment analysis of Alu elements with different spatial chromatin proximity in the human genome.

    Science.gov (United States)

    Gu, Zhuoya; Jin, Ke; Crabbe, M James C; Zhang, Yang; Liu, Xiaolin; Huang, Yanyan; Hua, Mengyi; Nan, Peng; Zhang, Zhaolei; Zhong, Yang

    2016-04-01

    Transposable elements (TEs) have no longer been totally considered as "junk DNA" for quite a time since the continual discoveries of their multifunctional roles in eukaryote genomes. As one of the most important and abundant TEs that still active in human genome, Alu, a SINE family, has demonstrated its indispensable regulatory functions at sequence level, but its spatial roles are still unclear. Technologies based on 3C (chromosome conformation capture) have revealed the mysterious three-dimensional structure of chromatin, and make it possible to study the distal chromatin interaction in the genome. To find the role TE playing in distal regulation in human genome, we compiled the new released Hi-C data, TE annotation, histone marker annotations, and the genome-wide methylation data to operate correlation analysis, and found that the density of Alu elements showed a strong positive correlation with the level of chromatin interactions (hESC: r = 0.9, P elements like enhancers and promoters (Enhancer: hESC: r = 0.997, P = 2.3 × 10(-4); IMR90: r = 0.934, P = 2 × 10(-2); Promoter: hESC: r = 0.995, P = 3.8 × 10(-4); IMR90: r = 0.996, P = 3.2 × 10(-4)). Further investigation involving GC content and methylation status showed the GC content of Alu covered sequences shared a similar pattern with that of the overall sequence, suggesting that Alu elements also function as the GC nucleotide and CpG site provider. In all, our results suggest that the Alu elements may act as an alternative parameter to evaluate the Hi-C data, which is confirmed by the correlation analysis of Alu elements and histone markers. Moreover, the GC-rich Alu sequence can bring high GC content and methylation flexibility to the regions with more distal chromatin contact, regulating the transcription of tissue-specific genes.

  4. The pivotal roles of TIA proteins in 5' splice-site selection of alu exons and across evolution.

    Directory of Open Access Journals (Sweden)

    Nurit Gal-Mark

    2009-11-01

    Full Text Available More than 5% of alternatively spliced internal exons in the human genome are derived from Alu elements in a process termed exonization. Alus are comprised of two homologous arms separated by an internal polypyrimidine tract (PPT. In most exonizations, splice sites are selected from within the same arm. We hypothesized that the internal PPT may prevent selection of a splice site further downstream. Here, we demonstrate that this PPT enhanced the selection of an upstream 5' splice site (5'ss, even in the presence of a stronger 5'ss downstream. Deletion of this PPT shifted selection to the stronger downstream 5'ss. This enhancing effect depended on the strength of the downstream 5'ss, on the efficiency of base-pairing to U1 snRNA, and on the length of the PPT. This effect of the PPT was mediated by the binding of TIA proteins and was dependent on the distance between the PPT and the upstream 5'ss. A wide-scale evolutionary analysis of introns across 22 eukaryotes revealed an enrichment in PPTs within approximately 20 nt downstream of the 5'ss. For most metazoans, the strength of the 5'ss inversely correlated with the presence of a downstream PPT, indicative of the functional role of the PPT. Finally, we found that the proteins that mediate this effect, TIA and U1C, and in particular their functional domains, are highly conserved across evolution. Overall, these findings expand our understanding of the role of TIA1/TIAR proteins in enhancing recognition of exons, in general, and Alu exons, in particular.

  5. 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.

  6. Cluster editing

    DEFF Research Database (Denmark)

    Böcker, S.; Baumbach, Jan

    2013-01-01

    . The 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......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...

  7. 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.

  8. 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...

  9. 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.

  10. Age-Associated ALU Element Instability in White Blood Cells Is Linked to Lower Survival in Elderly Adults: A Preliminary Cohort Study

    Science.gov (United States)

    Venturelli, Massimo; Gross, Cole; Tarperi, Cantor; Schena, Federico; Reggiani, Carlo; Naro, Fabio; Pedrinolla, Anna; Monaco, Lucia; Richardson, Russell S.; Donato, Anthony J.

    2017-01-01

    Background ALU element instability could contribute to gene function variance in aging, and may partly explain variation in human lifespan. Objective To assess the role of ALU element instability in human aging and the potential efficacy of ALU element content as a marker of biological aging and survival. Design Preliminary cohort study. Methods We measured two high frequency ALU element subfamilies, ALU-J and ALU-Sx, by a single qPCR assay and compared ALU-J/Sx content in white blood cell (WBCs) and skeletal muscle cell (SMCs) biopsies from twenty-three elderly adults with sixteen healthy sex-balanced young adults; all-cause survival rates of elderly adults predicted by ALU-J/Sx content in both tissues; and cardiovascular disease (CVD)- and cancer-specific survival rates of elderly adults predicted by ALU-J/Sx content in both tissues, as planned subgroup analyses. Results We found greater ALU-J/Sx content variance in WBCs from elderly adults than young adults (P < 0.001) with no difference in SMCs (P = 0.94). Elderly adults with low WBC ALU-J/Sx content had worse four-year all-cause and CVD-associated survival than those with high ALU-J/Sx content (both P = 0.03 and hazard ratios (HR) ≥ 3.40), while WBC ALU-J/Sx content had no influence on cancer-associated survival (P = 0.42 and HR = 0.74). SMC ALU-J/Sx content had no influence on all-cause, CVD- or cancer -associated survival (all P ≥ 0.26; HR ≤ 2.07). Conclusions These initial findings demonstrate that ALU element instability occurs with advanced age in WBCs, but not SMCs, and imparts greater risk of all-cause mortality that is likely driven by an increased risk for CVD and not cancer. PMID:28060910

  11. 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...

  12. 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

  13. 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

    Methylation of the cytosine is the most frequent epigenetic modification of DNA in mammalian cells. In humans, most of the methylated cytosines are found in CpG-rich sequences within tandem and interspersed repeats that make up to 45% of the human genome, being Alu repeats the most common family....

  14. Underwater video footage, March 2014, Faga'alu Bay, Tutuila Island, American Samoa

    Data.gov (United States)

    U.S. Geological Survey, Department of the Interior — Underwater video imagery was collected in March 2014 in the nearshore waters of Faga'alu Bay on the Island of Tutuila, American Samoa, as part of the U.S. Geological...

  15. 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 alon...

  16. Enrichment analysis of Alu elements with different spatial chromatin proximity in the human genome

    Directory of Open Access Journals (Sweden)

    Zhuoya Gu

    2016-02-01

    Full Text Available ABSTRACT Transposable elements (TEs have no longer been totally considered as “junk DNA” for quite a time since the continual discoveries of their multifunctional roles in eukaryote genomes. As one of the most important and abundant TEs that still active in human genome, Alu, a SINE family, has demonstrated its indispensable regulatory functions at sequence level, but its spatial roles are still unclear. Technologies based on 3C (chromosome conformation capture have revealed the mysterious three-dimensional structure of chromatin, and make it possible to study the distal chromatin interaction in the genome. To find the role TE playing in distal regulation in human genome, we compiled the new released Hi-C data, TE annotation, histone marker annotations, and the genome-wide methylation data to operate correlation analysis, and found that the density of Alu elements showed a strong positive correlation with the level of chromatin interactions (hESC: r = 0.9, P < 2.2 × 1016; IMR90 fibroblasts: r = 0.94, P < 2.2 × 1016 and also have a significant positive correlation with some remote functional DNA elements like enhancers and promoters (Enhancer: hESC: r = 0.997, P = 2.3 × 10−4; IMR90: r = 0.934, P = 2 × 10−2; Promoter: hESC: r = 0.995, P = 3.8 × 10−4; IMR90: r = 0.996, P = 3.2 × 10−4. Further investigation involving GC content and methylation status showed the GC content of Alu covered sequences shared a similar pattern with that of the overall sequence, suggesting that Alu elements also function as the GC nucleotide and CpG site provider. In all, our results suggest that the Alu elements may act as an alternative parameter to evaluate the Hi-C data, which is confirmed by the correlation analysis of Alu elements and histone markers. Moreover, the GC-rich Alu sequence can bring high GC content and methylation flexibility to the regions with more distal chromatin contact, regulating the transcription of tissue

  17. 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.

  18. 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

  19. AluScan: a method for genome-wide scanning of sequence and structure variations in the human genome

    Directory of Open Access Journals (Sweden)

    Mei Lingling

    2011-11-01

    Full Text Available Abstract Background To complement next-generation sequencing technologies, there is a pressing need for efficient pre-sequencing capture methods with reduced costs and DNA requirement. The Alu family of short interspersed nucleotide elements is the most abundant type of transposable elements in the human genome and a recognized source of genome instability. With over one million Alu elements distributed throughout the genome, they are well positioned to facilitate genome-wide sequence amplification and capture of regions likely to harbor genetic variation hotspots of biological relevance. Results Here we report on the use of inter-Alu PCR with an enhanced range of amplicons in conjunction with next-generation sequencing to generate an Alu-anchored scan, or 'AluScan', of DNA sequences between Alu transposons, where Alu consensus sequence-based 'H-type' PCR primers that elongate outward from the head of an Alu element are combined with 'T-type' primers elongating from the poly-A containing tail to achieve huge amplicon range. To illustrate the method, glioma DNA was compared with white blood cell control DNA of the same patient by means of AluScan. The over 10 Mb sequences obtained, derived from more than 8,000 genes spread over all the chromosomes, revealed a highly reproducible capture of genomic sequences enriched in genic sequences and cancer candidate gene regions. Requiring only sub-micrograms of sample DNA, the power of AluScan as a discovery tool for genetic variations was demonstrated by the identification of 357 instances of loss of heterozygosity, 341 somatic indels, 274 somatic SNVs, and seven potential somatic SNV hotspots between control and glioma DNA. Conclusions AluScan, implemented with just a small number of H-type and T-type inter-Alu PCR primers, provides an effective capture of a diversity of genome-wide sequences for analysis. The method, by enabling an examination of gene-enriched regions containing exons, introns, and

  20. CRISPR as a strong gene editing tool.

    Science.gov (United States)

    Shen, Shengfu; Loh, Tiing Jen; Shen, Hongling; Zheng, Xuexiu; Shen, Haihong

    2017-01-01

    Clustered regularly-interspaced short palindromic repeats (CRISPR) is a new and effective genetic editing tool. CRISPR was initially found in bacteria to protect it from virus invasions. In the first step, specific DNA strands of virus are identified by guide RNA that is composed of crRNA and tracrRNA. Then RNAse III is required for producing crRNA from pre-crRNA. In The second step, a crRNA:tracrRNA:Cas9 complex guides RNase III to cleave target DNA. After cleavage of DNA by CRISPR-Cas9, DNA can be fixed by Non- Homologous End Joining (NHEJ) and Homology Directed Repair (HDR). Whereas NHEJ is simple and random, HDR is much more complex and accurate. Gene editing by CRISPR is able to be applied to various biological field such as agriculture and treating genetic diseases in human. [BMB Reports 2017; 50(1): 20-24].

  1. 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.

  2. 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.

  3. Alu-mediated large deletion of the CDSN gene as a cause of peeling skin disease.

    Science.gov (United States)

    Wada, T; Matsuda, Y; Muraoka, M; Toma, T; Takehara, K; Fujimoto, M; Yachie, A

    2014-10-01

    Peeling skin disease (PSD) is an autosomal recessive skin disorder caused by mutations in CDSN and is characterized by superficial peeling of the upper epidermis. Corneodesmosin (CDSN) is a major component of corneodesmosomes that plays an important role in maintaining epidermis integrity. Herein, we report a patient with PSD caused by a novel homozygous large deletion in the 6p21.3 region encompassing the CDSN gene, which abrogates CDSN expression. Several genes including C6orf15, PSORS1C1, PSORS1C2, CCHCR1, and TCF19 were also deleted, however, the patient showed only clinical features typical of PSD. The deletion size was 59.1 kb. Analysis of the sequence surrounding the breakpoint showed that both telomeric and centromeric breakpoints existed within Alu-S sequences that were oriented in opposite directions. These results suggest an Alu-mediated recombination event as the mechanism underlying the deletion in our patient.

  4. 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.

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

    OpenAIRE

    Zulkharnaim; Jakaria; R. R. Noor

    2010-01-01

    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, whic...

  6. Editing modifies the GABA(A) receptor subunit alpha3

    DEFF Research Database (Denmark)

    Ohlson, Johan; Pedersen, Jakob Skou; Haussler, David

    2007-01-01

    Adenosine to inosine (A-to-I) pre-mRNA editing by the ADAR enzyme family has the potential to increase the variety of the proteome. This editing by adenosine deamination is essential in mammals for a functional brain. To detect novel substrates for A-to-I editing we have used an experimental method...... 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(A) receptor......, is a substrate for editing by both ADAR1 and ADAR2. Editing of the Gabra-3 mRNA recodes an isoleucine to a methionine. The extent of editing is low at birth but increases with age, reaching close to 100% in the adult brain. We therefore propose that editing of the Gabra-3 mRNA is important for normal brain...

  7. 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.

  8. 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.

  9. Design and implementation of low power clock gated 64-bit ALU on ultra scale FPGA

    Science.gov (United States)

    Gupta, Ashutosh; Murgai, Shruti; Gulati, Anmol; Kumar, Pradeep

    2016-03-01

    64-bit energy efficient Arithmetic and Logic Unit using negative latch based clock gating technique is designed in this paper. The 64-bit ALU is designed using multiplexer based full adder cell. We have designed a 64-bit ALU with a gated clock. We have used negative latch based circuit for generating gated clock. This gated clock is used to control the multiplexer based 64-bit ALU. The circuit has been synthesized on kintex FPGA through Xilinx ISE Design Suite 14.7 using 28 nm technology in Verilog HDL. The circuit has been simulated on Modelsim 10.3c. The design is verified using System Verilog on QuestaSim in UVM environment. We have achieved 74.07%, 92. 93% and 95.53% reduction in total clock power, 89.73%, 91.35% and 92.85% reduction in I/Os power, 67.14%, 62.84% and 74.34% reduction in dynamic power and 25.47%, 29.05% and 46.13% reduction in total supply power at 20 MHz, 200 MHz and 2 GHz frequency respectively. The power has been calculated using XPower Analyzer tool of Xilinx ISE Design Suite 14.3.

  10. 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

  11. 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.

  12. 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

  13. The X chromosome Alu insertions as a tool for human population genetics: data from European and African human groups.

    Science.gov (United States)

    Athanasiadis, Georgios; Esteban, Esther; Via, Marc; Dugoujon, Jean-Michel; Moschonas, Nicholas; Chaabani, Hassen; Moral, Pedro

    2007-05-01

    Alu elements are the most abundant mobile elements in the human genome (approximately 1,100,000 copies). Polymorphic Alu elements have been proved to be useful in studies of human origins and relationships owing to two important advantages: identity by descent and absence of the Alu element known to be the ancestral state. Alu variation in the X chromosome has been described previously in human populations but, as far as we know, these elements have not been used in population relationship studies. Here, we describe the allele frequencies of 13 'young' Alu elements of the X chromosome (Ya5DP62, Ya5DP57, Yb8DP49, Ya5a2DP1, Yb8DP2, Ya5DP3, Ya5NBC37, Yd3JX437, Ya5DP77, Ya5NBC491, Yb8NBC578, Ya5DP4 and Ya5DP13) in six human populations from sub-Saharan Africa (the Ivory Coast), North Africa (Moroccan High Atlas, Siwa oasis in Egypt, Tunisia), Greece (Crete Island) and Spain (Basque Country). Eight out of 13 Alu elements have shown remarkably high gene diversity values in all groups (average heterozygosities: 0.342 in the Ivory Coast, 0.250 in North Africa, 0.209 in Europe). Genetic relationships agree with a geographical pattern of differentiation among populations, with some peculiar features observed in North Africans. Crete Island and the Basque Country show the lowest genetic distance (0.0163) meanwhile Tunisia, in spite of its geographical location, lies far from the other two North African samples. The results of our work demonstrate that X chromosome Alu elements comprise a reliable set of genetic markers useful to describe human population relationships for fine-scale geographical studies.

  14. Crystallization of the Zalpha domain of the human editing enzyme ADAR1 complexed with a DNA-RNA chimeric oligonucleotide in the left-handed Z-conformation.

    Science.gov (United States)

    Brown, Bernard A; Athanasiadis, Alekos; Hanlon, Eugene B; Lowenhaupt, Ky; Wilbert, Christina M; Rich, Alexander

    2002-01-01

    The Zalpha domain of human double-stranded RNA adenosine deaminase (ADAR1) has been crystallized with a hexanucleotide containing alternating deoxyribose and ribose furanose sugars. Solution circular dichroism experiments show that this double-stranded chimera (dCrG)(3) initially adopts the right-handed A-conformation. However, addition of stoichiometric amounts of Zalpha causes a rapid transition to the Z-conformation. Raman spectroscopy of crystals of the Zalpha-(dCrG)(3) complex confirm that the chimeric oligonucleotide is stabilized in the Z-conformation. A complete data set has been obtained at 2.5 A resolution. The Zalpha-(dCrG)(3) crystals belong to the tetragonal I422 space group, with unit-cell parameters a = b = 104.2, c = 117.6 A. Work is under way to solve the structure by molecular replacement.

  15. 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)).

  16. ADAR1 forms a complex with Dicer to promote microRNA processing and RNA-induced gene silencing.

    NARCIS (Netherlands)

    Ota, H.; Sakurai, M.; Gupta, R; Valente, L.; Wulff, B.E.; Ariyoshi, K.; Iizasa, H.; Davuluri, R.V.; Nishikura, K.

    2013-01-01

    Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosine residues to inosine specifically in double-stranded RNAs. In this study, we investigated the interaction of the RNA editing mechanism with the RNA interference (RNAi) machinery and found that ADAR1 forms a

  17. 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.

  18. 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.

  19. 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.

  20. 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...... for editing nucleic acids and a cell comprising a stably integrated endonuclease....

  1. An AluI RFLP detected in the human prion protein (PrP) gene

    Energy Technology Data Exchange (ETDEWEB)

    Harris, M.S.; Devine-Gage, E. (New York State Institute for Basic Research in Developmental Disabilities, Staten Island (USA)); Robakis, N.K. (Mt. Sinai School of Medicine, New York, NY (USA))

    1990-01-25

    Probe pEA974 contains a 974 bp fragment present in Prp Clone XIV inserted into pBR322. A PvuII RFLP has been previously described. AluI identifies a two allele polymorphism of either a band at 965 bp, a band at 565 bp, or two bands at 965 bp and 565 bp. The allele frequency was studied in 14 European Caucasians. The PrP gene has been sublocalized to the p arm of chromosome 20. Mendelian pattern of inheritance was shown in an informative family.

  2. 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.

  3. 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....

  4. Genetic change in the polynesian population of Easter Island: evidence from Alu insertion polymorphisms.

    Science.gov (United States)

    González-Pérez, E; Esteban, E; Via, M; García-Moro, C; Hernández, M; Moral, P

    2006-11-01

    The origin of Pacific islanders is still an open issue in human population genetics. To address this topic we analyzed a set of 18 Alu insertion polymorphisms in a total of 176 chromosomes from native Easter Island inhabitants (Rapanui). Available genealogical records allowed us to subdivide the total island sample into two groups, representative of the native population living in the island around 1900, and another formed by individuals with some ancestors of non-Rapanui origin. Significant genetic differentiation was found between these groups, allowing us to make some biodemographic and historical inferences about the origin and evolution of this geographically isolated island population. Our data are consistent with equivalent and recent contributions from Amerindian and European migrants to the 1900s Rapanui population, with an accelerated increase in the European gene flow during the 20(th) century, especially since the 1960s. Comparative analysis of our results with other available Alu variation data on neighbouring populations supports the "Voyaging Corridor" model of Polynesian human settlement, which indicates that pre-Polynesians are mainly derived from Southeast Asian and Wallacean populations rather than from Taiwan or the Philippines. This study underlines the importance of sampling and taking into account historical information in genetic studies to unravel the recent evolution of human populations.

  5. 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.

  6. 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.

  7. 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.

  8. Genome editing with engineered nucleases in plants.

    Science.gov (United States)

    Osakabe, Yuriko; Osakabe, Keishi

    2015-03-01

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

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

    NARCIS (Netherlands)

    Peixoto, Ana; Santos, Catarina; Pinheiro, Manuela; Pinto, Pedro; Soares, Maria Jose; Rocha, Patricia; Gusmao, Leonor; Amorim, Antonio; van der Hout, Annemarie; Gerdes, Anne-Marie; Thomassen, Mads; Kruse, Torben A.; Cruger, Dorthe; Sunde, Lone; Bignon, Yves-Jean; Uhrhammer, Nancy; Cornil, Lucie; Rouleau, Etienne; Lidereau, Rosette; Yannoukakos, Drakoulis; Pertesi, Maroulio; Narod, Steven; Royer, Robert; Costa, Mauricio M.; Lazaro, Conxi; Feliubadalo, Lidia; Grana, Begona; Blanco, Ignacio; de la Hoya, Miguel; Caldes, Trinidad; Maillet, Philippe; Benais-Pont, Gaelle; Pardo, Bruno; Laitman, Yael; Friedman, Eitan; Velasco, Eladio A.; Duran, Mercedes; Miramar, Maria-Dolores; Rodriguez Valle, Ana; Calvo, Maria-Teresa; Vega, Ana; Blanco, Ana; Diez, Orland; Gutierrez-Enriquez, Sara; Balmana, Judith; Ramon y Cajal, Teresa; Alonso, Carmen; Baiget, Montserrat; Foulkes, William; Tischkowitz, Marc; Kyle, Rachel; Sabbaghian, Nelly; Ashton-Prolla, Patricia; Ewald, Ingrid P.; Rajkumar, Thangarajan; Mota-Vieira, Luisa; Giannini, Giuseppe; Gulino, Alberto; Achatz, Maria I.; Carraro, Dirce M.; de Paillerets, Brigitte Bressac; Remenieras, Audrey; Benson, Cindy; Casadei, Silvia; King, Mary-Claire; Teugels, Erik; Teixeira, Manuel R.

    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 to

  10. 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...

  11. Association of the Alu insertion polymorphism in the progesterone receptor gene with breast cancer in a Mexican population

    Science.gov (United States)

    Figuera, Luis E.; Flores-Ramos, Liliana Gómez; Puebla-Pérez, Ana María; Zúñiga-González, Guillermo Moisés

    2015-01-01

    Introduction The progesterone receptor (PR) gene plays an important role in reproduction-related events. Data on polymorphisms in the PR gene have revealed associations with cancer, particularly for the Alu insertion polymorphism, which has been suggested to affect progesterone receptor function and contribute to tumor promotion in the mammary gland. Material and methods We examined the role of the Alu insertion polymorphism in the PR gene by comparing the genotypes of 209 healthy Mexican women with those of 481 Mexican women with breast cancer (BC). Results The genotype frequencies observed in the controls and BC patients were 0% and 4% for T2/T2 (Alu insertion), 16% and 21% for T1/T2, and 84% and 75% for T1/T1 (Alu deletion), respectively. The obtained odds ratio (OR) was 1.7, with a 95% confidence interval (95% CI) of 1.1–2.6, p = 0.009, for the T1/T2–T2/T2 genotypes. The association was also evident when the distributions of the T1/T2–T2/T2 genotypes in patients in the following categories were compared: obesity grade II (OR = 1.81, 95% CI: 1.03–3.18, p = 0.039) and the chemotherapy response (OR = 1.91, 95% CI: 1.27–3.067, p = 0.002). Conclusions The T1/T2–T2/T2 genotypes of the Alu insertion polymorphism in the PR gene are associated with BC susceptibility in the analyzed Mexican population. PMID:26170848

  12. 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.

  13. Evolution of human alpha 1-acid glycoprotein genes and surrounding Alu repeats.

    Science.gov (United States)

    Merritt, C M; Easteal, S; Board, P G

    1990-04-01

    There is a mosaic pattern of variation between the two tandemly arranged human alpha 1-acid glycoprotein genes. Both the synonymous and the nonsynonymous sites of exons 3 and 4 are more divergent than the rest of the gene, suggesting that they have had a different evolutionary history. Comparisons of the two gene sequences with rat AGP indicate that exons 3 and 4 of AGP2 have been evolving without functional constraint since their divergence from AGP1. It is proposed that the conserved region of the gene has been homogenized recently by gene conversion with the homologous regions of AGP1. The Alu sequences surrounding the genes appear to have been involved in both the gene duplication and the gene conversion events.

  14. Antisense sequencing improves the accuracy and precision of A-to-I editing measurements using the peak height ratio method

    Directory of Open Access Journals (Sweden)

    Rinkevich Frank D

    2012-01-01

    Full Text Available Abstract Background A-to-I RNA editing is found in all phyla of animals and contributes to transcript diversity that may have profound impacts on behavior and physiology. Many transcripts of genes involved in axonal conductance, synaptic transmission and modulation are the targets of A-to-I RNA editing. There are a number of methods to measure the extent of A-to-I RNA editing, but they are generally costly and time consuming. One way to determine the frequency of A-to-I RNA editing is the peak height ratio method, which compares the size of peaks on electropherograms that represent unedited and edited sites. Findings Sequencing of 4 editing sites of the Dα6 nicotinic acetylcholine receptor subunit with an antisense primer (which uses T/C peaks to measure unedited and edited sites, respectively showed very accurate and precise measurements of A-to-I RNA editing. The accuracy and precision were excellent for all editing sites, including those edited with high or low frequencies. The frequency of A-to-I RNA editing was comparable to the editing frequency as measured by clone counting from the same sample. Sequencing these same sites with the sense primer (which uses A/G peaks yielded inaccurate and imprecise measurements. Conclusions We have validated and improved the accuracy and precision of the peak height ratio method to measure the frequency of A-to-I RNA editing, and shown that results are primer specific. Thus, the correct sequencing primer must be utilized for the most dependable data. When compared to other methods used to measure the frequency of A-to-I RNA editing, the major benefits of the peak height ratio are that this method is inexpensive, fast, non-labor intensive and easily adaptable to many laboratory and field settings.

  15. Developing new levels of edit

    Energy Technology Data Exchange (ETDEWEB)

    Prono, J.; DeLanoy, M.; Deupree, R.; Skiby, J.; Thompson, B.

    1998-07-01

    In 1985, the writing and editing group at Los Alamos National Laboratory established four levels of edit for technical reports. When a survey in 1994 showed that both authors and editors felt the levels were not meeting author needs, the authors set about revising them. Their goals were to simplify the editing process, focus editing on improving technical clarity, and ensure that value was added in editing. This paper describes the revision process and product -- three author-based levels of edit.

  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. Learning string edit distance

    CERN Document Server

    Ristad, E S; Ristad, Eric Sven; Yianilos, Peter N.

    1996-01-01

    In many applications, it is necessary to determine the similarity of two strings. A widely-used notion of string similarity is the edit distance: the minimum number of insertions, deletions, and substitutions required to transform one string into the other. In this report, we provide a stochastic model for string edit distance. Our stochastic model allows us to learn a string edit distance function from a corpus of examples. We illustrate the utility of our approach by applying it to the difficult problem of learning the pronunciation of words in conversational speech. In this application, we learn a string edit distance with one fourth the error rate of the untrained Levenshtein distance. Our approach is applicable to any string classification problem that may be solved using a similarity function against a database of labeled prototypes. Keywords: string edit distance, Levenshtein distance, stochastic transduction, syntactic pattern recognition, prototype dictionary, spelling correction, string correction, ...

  18. 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.

  19. Designed nucleases for targeted genome editing.

    Science.gov (United States)

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

    2016-02-01

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

  20. Plastid mRNAs are neither spliced nor edited in maize and cauliflower mitochondrial in organello systems

    OpenAIRE

    Bolle, Nina; Hinrichsen, Inga; Kempken, Frank

    2007-01-01

    The process of RNA editing in chloroplasts and higher plant mitochondria displays some similarities, raising the question of common or similar components in editing apparatus of these two organelles. To investigate the ability of plant mitochondria to edit plastid transcripts, we employed a previously established mitochondrial maize and cauliflower in organello system. Two plastid genes, Zea mays ndhB and ycf3 containing group II introns and several editing sites, were introduced into mitocho...

  1. 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.

  2. 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.

  3. Developing new levels of edit

    Energy Technology Data Exchange (ETDEWEB)

    Prono, J.K.

    1997-06-01

    Since 1985, Los Alamos National Laboratory (LANL) staff have had four levels of edit to choose from for technical reports. When a CQI survey showed that both authors and editors felt the levels were not meeting author needs, LANL set about revising them. The goals were to simplify the editing process, focus editing on improving technical clarity, and ensure value added in editing. This paper describes the revision process and product--three author-based levels of edit.

  4. 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.

  5. Alu insertion polymorphisms in the Balkans and the origins of the Aromuns.

    Science.gov (United States)

    Comas, D; Schmid, H; Braeuer, S; Flaiz, C; Busquets, A; Calafell, F; Bertranpetit, J; Scheil, H-G; Huckenbeck, W; Efremovska, L; Schmidt, H

    2004-03-01

    We have analysed 11 human-specific Alu insertion polymorphisms in the Balkans to elucidate the origins of the Aromuns, a linguistic isolate inhabiting scattered areas in the Balkan Peninsula. Four Aromun samples (two from the Republic of Macedonia, one from Albania, and one from Romania) and five neighbouring populations (Macedonians, Albanians, Romanians, Greeks, and Turks) were analysed by means of genetic distances, principal components and analyses of the molecular variance (AMOVA). Three hypotheses were tested: Aromuns are Romanophonic Greeks; the result of a Romanian southward migration; or local descendants of the Thracians. The analyses show that the Aromuns do not constitute a homogeneous group separated from the rest of the Balkan populations. Grouping by language or geography does not explain the genetic differences observed in the region, suggesting a lack of genetic structure in the area. Aromuns do not seem to be particularly related to Greeks, Romanians, or to other Romance speakers. The Aromuns might have their origin to the south of the Danube river, with extensive gene flow with the neighbouring populations. The present results suggest a common ancestry of all Balkan populations, including Aromuns, with a lack of correlation between genetic differentiation and language or ethnicity, stressing that no major migration barriers have existed in the making of the complex Balkan human puzzle.

  6. 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.

  7. 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.

  8. Generation of gene edited birds in one generation using sperm transfection assisted gene editing (STAGE).

    Science.gov (United States)

    Cooper, Caitlin A; Challagulla, Arjun; Jenkins, Kristie A; Wise, Terry G; O'Neil, Terri E; Morris, Kirsten R; Tizard, Mark L; Doran, Timothy J

    2016-11-28

    Generating transgenic and gene edited mammals involves in vitro manipulation of oocytes or single cell embryos. Due to the comparative inaccessibility of avian oocytes and single cell embryos, novel protocols have been developed to produce transgenic and gene edited birds. While these protocols are relatively efficient, they involve two generation intervals before reaching complete somatic and germline expressing transgenic or gene edited birds. Most of this work has been done with chickens, and many protocols require in vitro culturing of primordial germ cells (PGCs). However, for many other bird species no methodology for long term culture of PGCs exists. Developing methodologies to produce germline transgenic or gene edited birds in the first generation would save significant amounts of time and resource. Furthermore, developing protocols that can be readily adapted to a wide variety of avian species would open up new research opportunities. Here we report a method using sperm as a delivery mechanism for gene editing vectors which we call sperm transfection assisted gene editing (STAGE). We have successfully used this method to generate GFP knockout embryos and chickens, as well as generate embryos with mutations in the doublesex and mab-3 related transcription factor 1 (DMRT1) gene using the CRISPR/Cas9 system. The efficiency of the method varies from as low as 0% to as high as 26% with multiple factors such as CRISPR guide efficiency and mRNA stability likely impacting the outcome. This straightforward methodology could simplify gene editing in many bird species including those for which no methodology currently exists.

  9. Genome Editing in Sugarcane: Challenges ahead

    Directory of Open Access Journals (Sweden)

    Chakravarthi Mohan

    2016-10-01

    Full Text Available 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.

  10. 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

  11. Editing Audio with Audacity

    Directory of Open Access Journals (Sweden)

    Brandon Walsh

    2016-08-01

    Full Text Available For those interested in audio, basic sound editing skills go a long way. Being able to handle and manipulate the materials can help you take control of your object of study: you can zoom in and extract particular moments to analyze, process the audio, and upload the materials to a server to compliment a blog post on the topic. On a more practical level, these skills could also allow you to record and package recordings of yourself or others for distribution. That guest lecture taking place in your department? Record it and edit it yourself! Doing so is a lightweight way to distribute resources among various institutions, and it also helps make the materials more accessible for readers and listeners with a wide variety of learning needs. In this lesson you will learn how to use Audacity to load, record, edit, mix, and export audio files. Sound editing platforms are often expensive and offer extensive capabilities that can be overwhelming to the first-time user, but Audacity is a free and open source alternative that offers powerful capabilities for sound editing with a low barrier for entry. For this lesson we will work with two audio files: a recording of Bach’s Goldberg Variations available from MusOpen and another recording of your own voice that will be made in the course of the lesson. This tutorial uses Audacity 2.1.2, released January 2016.

  12. Association of AluYb8 insertion/deletion polymorphism in the MUTYH gene with mtDNA maintain in the type 2 diabetes mellitus patients.

    Science.gov (United States)

    Guo, Wenwen; Zheng, Bixia; Guo, Dong; Cai, Zhenming; Wang, Yaping

    2015-07-05

    A common AluYb8-element insertion/deletion polymorphism of the MUTYH gene (AluYb8MUTYH) is a novel genetic risk factor for type 2 diabetes mellitus (T2DM). In the present study, mtDNA sequencing analysis indicated that the mtDNA sequence heteroplasmy was not associated with AluYb8MUTYH polymorphism. To better understand the genetic risk for T2DM, we investigated the association of this polymorphism with mtDNA content, mtDNA breakage and mtDNA transcription in the leukocytes of T2DM patients. The mtDNA content and unbroken mtDNA were significantly increased in the mutant patients than in the wild-type patients (P <0.05, respectively). However, no association between mtDNA transcription and AluYb8MUTYH variant was observed. The results suggested that the AluYb8MUTYH variant was associated with an altered mtDNA maintain in T2DM patients. The high level of mtDNA content observed in the mutant patients may have resulted from inefficient base excision repair of mitochondrial MUTYH and a compensatory mechanism that is triggered by elevated oxidative stress.

  13. CRISPR/Cas9-Mediated Genome Editing of Mouse Small Intestinal Organoids

    NARCIS (Netherlands)

    Schwank, Gerald; Clevers, Hans

    2016-01-01

    The CRISPR/Cas9 system is an RNA-guided genome-editing tool that has been recently developed based on the bacterial CRISPR-Cas immune defense system. Due to its versatility and simplicity, it rapidly became the method of choice for genome editing in various biological systems, including mammalian ce

  14. Precision genome editing

    DEFF Research Database (Denmark)

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

    2014-01-01

    Precise and stable gene editing in mammalian cell lines has until recently been hampered by the lack of efficient targeting methods. While different gene silencing strategies have had tremendous impact on many biological fields, they have generally not been applied with wide success in the field...... 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...

  15. 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.

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

    OpenAIRE

    2012-01-01

    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 90n...

  17. Physical mapping of BK virus DNA with SacI, MboII, and AluI restriction endonucleases.

    Science.gov (United States)

    Yang, R C; Wu, R

    1978-12-01

    A new restriction endonuclease, SacI from Streptomyces achromogenes cleaves BK virus (strain MM) DNA into 3 fragments, whereas MboII from Moraxella bovis and AluI from Arthrobacter luteus give 22 and 30 fragments, respectively. All these specific DNA fragments were ordered and mapped on the viral genome by two methods first, by the reciprocal digestion method using uniformly 32P-labeled DNA; and second, by the partial digestion technique using the single-end 32P-labeled DNA. This study, together with those reported earlier, defined the location of 90 cleavage sites on the BK virus DNA.

  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. 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.

  20. 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...

  1. The CRISPR/Cas Genome-Editing Tool: Application in Improvement of Crops

    OpenAIRE

    2016-01-01

    The Clustered Regularly Interspaced Short Palindromic Repeats associated Cas9/sgRNA system is a novel targeted genome-editing technique derived from bacterial immune system. It is an inexpensive, easy, most user friendly and rapidly adopted genome editing tool transforming to revolutionary paradigm. This technique enables precise genomic modifications in many different organisms and tissues. Cas9 protein is an RNA guided endonuclease utilized for creating targeted double-stranded breaks with ...

  2. Genome editing: The efficient tool CRISPR–Cpf1

    KAUST Repository

    Mahfouz, Magdy M.

    2017-03-01

    The novel features of the CRISPR–Cpf1 RNA-guided endonuclease system facilitate precise and efficient genome engineering. Application of CRISPR–Cpf1 in plants shows promise for robust gene editing and regulation, opening exciting possibilities for targeted trait improvement in crops.

  3. 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.

  4. Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing.

    Science.gov (United States)

    Liang, Gang; Zhang, Huimin; Lou, Dengji; Yu, Diqiu

    2016-02-19

    The CRISPR/Cas9-sgRNA system has been developed to mediate genome editing and become a powerful tool for biological research. Employing the CRISPR/Cas9-sgRNA system for genome editing and manipulation has accelerated research and expanded researchers' ability to generate genetic models. However, the method evaluating the efficiency of sgRNAs is lacking in plants. Based on the nucleotide compositions and secondary structures of sgRNAs which have been experimentally validated in plants, we instituted criteria to design efficient sgRNAs. To facilitate the assembly of multiple sgRNA cassettes, we also developed a new strategy to rapidly construct CRISPR/Cas9-sgRNA system for multiplex editing in plants. In theory, up to ten single guide RNA (sgRNA) cassettes can be simultaneously assembled into the final binary vectors. As a proof of concept, 21 sgRNAs complying with the criteria were designed and the corresponding Cas9/sgRNAs expression vectors were constructed. Sequencing analysis of transgenic rice plants suggested that 82% of the desired target sites were edited with deletion, insertion, substitution, and inversion, displaying high editing efficiency. This work provides a convenient approach to select efficient sgRNAs for target editing.

  5. Fan edits and the legacy of The Phantom Edit

    Directory of Open Access Journals (Sweden)

    Joshua Wille

    2014-09-01

    Full Text Available A fan edit can generally be defined as an alternative version of a film or television text created by a fan. It offers a different viewing experience, much as a song remix offers a different listening experience. The contemporary wave of fan edits has emerged during the remix zeitgeist of digital media and at a time when digital video editing technology has become more affordable and popular. The increasing number of alternative versions of films and the works of revisionist Hollywood filmmakers such as George Lucas have contributed to a greater public understanding of cinema as a fluid medium instead of one that exists in a fixed form. The Phantom Edit (2000, a seminal fan edit based on Lucas's Star Wars Episode I: The Phantom Menace (1999, inspired new ranks of fan editors. However, critics have misunderstood fan edits as merely the work of disgruntled fans. In order to provide a critical and historical basis for studies in fan editing as a creative practice, I examine previous interpretations of fan edits in the context of relevant contemporary works, and I use an annotated chronology of The Phantom Edit to trace its influence on subsequent fan editing communities and uncover their relationship with intellectual property disputes.

  6. 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.

  7. 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.

  8. Characterization of Alu and recombination-associated motifs mediating a large homozygous SPG7 gene rearrangement causing hereditary spastic paraplegia.

    Science.gov (United States)

    López, Eva; Casasnovas, Carlos; Giménez, Javier; Matilla-Dueñas, Antoni; Sánchez, Ivelisse; Volpini, Víctor

    2015-04-01

    Spastic paraplegia type 7 (SPG7) is one of the most common forms of autosomal recessive hereditary spastic paraplegia (AR-HSP). Although over 77 different mutations have been identified in SPG7 patients, only 9 gross deletions have been reported with only a few of them being fully characterized. Here, we present a detailed description of a large homozygous intragenic SPG7 gene rearrangement involving a 5144-base pair (bp) genomic loss (c. 1450-446_1779 + 746 delinsAAAGTGCT) encompassing exons 11 to 13, identified in a Spanish AR-HSP family. Analysis of the deletion junction sequences revealed that the 5' breakpoint of this SPG7 gene deletion was located within highly homologous Alu sequences where the 3' breakpoint appears to be flanked by the core crossover hotspot instigator (chi)-like sequence (GCTGG). Furthermore, an 8-bp (AAAGTTGCT) conserved sequence at the breakpoint junction was identified, suggesting that the most likely mechanism for the occurrence of this rearrangement is by Alu microhomology and chi-like recombination-associated motif-mediated multiple exon deletion. Our results are consistent with non-allelic homologous recombination and non-homologous end joining in deletion mutagenesis for the generation of rearrangements. This study provides more evidence associating repeated elements as a genetic mechanism underlying neurodegenerative disorders, highlighting their importance in human diseases.

  9. Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes.

    Science.gov (United States)

    Chen, Sean; Lee, Benjamin; Lee, Angus Yiu-Fai; Modzelewski, Andrew J; He, Lin

    2016-07-08

    The CRISPR/Cas9 system has been employed to efficiently edit the genomes of diverse model organisms. CRISPR-mediated mouse genome editing is typically accomplished by microinjection of Cas9 DNA/RNA and single guide RNA (sgRNA) into zygotes to generate modified animals in one step. However, microinjection is a technically demanding, labor-intensive, and costly procedure with poor embryo viability. Here, we describe a simple and economic electroporation-based strategy to deliver Cas9/sgRNA ribonucleoproteins into mouse zygotes with 100% efficiency for in vivo genome editing. Our methodology, designated as CRISPR RNP Electroporation of Zygotes (CRISPR-EZ), enables highly efficient and high-throughput genome editing in vivo, with a significant improvement in embryo viability compared with microinjection. Using CRISPR-EZ, we generated a variety of editing schemes in mouse embryos, including indel (insertion/deletion) mutations, point mutations, large deletions, and small insertions. In a proof-of-principle experiment, we used CRISPR-EZ to target the tyrosinase (Tyr) gene, achieving 88% bi-allelic editing and 42% homology-directed repair-mediated precise sequence modification in live mice. Taken together, CRISPR-EZ is simple, economic, high throughput, and highly efficient with the potential to replace microinjection for in vivo genome editing in mice and possibly in other mammals.

  10. 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.

  11. Understanding Physics, First Edition

    Science.gov (United States)

    Cummings, Karen; Laws, Priscilla W.; Redish, Edward F.; Cooney, Patrick J.

    2004-03-01

    Built on the foundations of Halliday, Resnick, and Walker's Fundamentals of Physics Sixth Edition, this text is designed to work with interactive learning strategies that are increasingly being used in physics instruction (for example, microcomputer-based labs, interactive lectures, etc. ). In doing so, it incorporates new approaches based upon Physics Education Research (PER), aligns with courses that use computer-based laboratory tools, and promotes Activity Based Physics in lectures, labs, and recitations.

  12. 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.

  13. 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.

  14. Semiautomated improvement of RNA alignments

    DEFF Research Database (Denmark)

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

    2007-01-01

    We have developed a semiautomated RNA sequence editor (SARSE) that integrates tools for analyzing RNA alignments. The editor highlights different properties of the alignment by color, and its integrated analysis tools prevent the introduction of errors when doing alignment editing. SARSE readily...... connects to external tools to provide a flexible semiautomatic editing environment. A new method, Pcluster, is introduced for dividing the sequences of an RNA alignment into subgroups with secondary structure differences. Pcluster was used to evaluate 574 seed alignments obtained from the Rfam database...... and we identified 71 alignments with significant prediction of inconsistent base pairs and 102 alignments with significant prediction of novel base pairs. Four RNA families were used to illustrate how SARSE can be used to manually or automatically correct the inconsistent base pairs detected by Pcluster...

  15. 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.

  16. Signal recognition particle RNA in dinoflagellates and the Perkinsid Perkinsus marinus.

    Science.gov (United States)

    Zhang, Huan; Campbell, David A; Sturm, Nancy R; Rosenblad, Magnus A; Dungan, Christopher F; Lin, Senjie

    2013-09-01

    In dinoflagellates and perkinsids, the molecular structure of the protein translocating machinery is unclear. Here, we identified several types of full-length signal recognition particle (SRP) RNA genes from Karenia brevis (dinoflagellate) and Perkinsus marinus (perkinsid). We also identified the four SRP S-domain proteins, but not the two Alu domain proteins, from P. marinus and several dinoflagellates. We mapped both ends of SRP RNA transcripts from K. brevis and P. marinus, and obtained the 3' end from four other dinoflagellates. The lengths of SRP RNA are predicted to be ∼260-300 nt in dinoflagellates and 280-285 nt in P. marinus. Although these SRP RNA sequences are substantially variable, the predicted structures are similar. The genomic organization of the SRP RNA gene differs among species. In K. brevis, this gene is located downstream of the spliced leader (SL) RNA, either as SL RNA-SRP RNA-tRNA gene tandem repeats, or within a SL RNA-SRP RNA-tRNA-U6-5S rRNA gene cluster. In other dinoflagellates, SRP RNA does not cluster with SL RNA or 5S rRNA genes. The majority of P. marinus SRP RNA genes array as tandem repeats without the above-mentioned small RNA genes. Our results capture a snapshot of a potentially complex evolutionary history of SRP RNA in alveolates.

  17. Still-image frame grabs and benthic habitat interpretation of underwater video footage, March 2014, Faga`alu Bay, American Samoa

    Data.gov (United States)

    U.S. Geological Survey, Department of the Interior — Underwater video was collected in March 2014 in the nearshore waters of Faga`alu Bay on the island of Tutuila, American Samoa, as part of the U.S. Geological Survey...

  18. [Conformational polymorphysm of G-rich fragments of DNA Alu-repeats. II. the putative role of G-quadruplex structures in genomic rearrangements].

    Science.gov (United States)

    Varizhuk, A M; Sekridova, A V; Tankevich, M V; Podgorsky, V S; Smirnov, I P; Pozmogova, G E

    2016-11-01

    Three evolutionary conserved sites of Alu repeats (PQS2, PQS3 and PQS4) were shown to form stable inter- and intramolecular G-quadruplexes (GQs) in vitro. Structures and topologies of these GQs were elucidated using spectral methods. Self-association of G-rich Alu fragments was studied. Dimeric GQ formation from two distal identical or different putative quadruplex sites - (PQS2)2, (PQS3)2 or PQS2-PQS3 - within one lengthy DNA strand was demonstrated by a FRET-based method. Oligomer PQS4 (folded into a parallel intramolecular GQ) was shown to form stacks of quadruplexes that are stabilized by stacking interactions of external G-tetrads (this was confirmed by DOSY NMR, AFM microscopy and differential CD spectroscopy). Comparative analysis of the properties of various GQs allowed us to put forward a hypothesis of two general mechanisms of intermolecular GQ-dependant genomic rearrangements: 1) formation of a dimeric GQs; 2) association of pre-folded intramolecular parallel GQs from different strands into GQ-stacks. Thus, the observed co-localization of G-rich motifs of Alu elements with double-strand break hotspots and rearrangement hotspots may be accounted for by the specific secondary structure of these motifs. At the same time, this is likely primarily due to high abundance of such G-rich Alu fragments in the genome.

  19. 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.

  20. 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...

  1. 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.

  2. Antiviral Defenses in Plants through Genome Editing

    Science.gov (United States)

    Romay, Gustavo; Bragard, Claude

    2017-01-01

    Plant–virus interactions based-studies have contributed to increase our understanding on plant resistance mechanisms, providing new tools for crop improvement. In the last two decades, RNA interference, a post-transcriptional gene silencing approach, has been used to induce antiviral defenses in plants with the help of genetic engineering technologies. More recently, the new genome editing systems (GES) are revolutionizing the scope of tools available to confer virus resistance in plants. The most explored GES are zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats/Cas9 endonuclease. GES are engineered to target and introduce mutations, which can be deleterious, via double-strand breaks at specific DNA sequences by the error-prone non-homologous recombination end-joining pathway. Although GES have been engineered to target DNA, recent discoveries of GES targeting ssRNA molecules, including virus genomes, pave the way for further studies programming plant defense against RNA viruses. Most of plant virus species have an RNA genome and at least 784 species have positive ssRNA. Here, we provide a summary of the latest progress in plant antiviral defenses mediated by GES. In addition, we also discuss briefly the GES perspectives in light of the rebooted debate on genetic modified organisms (GMOs) and the current regulatory frame for agricultural products involving the use of such engineering technologies. PMID:28167937

  3. The unstable CCTG repeat responsible for myotonic dystrophy type 2 originates from an AluSx element insertion into an early primate genome.

    Directory of Open Access Journals (Sweden)

    Tatsuaki Kurosaki

    Full Text Available Myotonic dystrophy type 2 (DM2 is a subtype of the myotonic dystrophies, caused by expansion of a tetranucleotide CCTG repeat in intron 1 of the zinc finger protein 9 (ZNF9 gene. The expansions are extremely unstable and variable, ranging from 75-11,000 CCTG repeats. This unprecedented repeat size and somatic heterogeneity make molecular diagnosis of DM2 difficult, and yield variable clinical phenotypes. To better understand the mutational origin and instability of the ZNF9 CCTG repeat, we analyzed the repeat configuration and flanking regions in 26 primate species. The 3'-end of an AluSx element, flanked by target site duplications (5'-ACTRCCAR-3'or 5'-ACTRCCARTTA-3', followed the CCTG repeat, suggesting that the repeat was originally derived from the Alu element insertion. In addition, our results revealed lineage-specific repetitive motifs: pyrimidine (CT-rich repeat motifs in New World monkeys, dinucleotide (TG repeat motifs in Old World monkeys and gibbons, and dinucleotide (TG and tetranucleotide (TCTG and/or CCTG repeat motifs in great apes and humans. Moreover, these di- and tetra-nucleotide repeat motifs arose from the poly (A tail of the AluSx element, and evolved into unstable CCTG repeats during primate evolution. Alu elements are known to be the source of microsatellite repeats responsible for two other repeat expansion disorders: Friedreich ataxia and spinocerebellar ataxia type 10. Taken together, these findings raise questions as to the mechanism(s by which Alu-mediated repeats developed into the large, extremely unstable expansions common to these three disorders.

  4. 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...

  5. 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.

  6. 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.

  7. 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.

  8. Gene Editing: Powerful New Tools for Nephrology Research and Therapy.

    Science.gov (United States)

    Miyagi, Ayano; Lu, Aiwu; Humphreys, Benjamin D

    2016-10-01

    Biologic research is experiencing a transformation brought about by the ability of programmable nucleases to manipulate the genome. In the recently developed CRISPR/Cas system, short RNA sequences guide the endonuclease Cas9 to any location in the genome, causing a DNA double-strand break (DSB). Repair of DSBs allows the introduction of targeted genetic manipulations with high precision. Cas9-mediated gene editing is simple, scalable, and rapid, and it can be applied to virtually any organism. Here, we summarize the development of modern gene editing techniques and the biology of DSB repair on which these techniques are based. We discuss technical points in applying this technology and review its use in model organisms. Finally, we describe prospects for the use of gene editing to treat human genetic diseases. This technology offers tremendous promise for equipping the nephrology research community to better model and ultimately, treat kidney diseases.

  9. Control of gene editing by manipulation of DNA repair mechanisms.

    Science.gov (United States)

    Danner, Eric; Bashir, Sanum; Yumlu, Saniye; Wurst, Wolfgang; Wefers, Benedikt; Kühn, Ralf

    2017-04-03

    DNA double-strand breaks (DSBs) are produced intentionally by RNA-guided nucleases to achieve genome editing through DSB repair. These breaks are repaired by one of two main repair pathways, classic non-homologous end joining (c-NHEJ) and homology-directed repair (HDR), the latter being restricted to the S/G2 phases of the cell cycle and notably less frequent. Precise genome editing applications rely on HDR, with the abundant c-NHEJ formed mutations presenting a barrier to achieving high rates of precise sequence modifications. Here, we give an overview of HDR- and c-NHEJ-mediated DSB repair in gene editing and summarize the current efforts to promote HDR over c-NHEJ.

  10. Genome-editing tools for stem cell biology.

    Science.gov (United States)

    Vasileva, E A; Shuvalov, O U; Garabadgiu, A V; Melino, G; Barlev, N A

    2015-07-23

    Human pluripotent stem cells provide a versatile platform for regenerative studies, drug testing and disease modeling. That the expression of only four transcription factors, Oct4, Klf4, Sox2 and c-Myc (OKSM), is sufficient for generation of induced pluripotent stem cells (iPSCs) from differentiated somatic cells has revolutionized the field and also highlighted the importance of OKSM as targets for genome editing. A number of novel genome-editing systems have been developed recently. In this review, we focus on successful applications of several such systems for generation of iPSCs. In particular, we discuss genome-editing systems based on zinc-finger fusion proteins (ZFs), transcription activator-like effectors (TALEs) and an RNA-guided DNA-specific nuclease, Cas9, derived from the bacterial defense system against viruses that utilizes clustered regularly interspaced short palindromic repeats (CRISPR).

  11. 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.

  12. 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.

  13. The CRISPR/Cas Genome-Editing Tool: Application in Improvement of Crops.

    Science.gov (United States)

    Khatodia, Surender; Bhatotia, Kirti; Passricha, Nishat; Khurana, S M P; Tuteja, Narendra

    2016-01-01

    The Clustered Regularly Interspaced Short Palindromic Repeats associated Cas9/sgRNA system is a novel targeted genome-editing technique derived from bacterial immune system. It is an inexpensive, easy, most user friendly and rapidly adopted genome editing tool transforming to revolutionary paradigm. This technique enables precise genomic modifications in many different organisms and tissues. 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 a changing climate. The emerging areas of research for the genome editing in plants include interrogating gene function, rewiring the regulatory signaling networks and sgRNA library for high-throughput loss-of-function screening. In this review, we have described 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 described. With this powerful and innovative technique the designer GE non-GM plants could further advance climate resilient and sustainable agriculture in the future and maximizing yield by combating abiotic and biotic stresses.

  14. Targeted genome editing in human cells using CRISPR/Cas nucleases and truncated guide RNAs.

    Science.gov (United States)

    Fu, Yanfang; Reyon, Deepak; Joung, J Keith

    2014-01-01

    CRISPR RNA-guided nucleases have recently emerged as a robust genome-editing platform that functions in a wide range of organisms. To reduce off-target effects of these nucleases, we developed and validated a modified system that uses truncated guide RNAs (tru-gRNAs). The use of tru-gRNAs leads to decreases in off-target effects and does not generally compromise the on-target efficiencies of these genome-editing nucleases. In this chapter, we describe guidelines for identifying potential tru-gRNA target sites and protocols for measuring the on-target efficiencies of CRISPR RNA-guided nucleases in human cells.

  15. Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing.

    Science.gov (United States)

    Goldfarb, Katherine C; Cech, Thomas R

    2017-01-01

    MRP RNA is an abundant, essential noncoding RNA whose functions have been proposed in yeast but are incompletely understood in humans. Mutations in the genomic locus for MRP RNA cause pleiotropic human diseases, including cartilage hair hypoplasia (CHH). Here we applied CRISPR-Cas9 genome editing to disrupt the endogenous human MRP RNA locus, thereby attaining what has eluded RNAi and RNase H experiments: elimination of MRP RNA in the majority of cells. The resulting accumulation of ribosomal RNA (rRNA) precursor-analyzed by RNA fluorescent in situ hybridization (FISH), Northern blots, and RNA sequencing-implicates MRP RNA in pre-rRNA processing. Amelioration of pre-rRNA imbalance is achieved through rescue of MRP RNA levels by ectopic expression. Furthermore, affinity-purified MRP ribonucleoprotein (RNP) from HeLa cells cleaves the human pre-rRNA in vitro at at least one site used in cells, while RNP isolated from cells with CRISPR-edited MRP loci loses this activity, and ectopic MRP RNA expression restores cleavage activity. Thus, a role for RNase MRP in human pre-rRNA processing is established. As demonstrated here, targeted CRISPR disruption is a valuable tool for functional studies of essential noncoding RNAs that are resistant to RNAi and RNase H-based degradation.

  16. 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.

  17. 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…

  18. 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…

  19. 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…

  20. 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";…

  1. The Bacterial Origins of the CRISPR Genome-Editing Revolution.

    Science.gov (United States)

    Sontheimer, Erik J; Barrangou, Rodolphe

    2015-07-01

    Like most of the tools that enable modern life science research, the recent genome-editing revolution has its biological roots in the world of bacteria and archaea. Clustered, regularly interspaced, short palindromic repeats (CRISPR) loci are found in the genomes of many bacteria and most archaea, and underlie an adaptive immune system that protects the host cell against invasive nucleic acids such as viral genomes. In recent years, engineered versions of these systems have enabled efficient DNA targeting in living cells from dozens of species (including humans and other eukaryotes), and the exploitation of the resulting endogenous DNA repair pathways has provided a route to fast, easy, and affordable genome editing. In only three years after RNA-guided DNA cleavage was first harnessed, the ability to edit genomes via simple, user-defined RNA sequences has already revolutionized nearly all areas of biological science. CRISPR-based technologies are now poised to similarly revolutionize many facets of clinical medicine, and even promise to advance the long-term goal of directly editing genomic sequences of patients with inherited disease. In this review, we describe the biological and mechanistic basis for these remarkable immune systems, and how their engineered derivatives are revolutionizing basic and clinical research.

  2. Aluísio de Azevedo e o Japão: uma apreciação crítica Aluísio de Azevedo and Japan: a critical appreciation

    Directory of Open Access Journals (Sweden)

    Renato Ortiz

    1997-10-01

    Full Text Available Este artigo analisa o livro O Japão, de Aluísio de Azevedo, que foi vice-cônsul do Brasil em Yokohama entre 1987 e 1989. Busca inserir as opiniões do autor e o fascínio que ele parece demonstrar pelo Imperador no contexto complexo do processo de japonização que atravessava o Japão no final dos anos 80 do século passado. Imerso num debate que contrapunha modernização e tradição dentro dos parâmetros de um nacionalismo exaltado, busca-se investigar os fundamentos da visão proposta por um autor marcado pela brasilidade e por uma percepção idílica do isolacionismo nipônico, medida de pureza em relação à Europa. Ressalta-se a oscilação entre um olhar que tende a ver o Oriente como um bloco homogêneo, em contraste com o Ocidente, ao mesmo tempo que coloca em relevo a questão nacional que justamente é a afirmação de uma especificidade.The article analyses the book Japan by Aluísio de Azevedo, who was Brazils vice-consul in Yokohama, between 1887 and 1889. It tries to insert the author's opinions and the fascination he seems to have for the Emperor in the complex context of japonization-process which ran throughout Japan at the end of the 80ies of the last century. Talking into account that the country of that time is immersed in a debate which opposes modernization to tradition inside the parameters of an exalted nationalism, the aim here is to investigate the fundaments of a perspective suggested by an author characterized by his brasility and by an idyllic perception of Japanese isolationism, a measure of purity in relation to Europe. The analysis emphasizes the oscillation between a look which tends to see the Orient as a homogeneous block contrasting with the Occident as well as the national question, which precisely is the assertion of a specificity.

  3. Tools of Radio Astronomy, 5th edition

    Science.gov (United States)

    Wilson, Thomas L.; Rohlfs, Kristian; Huttemeister, Susanne

    2012-12-01

    New 5th corrected edition of the book http://adsabs.harvard.edu/abs/2009tra..book.....W in Russian, translated by O. Verkhodanov and S. Trushkin, editing S.A. Trushkin from Special astrophysical observatory RAS. This edition contains the translation of the 5th Springer edition of 2009 and new additional chapter (wrote by authors) of Solutions of the problems.

  4. 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.

  5. 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. 

  6. Hypermethylation of CpG island loci and hypomethylation of LINE-1 and Alu repeats in prostate adenocarcinoma and their relationship to clinicopathological features.

    Science.gov (United States)

    Cho, N-Y; Kim, B-H; Choi, M; Yoo, E J; Moon, K C; Cho, Y-M; Kim, D; Kang, G H

    2007-02-01

    Promoter CpG island hypermethylation is an important carcinogenic event in prostate adenocarcinoma. Regardless of tissue type, human cancers have in common both focal CpG island hypermethylation and global genomic hypomethylation. The present study evaluated CpG island loci hypermethylation and LINE-1 and Alu repeat hypomethylation in prostate adenocarcinoma, analysed the relationship between them, and correlated these findings with clinicopathological features. We examined 179 cases of prostate adenocarcinoma and 30 cases of benign prostate hypertrophy for the methylation status of 22 CpG island loci and the methylation levels of LINE-1 and Alu repeats using methylation-specific polymerase chain reaction and combined bisulphite restriction analysis, respectively. The following 16 CpG island loci were found to display cancer-related hypermethylation: RASSF1A, GSTP1, RARB, TNFRSF10C, APC, BCL2, MDR1, ASC, TIG1, RBP1, COX2, THBS1, TNFRSF10D, CD44, p16, and RUNX3. Except for the last four CpG island loci, hypermethylation of each of the remaining 12 CpG island loci displayed a close association with one or more of the prognostic parameters (ie preoperative serum prostate specific antigen level, Gleason score sum, and clinical stage). Prostate adenocarcinoma with hypermethylation of each of ASC, COX2, RARB, TNFRSF10C, MDR1, TIG1, RBP1, NEUROG1, RASSF1A, and GSTP1 showed a significantly lower methylation level of Alu or LINE-1 than prostate adenocarcinoma without hypermethylation. In addition, hypomethylation of Alu or LINE-1 was closely associated with one or more of the above prognostic parameters. These data suggest that in tumour progression a close relationship exists between CpG island hypermethylation and the hypomethylation of repetitive elements, and that CpG island hypermethylation and DNA hypomethylation contribute to cancer progression.

  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. Water quality management library. 2. edition

    Energy Technology Data Exchange (ETDEWEB)

    Eckenfelder, W.W.; Malina, J.F.; Patterson, J.W. [eds.

    1998-12-31

    A series of ten books offered in conjunction with Water Quality International, the Biennial Conference and Exposition of the International Association on Water Pollution Research and Control (IAWPRC). Volume 1, Activated Sludge Process, Design and Control, 2nd edition, 1998: Volume 2, Upgrading Wastewater Treatment Plants, 2nd edition, 1998: Volume 3, Toxicity Reduction, 2nd edition, 1998: Volume 4, Municipal Sewage Sludge Management, 2nd edition, 1998: Volume 5, Design and Retrofit of Wastewater Treatment Plants for Biological Nutrient Removal, 1st edition, 1992: Volume 6, Dynamics and Control of the Activated Sludge Process, 2nd edition, 1998: Volume 7: Design of Anaerobic Processes for the Treatment of Industrial and Municipal Wastes, 1st edition, 1992: Volume 8, Groundwater Remediation, 1st edition, 1992: Volume 9, Nonpoint Pollution and Urban Stormwater Management, 1st edition, 1995: Volume 10, Wastewater Reclamation and Reuse, 1st edition, 1998.

  9. Identification of region-specific yeast artificial chromosomes using pools of Alu element-mediated polymerase chain reaction probes labeled via linear amplification

    Energy Technology Data Exchange (ETDEWEB)

    Cole, C.G.; Bobrow, M.; Bentley, D.R.; Dunham, I. (United Medical and Dental Schools of Guy' s and St. Thomas Hospitals, London Bridge, London, England (United Kingdom)); Patel, K.; Shipley, J.; Sheer, D. (Imperial Cancer Research Fund, London (United Kingdom))

    1992-12-01

    The ability to identify large numbers of yeast artificial chromosomes (YACS) specific to any given genomic region rapidly and efficiently enhances both the construction of clone maps and the isolation of region-specific landmarks (e.g., polymorphic markers). The authors describe a method of preparing region-specific single-stranded hybridization probes from Alu element-mediated polymerase chain reaction (Alu-PCR) products of somatic cell hybrids for YAC library screening. Pools of up to 50 cloned Alu-PCR products from an irradiation-reduced hybrid containing 22q11.2-q13.1 were labeled to high specific activity by linear amplification using a single vector primer. The resulting single-stranded probes were extensively competed to remove repetitive sequences, while retaining the full complexity of the probe. Extensive coverage of the region by YACs using multiple probe pools was demonstrated as many YACs were detected more than once. In situ analysis using chosen YACs confirmed that the clones were specific for the region. Thus, this pooled probe approach constitutes a rapid method to identify large numbers of YACs relevant to a large chromosomal region. 29 refs., 4 figs.

  10. Identification of region-specific yeast artificial chromosomes using pools of Alu element-mediated polymerase chain reaction probes labeled via linear amplification.

    Science.gov (United States)

    Cole, C G; Patel, K; Shipley, J; Sheer, D; Bobrow, M; Bentley, D R; Dunham, I

    1992-12-01

    The ability to identify large numbers of yeast artificial chromosomes (YACs) specific to any given genomic region rapidly and efficiently enhances both the construction of clone maps and the isolation of region-specific landmarks (e.g., polymorphic markers). We describe a method of preparing region-specific single-stranded hybridization probes from Alu element-mediated polymerase chain reaction (Alu-PCR) products of somatic cell hybrids for YAC library screening. Pools of up to 50 cloned Alu-PCR products from an irradiation-reduced hybrid containing 22q11.2-q13.1 were labeled to high specific activity by linear amplification using a single vector primer. The resulting single-stranded probes were extensively competed to remove repetitive sequences, while retaining the full complexity of the probe. Extensive coverage of the region by YACs using multiple probe pools was demonstrated as many YACs were detected more than once. In situ analysis using chosen YACs confirmed that the clones were specific for the region. Thus, this pooled probe approach constitutes a rapid method to identify large numbers of YACs relevant to a large chromosomal region.

  11. 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

  12. 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.

  13. RNA Interference

    Science.gov (United States)

    ... NIGMS Home > Science Education > RNA Interference Fact Sheet RNA Interference Fact Sheet Tagline (Optional) Middle/Main Content Area What is RNA interference? RNA interference (RNAi) is a natural process ...

  14. Modern Physics, 4th edition

    Science.gov (United States)

    Tipler, Paul A.; Llewellyn, Ralph

    The new edition of the classic text for the intermediate-level modern physics course, revised and updated to take students to the forefront of contemporary research and applications across the full spectrum of science and technology."

  15. 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

  16. RNA helicases

    OpenAIRE

    Owttrim, George W.

    2013-01-01

    Similar to proteins, RNA molecules must fold into the correct conformation and associate with protein complexes in order to be functional within a cell. RNA helicases rearrange RNA secondary structure and RNA-protein interactions in an ATP-dependent reaction, performing crucial functions in all aspects of RNA metabolism. In prokaryotes, RNA helicase activity is associated with roles in housekeeping functions including RNA turnover, ribosome biogenesis, translation and small RNA metabolism. In...

  17. Foundations of Mechanics, Second Edition

    OpenAIRE

    1987-01-01

    Preface to the Second Edition. Since the first edition of this book appeared in 1967, there has been a great deal of activity in the field of symplectic geometry and Hamiltonian systems. In addition to the recent textbooks of Arnold, Arnold-Avez, Godbillon, Guillemin-Sternberg, Siegel-Moser, and Souriau, there have been many research articles published. Two good collections are "Symposia Mathematica," vol. XIV, and "Géométrie Symplectique el Physique Mathématique," CNRS, Colloque Intern...

  18. Genome editing in cardiovascular diseases.

    Science.gov (United States)

    Strong, Alanna; Musunuru, Kiran

    2017-01-01

    Genome-editing tools, which include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) systems, have emerged as an invaluable technology to achieve somatic and germline genomic manipulation in cells and model organisms for multiple applications, including the creation of knockout alleles, introducing desired mutations into genomic DNA, and inserting novel transgenes. Genome editing is being rapidly adopted into all fields of biomedical research, including the cardiovascular field, where it has facilitated a greater understanding of lipid metabolism, electrophysiology, cardiomyopathies, and other cardiovascular disorders, has helped to create a wider variety of cellular and animal models, and has opened the door to a new class of therapies. In this Review, we discuss the applications of genome-editing technology throughout cardiovascular disease research and the prospect of in vivo genome-editing therapies in the future. We also describe some of the existing limitations of genome-editing tools that will need to be addressed if cardiovascular genome editing is to achieve its full scientific and therapeutic potential.

  19. RNA topology

    OpenAIRE

    Frank-Kamenetskii, Maxim D.

    2013-01-01

    A new variety on non-coding RNA has been discovered by several groups: circular RNA (circRNA). This discovery raises intriguing questions about the possibility of the existence of knotted RNA molecules and the existence of a new class of enzymes changing RNA topology, RNA topoisomerases.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. Alu insertion polymorphisms in NW Africa and the Iberian Peninsula: evidence for a strong genetic boundary through the Gibraltar Straits.

    Science.gov (United States)

    Comas, D; Calafell, F; Benchemsi, N; Helal, A; Lefranc, G; Stoneking, M; Batzer, M A; Bertranpetit, J; Sajantila, A

    2000-10-01

    An analysis of 11 I Alu insertion polymorphisms (ACE, TPA25, PV92, APO, FXIIIB, D1, A25, B65, HS2.43, HS3.23, and HS4.65) has been performed in several NW African (Northern, Western, and Southeastern Moroccans, Saharawi; Algerians; Tunisians) and Iberian (Basques, Catalans, and Andalusians) populations. Genetic distances and principal component analyses show a clear differentiation of NW African and Iberian groups of samples, suggesting a strong genetic barrier matching the geographical Mediterranean Sea barrier. The restriction to gene flow may be attributed to the navigational hazards across the Straits, but cultural factors must also have played a role. Some degree of gene flow from sub-Saharan Africa can be detected in the southern part of North Africa and in Saharawi and Southeastern Moroccans, as a result of a continuous gene flow across the Sahara desert that has created a south-north cline of sub-Saharan Africa influence in North Africa. Iberian samples show a substantial degree of homogeneity and fall within the cluster of European-based genetic diversity.

  5. Direct Cytosolic Delivery of CRISPR/Cas9-Ribonucleoprotein for Efficient Gene Editing.

    Science.gov (United States)

    Mout, Rubul; Ray, Moumita; Yesilbag Tonga, Gulen; Lee, Yi-Wei; Tay, Tristan; Sasaki, Kanae; Rotello, Vincent M

    2017-03-28

    Genome editing through the delivery of CRISPR/Cas9-ribonucleoprotein (Cas9-RNP) reduces unwanted gene targeting and avoids integrational mutagenesis that can occur through gene delivery strategies. Direct and efficient delivery of Cas9-RNP into the cytosol followed by translocation to the nucleus remains a challenge. Here, we report a remarkably highly efficient (∼90%) direct cytoplasmic/nuclear delivery of Cas9 protein complexed with a guide RNA (sgRNA) through the coengineering of Cas9 protein and carrier nanoparticles. This construct provides effective (∼30%) gene editing efficiency and opens up opportunities in studying genome dynamics.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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).

  11. [Genome editing of industrial microorganism].

    Science.gov (United States)

    Zhu, Linjiang; Li, Qi

    2015-03-01

    Genome editing is defined as highly-effective and precise modification of cellular genome in a large scale. In recent years, such genome-editing methods have been rapidly developed in the field of industrial strain improvement. The quickly-updating methods thoroughly change the old mode of inefficient genetic modification, which is "one modification, one selection marker, and one target site". Highly-effective modification mode in genome editing have been developed including simultaneous modification of multiplex genes, highly-effective insertion, replacement, and deletion of target genes in the genome scale, cut-paste of a large DNA fragment. These new tools for microbial genome editing will certainly be applied widely, and increase the efficiency of industrial strain improvement, and promote the revolution of traditional fermentation industry and rapid development of novel industrial biotechnology like production of biofuel and biomaterial. The technological principle of these genome-editing methods and their applications were summarized in this review, which can benefit engineering and construction of industrial microorganism.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Newer gene editing technologies toward HIV gene therapy.

    Science.gov (United States)

    Manjunath, N; Yi, Guohua; Dang, Ying; Shankar, Premlata

    2013-11-14

    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.

  17. 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....

  18. 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

    2016-01-28

    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.

  19. 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…

  20. Gas Tungsten Arc Welding and Plasma Arc Cutting. Teacher Edition [and] Student Edition [and] Student Workbook. Second Edition.

    Science.gov (United States)

    Harper, Eddie; Knapp, John

    This packet of instructional materials for a gas tungsten arc welding (GTAW) and plasma arc cutting course is comprised of a teacher edition, student edition, and student workbook. The teacher edition consists of introductory pages and teacher pages. Introductory pages include training and competency profile, state duty/task crosswalk,…

  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. Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes

    Science.gov (United States)

    Svitashev, Sergei; Schwartz, Christine; Lenderts, Brian; Young, Joshua K.; Mark Cigan, A.

    2016-01-01

    Targeted DNA double-strand breaks have been shown to significantly increase the frequency and precision of genome editing. In the past two decades, several double-strand break technologies have been developed. CRISPR–Cas9 has quickly become the technology of choice for genome editing due to its simplicity, efficiency and versatility. Currently, genome editing in plants primarily relies on delivering double-strand break reagents in the form of DNA vectors. Here we report biolistic delivery of pre-assembled Cas9–gRNA ribonucleoproteins into maize embryo cells and regeneration of plants with both mutated and edited alleles. Using this method of delivery, we also demonstrate DNA- and selectable marker-free gene mutagenesis in maize and recovery of plants with mutated alleles at high frequencies. These results open new opportunities to accelerate breeding practices in a wide variety of crop species. PMID:27848933

  3. 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...

  4. 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.

  5. 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

  6. 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

  7. Language Editing at Astronomy & Astrophysics

    Science.gov (United States)

    Adams, J.

    2011-07-01

    In 2002, the A&A Board of Directors voted that all articles must be written in English and decided to improve the overall quality of the language in the articles with the help of a team of language editors. This article reviews the general advantages of editing the English expression and describes both the aims of this effort and its place in the full publication process. This is followed by the Guide to language editing that has been available on the Journal's website for several years now.

  8. Delivery technologies for genome editing.

    Science.gov (United States)

    Yin, Hao; Kauffman, Kevin J; Anderson, Daniel G

    2017-03-24

    With the recent development of CRISPR technology, it is becoming increasingly easy to engineer the genome. Genome-editing systems based on CRISPR, as well as transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs), are becoming valuable tools for biomedical research, drug discovery and development, and even gene therapy. However, for each of these systems to effectively enter cells of interest and perform their function, efficient and safe delivery technologies are needed. This Review discusses the principles of biomacromolecule delivery and gene editing, examines recent advances and challenges in non-viral and viral delivery methods, and highlights the status of related clinical trials.

  9. Power Product Equipment Technician: Equipment Systems. Teacher Edition. Student Edition.

    Science.gov (United States)

    Hilley, Robert

    This packet contains teacher and student editions on the topic of equipment systems, intended for the preparation of power product equipment technicians. This publication contains seven units: (1) principles of power transmission; (2) mechanical drive systems; (3) principles of fluid power; (4) hydraulic and pneumatic drive systems; (5) wheel and…

  10. 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

  11. 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

  12. Human Genome Editing and Ethical Considerations.

    Science.gov (United States)

    Krishan, Kewal; Kanchan, Tanuj; Singh, Bahadur

    2016-04-01

    Editing human germline genes may act as boon in some genetic and other disorders. Recent editing of the genome of the human embryo with the CRISPR/Cas9 editing tool generated a debate amongst top scientists of the world for the ethical considerations regarding its effect on the future generations. It needs to be seen as to what transformation human gene editing brings to humankind in the times to come.

  13. Alu insertion polymorphisms and an assessment of the genetic contribution of Central Asia to Anatolia with respect to the Balkans.

    Science.gov (United States)

    Berkman, Ceren Caner; Dinc, Havva; Sekeryapan, Ceran; Togan, Inci

    2008-05-01

    In the evolutionary history of modern humans, Anatolia acted as a bridge between the Caucasus, the Near East, and Europe. Because of its geographical location, Anatolia was subject to migrations from multiple different regions throughout time. The last, well-known migration was the movement of Turkic speaking, nomadic groups from Central Asia. They invaded Anatolia and then the language of the region was gradually replaced by the Turkic language. In the present study, insertion frequencies of 10 Alu loci (A25 = 0.07, APO = 0.96, TPA25 = 0.44, ACE = 0.37, B65 = 0.57, PV92 = 0.18, FXIIIB = 0.52, D1 = 0.40, HS4.32 = 0.66, and HS4.69 = 0.30) have been determined in the Anatolian population. Together with the data compiled from other databases, the similarity of the Anatolian population to that of the Balkans and Central Asia has been visualized by multidimensional scaling method. Analysis suggested that, genetically, Anatolia is more closely related with the Balkan populations than to the Central Asian populations. Central Asian contribution to Anatolia with respect to the Balkans was quantified with an admixture analysis. Furthermore, the association between the Central Asian contribution and the language replacement episode was examined by comparative analysis of the Central Asian contribution to Anatolia, Azerbaijan (another Turkic speaking country) and their neighbors. In the present study, the Central Asian contribution to Anatolia was estimated as 13%. This was the lowest value among the populations analyzed. This observation may be explained by Anatolia having the lowest migrant/resident ratio at the time of migrations.

  14. Genome Editing in Human Cells Using CRISPR/Cas Nucleases.

    Science.gov (United States)

    Wyvekens, Nicolas; Tsai, Shengdar Q; Joung, J Keith

    2015-10-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been broadly adopted for highly efficient genome editing in a variety of model organisms and human cell types. Unlike previous genome editing technologies such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas technology does not require complex protein engineering and can be utilized by any researcher proficient in basic molecular biology and cell culture techniques. This unit describes protocols for design and cloning of vectors expressing single or multiplex gRNAs, for transient transfection of human cell lines, and for quantitation of mutation frequencies by T7 endonuclease I assay. These protocols also include guidance for using two improvements that increase the specificity of CRISPR/Cas nucleases: truncated gRNAs and dimeric RNA-guided FokI nucleases.

  15. GENOME EDITING IN HUMAN CELLS USING CRISPR/CAS NUCLEASES

    Science.gov (United States)

    Wyvekens, Nicolas; Tsai, Shengdar; Joung, J. Keith

    2016-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been broadly adopted for highly efficient genome editing in a variety of model organisms and human cell types. Unlike previous genome editing technologies such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR/Cas technology does not require complex protein engineering and can be utilized by any researcher proficient in basic molecular biology and cell culture techniques. Here we describe protocols for design and cloning of vectors expressing single or multiplex gRNAs, for transient transfection of human cell lines, and for quantitation of mutation frequencies by T7 Endonuclease I assay. These protocols also include guidance for using two improvements that increase the specificity of CRISPR/Cas nucleases: truncated gRNAs and dimeric RNA-guided FokI nucleases. PMID:26423589

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

    Science.gov (United States)

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

    2016-08-04

    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.

  17. 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…

  18. 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…

  19. 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.

  20. 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.

  1. 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.

  2. 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 ...

  3. 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...

  4. 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…

  5. Characterizing the transcriptome upon depletion of RNA processing factors

    DEFF Research Database (Denmark)

    Herudek, Jan

    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....

  6. Transgene-free genome editing in Caenorhabditis elegans using CRISPR-Cas.

    Science.gov (United States)

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

    2013-11-01

    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.

  7. Efficient Video Editing for Mobile Applications

    Directory of Open Access Journals (Sweden)

    Ignasi Vegas Pajaro

    2017-01-01

    Full Text Available Recording, storing and sharing video content has become one of the most popular usages of smartphones. This has resulted in demand for video editing apps that the users can use to edit their videos before sharing on various social networks. This study describes a technique to create a video editing application that uses the processing power of both GPU and CPU to process various editing tasks. The results and subsequent discussion shows that using the processing power of both the GPU and CPU in the video editing process makes the application much more time-efficient and responsive as compared to just the CPU-based processing.

  8. 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.

  9. 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.

  10. Inhibition of HSV-1 Replication by Gene Editing Strategy

    OpenAIRE

    Roehm, Pamela C.; Masoud Shekarabi; Wollebo, Hassen S.; Anna Bellizzi; Lifan He; Julian Salkind; Kamel Khalili

    2016-01-01

    HSV-1 induced illness affects greater than 85% of adults worldwide with no permanent curative therapy. We used RNA-guided CRISPR/Cas9 gene editing to specifically target for deletion of DNA sequences of the HSV-1 genome that span the region directing expression of ICP0, a key viral protein that stimulates HSV-1 gene expression and replication. We found that CRISPR/Cas9 introduced InDel mutations into exon 2 of the ICP0 gene profoundly reduced HSV-1 infectivity in permissive human cell culture...

  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. RNA structure. Structure of the HIV-1 RNA packaging signal.

    Science.gov (United States)

    Keane, Sarah C; Heng, Xiao; Lu, Kun; Kharytonchyk, Siarhei; Ramakrishnan, Venkateswaran; Carter, Gregory; Barton, Shawn; Hosic, Azra; Florwick, Alyssa; Santos, Justin; Bolden, Nicholas C; McCowin, Sayo; Case, David A; Johnson, Bruce A; Salemi, Marco; Telesnitsky, Alice; Summers, Michael F

    2015-05-22

    The 5' leader of the HIV-1 genome contains conserved elements that direct selective packaging of the unspliced, dimeric viral RNA into assembling particles. By using a (2)H-edited nuclear magnetic resonance (NMR) approach, we determined the structure of a 155-nucleotide region of the leader that is independently capable of directing packaging (core encapsidation signal; Ψ(CES)). The RNA adopts an unexpected tandem three-way junction structure, in which residues of the major splice donor and translation initiation sites are sequestered by long-range base pairing and guanosines essential for both packaging and high-affinity binding to the cognate Gag protein are exposed in helical junctions. The structure reveals how translation is attenuated, Gag binding promoted, and unspliced dimeric genomes selected, by the RNA conformer that directs packaging.

  14. Recent Advances in Genome Editing Using CRISPR/Cas9.

    Science.gov (United States)

    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 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.

  15. 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.

  16. Electromagnetic Fields, 2nd Edition

    Science.gov (United States)

    Wangsness, Roald K.

    1986-07-01

    This revised edition provides patient guidance in its clear and organized presentation of problems. It is rich in variety, large in number and provides very careful treatment of relativity. One outstanding feature is the inclusion of simple, standard examples demonstrated in different methods that will allow students to enhance and understand their calculating abilities. There are over 145 worked examples; virtually all of the standard problems are included.

  17. Strategic Leadership Primer (Third Edition)

    Science.gov (United States)

    2010-01-01

    Meinhart, while also acknowledging previous edition contributions from Dr� Lenny Wong, Dr� Craig Bullis , and Colonel (Ret) George Reed� 1 CHAPTER 1...the cyber war� Although the strategies are different between commercial and government entities, at the end of the day both seek competitive...and everyone else—to 140 characters per tweet, and he tweets once or more almost daily�4 400 million people have established accounts on Facebook �5

  18. 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

    2016-05-23

    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.

  19. 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.

  20. Genetic spell-checking: gene editing using single-stranded DNA oligonucleotides.

    Science.gov (United States)

    Rivera-Torres, Natalia; Kmiec, Eric B

    2016-02-01

    Single-stranded oligonucleotides (ssODNs) can be used to direct the exchange of a single nucleotide or the repair of a single base within the coding region of a gene in a process that is known, generically, as gene editing. These molecules are composed of either all DNA residues or a mixture of RNA and DNA bases and utilize inherent metabolic functions to execute the genetic alteration within the context of a chromosome. The mechanism of action of gene editing is now being elucidated as well as an understanding of its regulatory circuitry, work that has been particularly important in establishing a foundation for designing effective gene editing strategies in plants. Double-strand DNA breakage and the activation of the DNA damage response pathway play key roles in determining the frequency with which gene editing activity takes place. Cellular regulators respond to such damage and their action impacts the success or failure of a particular nucleotide exchange reaction. A consequence of such activation is the natural slowing of replication fork progression, which naturally creates a more open chromatin configuration, thereby increasing access of the oligonucleotide to the DNA template. Herein, how critical reaction parameters influence the effectiveness of gene editing is discussed. Functional interrelationships between DNA damage, the activation of DNA response pathways and the stalling of replication forks are presented in detail as potential targets for increasing the frequency of gene editing by ssODNs in plants and plant cells.

  1. Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing.

    Science.gov (United States)

    Hendel, Ayal; Kildebeck, Eric J; Fine, Eli J; Clark, Joseph T; Punjya, Niraj; Sebastiano, Vittorio; Bao, Gang; Porteus, Matthew H

    2014-04-10

    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.

  2. Non-viral delivery of genome-editing nucleases for gene therapy.

    Science.gov (United States)

    Wang, M; Glass, Z A; Xu, Q

    2016-12-01

    Manipulating the genetic makeup of mammalian cells using programmable nuclease-based genome-editing technology has recently evolved into a powerful avenue that holds great potential for treating genetic disorders. There are four types of genome-editing nucleases, including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases and clustered, regularly interspaced, short palindromic repeat-associated nucleases such as Cas9. These nucleases have been harnessed to introduce precise and specific changes of the genome sequence at virtually any genome locus of interest. The therapeutic relevance of these genome-editing technologies, however, is challenged by the safe and efficient delivery of nuclease into targeted cells. Herein, we summarize recent advances that have been made on non-viral delivery of genome-editing nucleases. In particular, we focus on non-viral delivery of Cas9/sgRNA ribonucleoproteins for genome editing. In addition, the future direction for developing non-viral delivery of programmable nucleases for genome editing is discussed.Gene Therapy advance online publication, 1 December 2016; doi:10.1038/gt.2016.72.

  3. Editing as a psychological practice.

    Science.gov (United States)

    Beebe, John

    2006-06-01

    The experience of the Jungian analyst in the role of editor of manuscripts by creative colleagues is examined. Historical precedents include Michael Fordham's editorial correspondence with Jung around the latter's synchronicity essay; Jung's handling of manuscripts submitted by Sabina Spielrein to the Jahrbuch für psychoanalytische und psychopathologische Forschungen and various authors to the Zentralblatt für Psychotherapie und ihre Grenzgebiete, and the author's close editing of a paper submitted by Andrew Samuels to the Journal of Analytical Psychology. In addition to mustering an adequate amount of generosity, erudition, and availability, the analytic editor must know how to clarify a psychological argument and to gauge the psychological impact of the written text. Notwithstanding transference/countertransference phenomena that can emerge around issues of competition, envy, and territoriality when author and editor are also fellow-authors working in the same field, the editor needs to be comfortable about serving as the author's selfobject and midwife. From an analytic perspective, although communicating decisions about the best way to put ideas into words can sometimes attract transference to the editor, the more profound transference that analysts experience in the editing situation is toward the text being edited, which helps to motivate donated time spent caring for journal manuscripts.

  4. Research progress of genome editing and derivative technologies in plants.

    Science.gov (United States)

    Qiwei, Shan; Caixia, Gao

    2015-10-01

    Genome editing technologies using engineered nucleases have been widely used in many model organisms. Genome editing with sequence-specific nuclease (SSN) creates DNA double-strand breaks (DSBs) in the genomic target sites that are primarily repaired by the non-homologous end joining (NHEJ) or homologous recombination (HR) pathways, which can be employed to achieve targeted genome modifications such as gene mutations, insertions, replacements or chromosome rearrangements. There are three major SSNs─zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system. In contrast to ZFN and TALEN, which require substantial protein engineering to each DNA target, the CRISPR/Cas9 system requires only a change in the guide RNA. For this reason, the CRISPR/Cas9 system is a simple, inexpensive and versatile tool for genome engineering. Furthermore, a modified version of the CRISPR/Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression, such as activation, depression and epigenetic regulation. In this review, we summarize the development and applications of genome editing technologies for basic research and biotechnology, as well as highlight challenges and future directions, with particular emphasis on plants.

  5. RNA Origami

    DEFF Research Database (Denmark)

    Sparvath, Steffen Lynge

    2017-01-01

    for biosensorer,  der kan spore enten microRNA’er eller små molekyler, eksemplificeret ved S-adenosylmethionin (SAM). Slutteligt indikerer foreløbige resultater, at apta-FRET SAM sensoren kan udtrykkes i Escherichia coli-celler, hvilket viser, at RNA-origami arkitekturen muliggør cotransskriptionel foldning af......RNA-nanoteknologi feltet har demonstreret alsidigheden af RNA som byggemateriale, og rationelt designede RNA-nanostrukturer er blevet brugt i udviklingen af strukturelle platforme og dynamiske anordninger med anvendelser både in vitro og in vivo. Naturlige RNA-strukturer foldes cotransskriptionelt...... fra en enkelt RNA-streng, og udfører en lang række komplekse cellulære funktioner. Mange af funktionerne er blevet udnyttet til at skabe funktionelle RNA-baserede nanoapparater, men den nuværende litteratur giver kun få eksempler på cotranskriptionel produktion af RNA-nanostrukturer. I 2014...

  6. RNA granules

    OpenAIRE

    Anderson, Paul; Kedersha, Nancy

    2006-01-01

    Cytoplasmic RNA granules in germ cells (polar and germinal granules), somatic cells (stress granules and processing bodies), and neurons (neuronal granules) have emerged as important players in the posttranscriptional regulation of gene expression. RNA granules contain various ribosomal subunits, translation factors, decay enzymes, helicases, scaffold proteins, and RNA-binding proteins, and they control the localization, stability, and translation of their RNA cargo. We review the relationshi...

  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. RNA genetics

    Energy Technology Data Exchange (ETDEWEB)

    Domingo, E. (Instituto de Biologia Molecular, Facultad de Ciencias, Universidad Autonoma de Madrid, Canto Blanco, Madrid (ES)); Holland, J.J. (California Univ., San Diego, La Jolla, CA (USA). Dept. of Biology); Ahlquist, P. (Wisconsin Univ., Madison, WI (USA). Dept. of Plant Pathology)

    1988-01-01

    This book contains the proceedings on RNA genetics: RNA-directed virus replication Volume 1. Topics covered include: Replication of the poliovirus genome; Influenza viral RNA transcription and replication; and Relication of the reoviridal: Information derived from gene cloning and expression.

  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. 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.

  11. CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling

    OpenAIRE

    Platt, Randall J.; Chen, Sidi; ZHOU Yang; Yim, Michael J.; Swiech, Lukasz; Kempton, Hannah R.; Dahlman, James E.; Parnas, Oren; Eisenhaure, Thomas M.; Jovanovic, Marko; Graham, Daniel B.; Jhunjhunwala, Siddharth; Xavier, Ramnik J.; Langer, Robert; Anderson, Daniel G.

    2014-01-01

    CRISPR-Cas9 is a versatile genome editing technology for studying the functions of genetic elements. To broadly enable the application of Cas9 in vivo, we established a Cre-dependent Cas9 knockin mouse. We demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells. Using these mice, we simultaneously modeled the dynamics of KRAS, p53, and LKB1, the top three...

  12. 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.

  13. Review on Urban Sociology(2nd Edition)

    Institute of Scientific and Technical Information of China (English)

    Liu; Yuting; Li; Caige

    2016-01-01

    Urban Sociology(2nd Edition)Author:Gu Chaolin,Liu Jiayan,et al.Year:2013Publisher:Tsinghua University Press ISBN:9787302337591(446 pages,in Chinese)Urban Sociology is a textbook edited by Gu Chaolin,a wellk now n schola r of u rba n st udies i n Ch i na,a nd published by Tsinghua University Press in 2013.The first edition of this book,

  14. 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.

  15. Operational Transformation In Co-Operative Editing

    OpenAIRE

    Mandeep Kaur; Manpreet Singh; Harneet Kaur; Simran Kaur

    2015-01-01

    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.

  16. RNA epigenetics

    OpenAIRE

    Liu, Nian; Pan, Tao

    2014-01-01

    Mammalian messenger and long non-coding RNA contain tens of thousands of post-transcriptional chemical modifications. Among these, the N6-methyl-adenosine (m6A) modification is the most abundant and can be removed by specific mammalian enzymes. M6A modification is recognized by families of RNA binding proteins that affect many aspects of mRNA function. mRNA/lncRNA modification represents another layer of epigenetic regulation of gene expression, analogous to DNA methylation and histone modifi...

  17. Endonucleases: new tools to edit the mouse genome.

    Science.gov (United States)

    Wijshake, Tobias; Baker, Darren J; van de Sluis, Bart

    2014-10-01

    Mouse transgenesis has been instrumental in determining the function of genes in the pathophysiology of human diseases and modification of genes by homologous recombination in mouse embryonic stem cells remains a widely used technology. However, this approach harbors a number of disadvantages, as it is time-consuming and quite laborious. Over the last decade a number of new genome editing technologies have been developed, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas). These systems are characterized by a designed DNA binding protein or RNA sequence fused or co-expressed with a non-specific endonuclease, respectively. The engineered DNA binding protein or RNA sequence guides the nuclease to a specific target sequence in the genome to induce a double strand break. The subsequent activation of the DNA repair machinery then enables the introduction of gene modifications at the target site, such as gene disruption, correction or insertion. Nuclease-mediated genome editing has numerous advantages over conventional gene targeting, including increased efficiency in gene editing, reduced generation time of mutant mice, and the ability to mutagenize multiple genes simultaneously. Although nuclease-driven modifications in the genome are a powerful tool to generate mutant mice, there are concerns about off-target cleavage, especially when using the CRISPR/Cas system. Here, we describe the basic principles of these new strategies in mouse genome manipulation, their inherent advantages, and their potential disadvantages compared to current technologies used to study gene function in mouse models. This article is part of a Special Issue entitled: From Genome to Function.

  18. CRISPR/Cas9 Based Genome Editing of Penicillium chrysogenum.

    Science.gov (United States)

    Pohl, C; Kiel, J A K W; Driessen, A J M; Bovenberg, R A L; Nygård, Y

    2016-07-15

    CRISPR/Cas9 based systems have emerged as versatile platforms for precision genome editing in a wide range of organisms. Here we have developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. The developed CRISPR/Cas9 toolbox is highly flexible and allows editing of new targets with minimal cloning efforts. The Cas9 protein and the sgRNA can be either delivered during transformation, as preassembled CRISPR-Cas9 ribonucleoproteins (RNPs) or expressed from an AMA1 based plasmid within the cell. The direct delivery of the Cas9 protein with in vitro synthesized sgRNA to the cells allows for a transient method for genome engineering that may rapidly be applicable for other filamentous fungi. The expression of Cas9 from an AMA1 based vector was shown to be highly efficient for marker-free gene deletions.

  19. 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.

  20. 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,

  1. 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.

  2. 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

  3. 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.

  4. 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.

  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. Aluísio Azevedo: a “trapeiro” between Eça de Queirós and Machado de Assis

    Directory of Open Access Journals (Sweden)

    Monica do Nascimento Figueiredo

    2016-07-01

    Full Text Available Between Eça’s “realism” and Machado’s “modernism”, there is a great si­lence dedicated to the literary work of Aluísio Azevedo. Even before Lima Barreto and João do Rio, Azevedo was able to take the city of Rio de Janeiro as a landscape to be transformed by his work of fiction, which witnesses the existence of a city that would be destroyed by the future hygienist govern­ment of Pereira Passos. This essay will present the difference and the dislo­cation expressed in the work of an author for whom the urban geography is an obsessive theme.

  8. CRISPR/Cas9-Mediated Genome Editing of Mouse Small Intestinal Organoids.

    Science.gov (United States)

    Schwank, Gerald; Clevers, Hans

    2016-01-01

    The CRISPR/Cas9 system is an RNA-guided genome-editing tool that has been recently developed based on the bacterial CRISPR-Cas immune defense system. Due to its versatility and simplicity, it rapidly became the method of choice for genome editing in various biological systems, including mammalian cells. Here we describe a protocol for CRISPR/Cas9-mediated genome editing in murine small intestinal organoids, a culture system in which somatic stem cells are maintained by self-renewal, while giving rise to all major cell types of the intestinal epithelium. This protocol allows the study of gene function in intestinal epithelial homeostasis and pathophysiology and can be extended to epithelial organoids derived from other internal mouse and human organs.

  9. Experimental and Computational Considerations in the Study of RNA-Binding Protein-RNA Interactions.

    Science.gov (United States)

    Van Nostrand, Eric L; Huelga, Stephanie C; Yeo, Gene W

    2016-01-01

    After an RNA is transcribed, it undergoes a variety of processing steps that can change the encoded protein sequence (through alternative splicing and RNA editing), regulate the stability of the RNA, and control subcellular localization, timing, and rate of translation. The recent explosion in genomics techniques has enabled transcriptome-wide profiling of RNA processing in an unbiased manner. However, it has also brought with it both experimental challenges in developing improved methods to probe distinct processing steps, as well as computational challenges in data storage, processing, and analysis tools to enable large-scale interpretation in the genomics era. In this chapter we review experimental techniques and challenges in profiling various aspects of RNA processing, as well as recent efforts to develop analyses integrating multiple data sources and techniques to infer RNA regulatory networks.

  10. 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...

  11. Tax Justice Focus – The Whistleblower edition

    OpenAIRE

    Young, M A; Tax Justice Network

    2015-01-01

    Guest edited by Mary Alice Young of the University of the West of England, this edition is both an exploration of the difficulties confronting people who want to blow the whistle on companies engaged in providing financial services, and an acknowledgment of their personal courage.

  12. 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…

  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. 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...

  15. Caution required for handling genome editing technology.

    Science.gov (United States)

    Araki, Motoko; Nojima, Kumie; Ishii, Tetsuya

    2014-05-01

    Genome-editing technology, although a robust tool for genetic engineering, is creating indistinct regulatory boundaries between naturally occurring and modified organisms. However, researchers must act with caution in research and development to avoid misleading society. Furthermore, appropriate regulations should be proactively discussed and established for handling genome-editing technology.

  16. 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…

  17. 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)

  18. A New Perspective on Peer Editing.

    Science.gov (United States)

    Amores, Maria J.

    1997-01-01

    Describes the peer-editing behaviors of eight undergraduate students in a third-year Spanish composition and grammar review course. Data collected over four months through interviews, participant observation, artifact inventories, and questionnaires revealed a strong tendency among informants to define the peer-editing process in social and…

  19. 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.

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

    Science.gov (United States)

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

    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 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.

  1. 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.

  2. 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.

  3. Progress in Genome Editing Technology and Its Application in Plants.

    Science.gov (United States)

    Zhang, Kai; Raboanatahiry, Nadia; Zhu, Bin; Li, Maoteng

    2017-01-01

    Genome editing technology (GET) is a versatile approach that has progressed rapidly as a mechanism to alter the genotype and phenotype of organisms. However, conventional genome modification using GET cannot satisfy current demand for high-efficiency and site-directed mutagenesis, retrofitting of artificial nucleases has developed into a new avenue within this field. Based on mechanisms to recognize target genes, newly-developed GETs can generally be subdivided into three cleavage systems, protein-dependent DNA cleavage systems (i.e., zinc-finger nucleases, ZFN, and transcription activator-like effector nucleases, TALEN), RNA-dependent DNA cleavage systems (i.e., clustered regularly interspaced short palindromic repeats-CRISPR associated proteins, CRISPR-Cas9, CRISPR-Cpf1, and CRISPR-C2c1), and RNA-dependent RNA cleavage systems (i.e., RNA interference, RNAi, and CRISPR-C2c2). All these techniques can lead to double-stranded (DSB) or single-stranded breaks (SSB), and result in either random mutations via non-homologous end-joining (NHEJ) or targeted mutation via homologous recombination (HR). Thus, site-directed mutagenesis can be induced via targeted gene knock-out, knock-in, or replacement to modify specific characteristics including morphology-modification, resistance-enhancement, and physiological mechanism-improvement along with plant growth and development. In this paper, an non-comprehensive review on the development of different GETs as applied to plants is presented.

  4. Progress in Genome Editing Technology and Its Application in Plants

    Science.gov (United States)

    Zhang, Kai; Raboanatahiry, Nadia; Zhu, Bin; Li, Maoteng

    2017-01-01

    Genome editing technology (GET) is a versatile approach that has progressed rapidly as a mechanism to alter the genotype and phenotype of organisms. However, conventional genome modification using GET cannot satisfy current demand for high-efficiency and site-directed mutagenesis, retrofitting of artificial nucleases has developed into a new avenue within this field. Based on mechanisms to recognize target genes, newly-developed GETs can generally be subdivided into three cleavage systems, protein-dependent DNA cleavage systems (i.e., zinc-finger nucleases, ZFN, and transcription activator-like effector nucleases, TALEN), RNA-dependent DNA cleavage systems (i.e., clustered regularly interspaced short palindromic repeats-CRISPR associated proteins, CRISPR-Cas9, CRISPR-Cpf1, and CRISPR-C2c1), and RNA-dependent RNA cleavage systems (i.e., RNA interference, RNAi, and CRISPR-C2c2). All these techniques can lead to double-stranded (DSB) or single-stranded breaks (SSB), and result in either random mutations via non-homologous end-joining (NHEJ) or targeted mutation via homologous recombination (HR). Thus, site-directed mutagenesis can be induced via targeted gene knock-out, knock-in, or replacement to modify specific characteristics including morphology-modification, resistance-enhancement, and physiological mechanism-improvement along with plant growth and development. In this paper, an non-comprehensive review on the development of different GETs as applied to plants is presented. PMID:28261237

  5. 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

    2015-10-05

    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.

  6. RNA. Introduction.

    Science.gov (United States)

    Bao, Marie Z; Kruger, Robert P; Rivas, Fabiola; Smith, Orla; Szewczak, Lara

    2009-02-20

    Two scientists walk into a bar. After a pint and an exchange of pleasantries, one says to the other, "Where do you come from? Scientifically, I mean." The queried scientist responds, "Out of the RNA world." "Don't we all," the asker responds chuckling. Fifteen years ago, the joke would have been made with a nod to the notion that life arose from an RNA-based precursor, the so-called "RNA world." Yet had this conversation happened last week, the scientists would also be grinning in appreciation of the extent to which contemporary cellular biology is steeped in all things RNA. Ours is truly an RNA world.In this year's special review issue, the Cell editorial team has brought together articles focused on RNA in the modern world, providing perspectives on classical and emerging areas of inquiry. We extend our thanks to the many distinguished experts who contributed their time and effort as authors and reviewers to make the issue informative, thought-provoking, and timely. We hope that this collection of articles, written as we stand on the verge of a new wave of RNA biology, edifies and inspires by revealing the inner workings of these versatile molecules and by highlighting the next key questions that need to be addressed as we strive to understand the full functional scope of RNA in cells.

  7. Delivery and therapeutic applications of gene editing technologies ZFNs, TALENs, and CRISPR/Cas9.

    Science.gov (United States)

    LaFountaine, Justin S; Fathe, Kristin; Smyth, Hugh D C

    2015-10-15

    In recent years, several new genome editing technologies have been developed. Of these the zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 RNA-guided endonuclease system are the most widely described. Each of these technologies utilizes restriction enzymes to introduce a DNA double stranded break at a targeted location with the guide of homologous binding proteins or RNA. Such targeting is viewed as a significant advancement compared to current gene therapy methods that lack such specificity. Proof-of-concept studies have been performed to treat multiple disorders, including in vivo experiments in mammals and even early phase human trials. Careful consideration and investigation of delivery strategies will be required so that the therapeutic potential for gene editing is achieved. In this review, the mechanisms of each of these gene editing technologies and evidence of therapeutic potential will be briefly described and a comprehensive list of past studies will be provided. The pharmaceutical approaches of each of these technologies are discussed along with the current delivery obstacles. The topics and information reviewed herein provide an outline of the groundbreaking research that is being performed, but also highlights the potential for progress yet to be made using these gene editing technologies.

  8. 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

  9. 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...

  10. Image editing with spatiograms transfer.

    Science.gov (United States)

    Papadakis, Nicolas; Bugeau, Aurélie; Caselles, Vicent

    2012-05-01

    Histogram equalization is a well-known method for image contrast enhancement. Nevertheless, as histograms do not include any information on the spatial repartition of colors, their application to local image editing problems remains limited. To cope with this lack of spatial information, spatiograms have been recently proposed for tracking purposes. A spatiogram is an image descriptor that combines a histogram with the mean and the variance of the position of each color. In this paper, we address the problem of local retouching of images by proposing a variational method for spatiogram transfer. More precisely, a reference spatiogram is used to modify the color value of a given region of interest of the processed image. Experiments on shadow removal and inpainting demonstrate the strength of the proposed approach.

  11. 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.

  12. 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.

  13. 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.

  14. RNA编辑功能和RNA干扰%Functions of RNA Editing and RNA Interference

    Institute of Scientific and Technical Information of China (English)

    代鹏; 沈辉; 马军武; 刘光远

    2011-01-01

    RNA编辑被认为是生命体一种新的基因加工与修饰现象,是指DNA转录成RNA后除RNA剪切外的其他加工过程,以核苷酸的删除、插入或替换等方式改变遗传信息,揭示生物进化过程中基因修饰和调控的另一个重要途径,是对中心法则的重要补充.而RNAi是一种由dsRNA介导的,在转录水平、转录后水平和翻译水平上阻断基因表达的基因调节途径.着重介绍 RNA编辑功能、RNA编辑与RNA干扰关系.

  15. 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...

  16. 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...

  17. 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.

  18. Data Director editor user's manual. [EDIT

    Energy Technology Data Exchange (ETDEWEB)

    McGoldrick, P.

    1977-03-21

    EDIT allows the user to edit or create text, either interactively or in the batch mode. The text may be a source program, program data, or documents. Special provisions permit the creation or editing of APT source-language programs. EDIT processes ASCII files from any accessible Data Director library and outputs files to any such library as specified by the user.

  19. 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.

  20. RNA conformational changes in the life cycles of RNA viruses, viroids, and virus-associated RNAs.

    Science.gov (United States)

    Simon, Anne E; Gehrke, Lee

    2009-01-01

    The rugged nature of the RNA structural free energy landscape allows cellular RNAs to respond to environmental conditions or fluctuating levels of effector molecules by undergoing dynamic conformational changes that switch on or off activities such as catalysis, transcription or translation. Infectious RNAs must also temporally control incompatible activities and rapidly complete their life cycle before being targeted by cellular defenses. Viral genomic RNAs must switch between translation and replication, and untranslated subviral RNAs must control other activities such as RNA editing or self-cleavage. Unlike well characterized riboswitches in cellular RNAs, the control of infectious RNA activities by altering the configuration of functional RNA domains has only recently been recognized. In this review, we will present some of these molecular rearrangements found in RNA viruses, viroids and virus-associated RNAs, relating how these dynamic regions were discovered, the activities that might be regulated, and what factors or conditions might cause a switch between conformations.

  1. Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants.

    Science.gov (United States)

    Osakabe, Yuriko; Watanabe, Takahito; Sugano, Shigeo S; Ueta, Risa; Ishihara, Ryosuke; Shinozaki, Kazuo; Osakabe, Keishi

    2016-05-26

    Genome editing using the CRISPR/Cas9 system can be used to modify plant genomes, however, improvements in specificity and applicability are still needed in order for the editing technique to be useful in various plant species. Here, using genome editing mediated by a truncated gRNA (tru-gRNA)/Cas9 combination, we generated new alleles for OST2, a proton pump in Arabidopsis, with no off-target effects. By following expression of Cas9 and the tru-gRNAs, newly generated mutations in CRIPSR/Cas9 transgenic plants were detected with high average mutation rates of up to 32.8% and no off-target effects using constitutive promoter. Reducing nuclear localization signals in Cas9 decreased the mutation rate. In contrast, tru-gRNA Cas9 cassettes driven by meristematic- and reproductive-tissue-specific promoters increased the heritable mutation rate in Arabidopsis, showing that high expression in the germ line can produce bi-allelic mutations. Finally, the new mutant alleles obtained for OST2 exhibited altered stomatal closing in response to environmental conditions. These results suggest further applications in molecular breeding to improve plant function using optimized plant CRISPR/Cas9 systems.

  2. Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants

    Science.gov (United States)

    Osakabe, Yuriko; Watanabe, Takahito; Sugano, Shigeo S; Ueta, Risa; Ishihara, Ryosuke; Shinozaki, Kazuo; Osakabe, Keishi

    2016-01-01

    Genome editing using the CRISPR/Cas9 system can be used to modify plant genomes, however, improvements in specificity and applicability are still needed in order for the editing technique to be useful in various plant species. Here, using genome editing mediated by a truncated gRNA (tru-gRNA)/Cas9 combination, we generated new alleles for OST2, a proton pump in Arabidopsis, with no off-target effects. By following expression of Cas9 and the tru-gRNAs, newly generated mutations in CRIPSR/Cas9 transgenic plants were detected with high average mutation rates of up to 32.8% and no off-target effects using constitutive promoter. Reducing nuclear localization signals in Cas9 decreased the mutation rate. In contrast, tru-gRNA Cas9 cassettes driven by meristematic- and reproductive-tissue-specific promoters increased the heritable mutation rate in Arabidopsis, showing that high expression in the germ line can produce bi-allelic mutations. Finally, the new mutant alleles obtained for OST2 exhibited altered stomatal closing in response to environmental conditions. These results suggest further applications in molecular breeding to improve plant function using optimized plant CRISPR/Cas9 systems. PMID:27226176

  3. 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.

  4. 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.

  5. Cas9-based genome editing in Arabidopsis and tobacco.

    Science.gov (United States)

    Li, Jian-Feng; Zhang, Dandan; Sheen, Jen

    2014-01-01

    Targeted modification of plant genome is key to elucidating and manipulating gene functions in plant research and biotechnology. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) technology is emerging as a powerful genome-editing method in diverse plants that traditionally lacked facile and versatile tools for targeted genetic engineering. This technology utilizes easily reprogrammable guide RNAs (sgRNAs) to direct Streptococcus pyogenes Cas9 endonuclease to generate DNA double-stranded breaks in targeted genome sequences, which facilitates efficient mutagenesis by error-prone nonhomologous end-joining (NHEJ) or sequence replacement by homology-directed repair (HDR). In this chapter, we describe the procedure to design and evaluate dual sgRNAs for plant codon-optimized Cas9-mediated genome editing using mesophyll protoplasts as model cell systems in Arabidopsis thaliana and Nicotiana benthamiana. We also discuss future directions in sgRNA/Cas9 applications for generating targeted genome modifications and gene regulations in plants.

  6. Bacterial delivery of TALEN proteins for human genome editing.

    Directory of Open Access Journals (Sweden)

    Jingyue Jia

    Full Text Available Transcription Activator-Like Effector Nucleases (TALENs are a novel class of sequence-specific nucleases that have recently gained prominence for its ease of production and high efficiency in genome editing. A TALEN pair recognizes specific DNA sequences and introduce double-strand break in the target site, triggering non-homologous end joining and homologous recombination. Current methods of TALEN delivery involves introduction of foreign genetic materials, such as plasmid DNA or mRNA, through transfection. Here, we show an alternative way of TALEN delivery, bacterial type III secretion system (T3SS mediated direct injection of the TALEN proteins into human cells. Bacterially injected TALEN was shown to efficiently target host cell nucleus where it persists for almost 12 hours. Using a pair of TALENs targeting venus gene, such injected nuclear TALENs were shown functional in introducing DNA mutation in the target site. Interestingly, S-phase cells seem to show greater sensitivity to the TALEN mediated target gene modification. Accordingly, efficiency of such genome editing can easily be manipulated by the infection dose, number of repeated infections as well as enrichment of S phase cells. This work further extends the utility of T3SS in the delivery of functional proteins into mammalian cells to alter their characters for biomedical applications.

  7. Bacterial delivery of TALEN proteins for human genome editing.

    Science.gov (United States)

    Jia, Jingyue; Jin, Yongxin; Bian, Ting; Wu, Donghai; Yang, Lijun; Terada, Naohiro; Wu, Weihui; Jin, Shouguang

    2014-01-01

    Transcription Activator-Like Effector Nucleases (TALENs) are a novel class of sequence-specific nucleases that have recently gained prominence for its ease of production and high efficiency in genome editing. A TALEN pair recognizes specific DNA sequences and introduce double-strand break in the target site, triggering non-homologous end joining and homologous recombination. Current methods of TALEN delivery involves introduction of foreign genetic materials, such as plasmid DNA or mRNA, through transfection. Here, we show an alternative way of TALEN delivery, bacterial type III secretion system (T3SS) mediated direct injection of the TALEN proteins into human cells. Bacterially injected TALEN was shown to efficiently target host cell nucleus where it persists for almost 12 hours. Using a pair of TALENs targeting venus gene, such injected nuclear TALENs were shown functional in introducing DNA mutation in the target site. Interestingly, S-phase cells seem to show greater sensitivity to the TALEN mediated target gene modification. Accordingly, efficiency of such genome editing can easily be manipulated by the infection dose, number of repeated infections as well as enrichment of S phase cells. This work further extends the utility of T3SS in the delivery of functional proteins into mammalian cells to alter their characters for biomedical applications.

  8. 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...

  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.

  11. Dynamic 5-HT2C receptor editing in a mouse model of obesity.

    Directory of Open Access Journals (Sweden)

    Harriët Schellekens

    Full Text Available The central serotonergic signalling system has been shown to play an important role in appetite control and the regulation of food intake. Serotonin exerts its anorectic effects mainly through the 5-HT(1B, 5-HT(2C and 5-HT(6 receptors and these are therefore receiving increasing attention as principal pharmacotherapeutic targets for the treatment of obesity. The 5-HT(2C receptor has the distinctive ability to be modified by posttranscriptional RNA editing on 5 nucleotide positions (A, B, C, D, E, having an overall decreased receptor function. Recently, it has been shown that feeding behaviour and fat mass are altered when the 5-HT(2C receptor RNA is fully edited, suggesting a potential role for 5-HT(2C editing in obesity. The present studies investigate the expression of serotonin receptors involved in central regulation of food intake, appetite and energy expenditure, with particular focus on the level of 5-HT(2C receptor editing. Using a leptin-deficient mouse model of obesity (ob/ob, we show increased hypothalamic 5-HT(1A receptor expression as well as increased hippocampal 5-HT(1A, 5-HT(1B, and 5-HT(6 receptor mRNA expression in obese mice compared to lean control mice. An increase in full-length 5-HT(2C expression, depending on time of day, as well as differences in 5-HT(2C receptor editing were found, independent of changes in total 5-HT(2C receptor mRNA expression. This suggests that a dynamic regulation exists of the appetite-suppressing effects of the 5-HT(2C receptor in both the hypothalamus and the hippocampus in the ob/ob mice model of obesity. The differential 5-HT(1A, 5-HT(1B and 5-HT(6 receptor expression and altered 5-HT(2C receptor editing profile reported here is poised to have important consequences for the development of novel anti-obesity therapies.

  12. 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.

  13. Mutvārdu kultūras mārketinga komunikācija: teātra izrādes "Alu cilvēks" gadījuma analīze

    OpenAIRE

    Lauva, Elīna

    2008-01-01

    Bakalaura darba „Mutvārdu kultūras marketinga komunikācija: teātra izrādes „Alu Cilvēks” gadījuma analīze” mērķis bija identificēt „Alu Cilvēka” marketinga komunikācijā izmantotos mutvārdu un baumu marketinga komunikācijas instrumentus, noskaidrot, cik lielā mērā „Alu Cilvēka” marketinga komunikācijā izmantotie mutvārdu marketinga instrumenti saistāmi ar izrādes popularitāti un apmeklētību, kā arī novērtēt, kā izrādes marketinga komunikācija tiek atspoguļota publiskā viedokļa telpā, konkrēti,...

  14. Looking forward to genetically edited fruit crops.

    Science.gov (United States)

    Nagamangala Kanchiswamy, Chidananda; Sargent, Daniel James; Velasco, Riccardo; Maffei, Massimo E; Malnoy, Mickael

    2015-02-01

    The availability of genome sequences for many fruit crops has redefined the boundaries of genetic engineering and genetically modified (GM) crop plants. However commercialization of GM crops is hindered by numerous regulatory and social hurdles. Here, we focus on recently developed genome-editing tools for fruit crop improvement and their importance from the consumer perspective. Challenges and opportunities for the deployment of new genome-editing tools for fruit plants are also discussed.

  15. Wetland Ecology Principles and Conservation, Second Edition

    OpenAIRE

    Richard Smardon

    2014-01-01

    This is a book review of Wetland Ecology Principles and Conservation, second edition, by Paul Keddy. This review focuses on the book’s content as it relates to wetland sustainability for both science and management. Besides overall comments, comparisons are made with the first edition of the book and then very specific chapter-by-chapter relationships to wetland sustainability are made to illustrate specific applications toward wetland sustainability.

  16. The commercialization of genome-editing technologies.

    Science.gov (United States)

    Brinegar, Katelyn; K Yetisen, Ali; Choi, Sun; Vallillo, Emily; Ruiz-Esparza, Guillermo U; Prabhakar, Anand M; Khademhosseini, Ali; Yun, Seok-Hyun

    2017-01-18

    The emergence of new gene-editing technologies is profoundly transforming human therapeutics, agriculture, and industrial biotechnology. Advances in clustered regularly interspaced short palindromic repeats (CRISPR) have created a fertile environment for mass-scale manufacturing of cost-effective products ranging from basic research to translational medicine. In our analyses, we evaluated the patent landscape of gene-editing technologies and found that in comparison to earlier gene-editing techniques, CRISPR has gained significant traction and this has established dominance. Although most of the gene-editing technologies originated from the industry, CRISPR has been pioneered by academic research institutions. The spinout of CRISPR biotechnology companies from academic institutions demonstrates a shift in entrepreneurship strategies that were previously led by the industry. These academic institutions, and their subsequent companies, are competing to generate comprehensive intellectual property portfolios to rapidly commercialize CRISPR products. Our analysis shows that the emergence of CRISPR has resulted in a fivefold increase in genome-editing bioenterprise investment over the last year. This entrepreneurial movement has spurred a global biotechnology revolution in the realization of novel gene-editing technologies. This global shift in bioenterprise will continue to grow as the demand for personalized medicine, genetically modified crops and environmentally sustainable biofuels increases. However, the monopolization of intellectual property, negative public perception of genetic engineering and ambiguous regulatory policies may limit the growth of these market segments.

  17. Genome editing systems in novel therapies.

    Science.gov (United States)

    Jang, Yoon-Young; Cai, Liuhong; Ye, Zhaohui

    2016-01-01

    Genome editing is the process in which DNA sequences at precise genomic locations are modified. In the past three decades, genome editing by homologous recombination has been successfully performed in mouse for generating genetic models. The low efficiency of this process in human cells, however, had prevented its clinical application until the recent advancements in designer endonuclease technologies. The significantly improved genome editing efficiencies aided by ZFN, TALEN, and CRISPR systems provide unprecedented opportunities not only for biomedical research, but also for developing novel therapies. Applications based on these genome editing tools to disrupt deleterious genes, correct genetic mutations, deliver functional transgenes more effectively or even modify the epigenetic landscape are being actively investigated for gene and cell therapy purposes. Encouraging results have been obtained in limited clinical trials in the past two years. While most of the applications are still in proof-of-principle or preclinical development stages, it is anticipated that the coming years will see increasing clinical success in novel therapies based on the modern genome editing technologies. It should be noted that critical issues still remain before the technologies can be translated into more reliable therapies. These key issues include off-target evaluation, establishing appropriate preclinical models and improving the currently low efficiency of homology-based precise gene replacement. In this review we discuss the preclinical and clinical studies aiming at translating the genome editing technologies as well as the issues that are important for more successful translation.

  18. Approximate Graph Edit Distance in Quadratic Time.

    Science.gov (United States)

    Riesen, Kaspar; Ferrer, Miquel; Bunke, Horst

    2015-09-14

    Graph edit distance is one of the most flexible and general graph matching models available. The major drawback of graph edit distance, however, is its computational complexity that restricts its applicability to graphs of rather small size. Recently the authors of the present paper introduced a general approximation framework for the graph edit distance problem. The basic idea of this specific algorithm is to first compute an optimal assignment of independent local graph structures (including substitutions, deletions, and insertions of nodes and edges). This optimal assignment is complete and consistent with respect to the involved nodes of both graphs and can thus be used to instantly derive an admissible (yet suboptimal) solution for the original graph edit distance problem in O(n3) time. For large scale graphs or graph sets, however, the cubic time complexity may still be too high. Therefore, we propose to use suboptimal algorithms with quadratic rather than cubic time for solving the basic assignment problem. In particular, the present paper introduces five different greedy assignment algorithms in the context of graph edit distance approximation. In an experimental evaluation we show that these methods have great potential for further speeding up the computation of graph edit distance while the approximated distances remain sufficiently accurate for graph based pattern classification.

  19. Precise genome editing by homologous recombination.

    Science.gov (United States)

    Hoshijima, K; Jurynec, M J; Grunwald, D J

    2016-01-01

    Simple and efficient methods are presented for creating precise modifications of the zebrafish genome. Edited alleles are generated by homologous recombination between the host genome and double-stranded DNA (dsDNA) donor molecules, stimulated by the induction of double-strand breaks at targeted loci in the host genome. Because several kilobase-long tracts of sequence can be exchanged, multiple genome modifications can be generated simultaneously at a single locus. Methods are described for creating: (1) alleles with simple sequence changes or in-frame additions, (2) knockin/knockout alleles that express a reporter protein from an endogenous locus, and (3) conditional alleles in which exons are flanked by recombinogenic loxP sites. Significantly, our approach to genome editing allows the incorporation of a linked reporter gene into the donor sequences so that successfully edited alleles can be identified by virtue of expression of the reporter. Factors affecting the efficiency of genome editing are discussed, including the finding that dsDNA products of I-SceI meganuclease enzyme digestion are particularly effective as donor molecules for gene-editing events. Reagents and procedures are described for accomplishing efficient genome editing in the zebrafish.

  20. Genome editing strategies: potential tools for eradicating HIV-1/AIDS.

    Science.gov (United States)

    Khalili, Kamel; Kaminski, Rafal; Gordon, Jennifer; Cosentino, Laura; Hu, Wenhui

    2015-06-01

    Current therapy for controlling human immunodeficiency virus (HIV-1) infection and preventing acquired immunodeficiency syndrome (AIDS) progression has profoundly decreased viral replication in cells susceptible to HIV-1 infection, but it does not eliminate the low level of viral replication in latently infected cells, which contain integrated copies of HIV-1 proviral DNA. There is an urgent need for the development of HIV-1 genome eradication strategies that will lead to a permanent or "sterile" cure of HIV-1/AIDS. In the past few years, novel nuclease-initiated genome editing tools have been developing rapidly, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system. These surgical knives, which can excise any genome, provide a great opportunity to eradicate the HIV-1 genome by targeting highly conserved regions of the HIV-1 long terminal repeats or essential viral genes. Given the time consuming and costly engineering of target-specific ZFNs and TALENs, the RNA-guided endonuclease Cas9 technology has emerged as a simpler and more versatile technology to allow permanent removal of integrated HIV-1 proviral DNA in eukaryotic cells, and hopefully animal models or human patients. The major unmet challenges of this approach at present include inefficient nuclease gene delivery, potential off-target cleavage, and cell-specific genome targeting. Nanoparticle or lentivirus-mediated delivery of next generation Cas9 technologies including nickase or RNA-guided FokI nuclease (RFN) will further improve the potential for genome editing to become a promising approach for curing HIV-1/AIDS.