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YEAST INTRAGENIC TRANSCRIPTIONAL CONTROL - ACTIVATION AND REPRESSION SITES WITHIN THE coding REGION OF THE SACCHAROMYCES-CEREVISIAE LPD1 GENE

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MOLECULAR AND CELLULAR BIOLOGY 1994年第1期14卷 214-225页

作者： SINCLAIR, DA KORNFELD, GD DAWES, IW School of Biochemistry and Molecular Genetics University of New South Wales Kensington Australia.

Though widely recognized in higher eukaryotes, the regulation of Saccharomyces cerevisiae genes transcribed by RNA polymerase II by proteins that bind within the coding sequence remains largely speculative. We have shown for the LPD1 gene, encoding lipoamide dehydrogenase, that the coding sequence between +13 and +469 activated gene expression of an LPD1=lacZ fusion by up to sixfold in the presence of the upstream promoter. This downstream region, inserted upstream of a promoterless CYC1=lacZ fusion, activated gene expression in a carbon source-dependent manner by a factor of 15 to 111, independent of orientation. Deletion and mutational analysis identified two downstream activation sites (DAS1 and DAS2) and two downstream repressor sites (DRS1 and DRS2) that influence the rate of LPD1 transcription rather than mRNA degradation or translation. Activation from the DAS1 region (positions +137 to +191), encompassing a CDEI-like element, is twofold under derepressive conditions. Activation from DAS2 (+291 to +296), a CRE-like motif, is 12-fold for both repressed and derepressed states. DRS1, a pair of adjacent and opposing ABF1 sites (+288 to +313), is responsible for a 1.3- to 2-fold repression of transcription, depending on the carbon source. DRS1 requires the concerted action of DRS2 (a RAP1 motif at position +406) for repression of transcription only when the gene is induced. Gel mobility shift analysis and in vitro footprinting have shown that proteins bind in vitro to these downstream elements.

关键词： .intragenic repressor repression coding sequence coding region transcriptional control DRs Abf Upstream Promoter downstream region lipoamide LPDJ LPDI lacZ

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The exon 13 duplication in the BRCA1 gene is a founder mutation present in geographically diverse populations

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AMERICAN JOURNAL OF HUMAN GENETICS 2000年第1期67卷 207-212页

作者： Mazoyer, S Leary, J Kirk, J Fleischmann, E Wagner, T Claes, K Messiaen, L Foulkes, W Desrochers, M Simard, J Phelan, CM Kwan, E Narod, SA Vahteristo, P Nevanlinna, H Durando, X Bignon, YJ Peyrat, JP Bonnardel, C Sinilnikova, OM Puget, N Lenoir, GM Mazoyer, S Audoynaud, C Goldgar, D Maugard, C Caux, V Gad, S Stoppa-Lyonnet, D Noguès, C Lidereau, R Machavoine, C Bressac-de Paillerets, B Kuschel, B Betz, B Niederacher, D Beckmann, MW Hamann, U Gayther, SA Ponder, BAP Robinson, M Taylor, GR Bishop, T Catteau, A Solomon, E Cohen, B Steel, M Collins, N Stratton, M van der Looij, M Oláh, E Miller, NJ Barton, DE Sverdlov, RS Friedman, E Radice, P Montagna, M Sensi, E Caligo, M van Eijk, R Devilee, P van der Luijt, R Heimdal, K Moller, P Borg, Å Diez, O Cortes, J Domenech, M Baiget, M Osorio, A Benítez, J Borg, Å Maillet, P Sappino, AP Özdag, H Özçelik, T Ozturk, M Rohlfs, EM Boyd, J McDermott, D Offit, K Unger, M Nathanson, K Weber, BL Sellers, TA Hampton, E Couch, FJ Neuhausen, S Fac Med UMR CNRS UCBL 5641 F-69373 Lyon 08 France

Recently a 6-kb duplication of exon 13, which creates a frameshift in the coding sequence of the BRCA1 gene, has been described in three unrelated U.S. families of European ancestry and in one Portuguese family. Here, our goal was to estimate the frequency and geographic diversity of carriers of this duplication. To do this, a collaborative screening study was set up that involved 39 institutions from 19 countries and included 3,580 unrelated individuals with a family history of the disease and 934 early-onset breast and/or ovarian cancer cases. A total of 11 additional families carrying this mutation were identified in Australia (1), Belgium (1), Canada (1), Great Britain (6), and the United States (2). Haplotyping showed that they are likely to derive from a common ancestor, possibly of northern British origin. Our results demonstrate that it is strongly advisable, for laboratories carrying out screening either in English-speaking countries or in countries with historical links with Britain, to include within their BRCA1 screening protocols the polymerase chain reaction-based assay described in this report.

关键词： .mainly Breast Cancer rearrangements carriers carrying unrelated ancestor coding sequence Great Britain Founder Mutation Exon Duplication BRCA Gene BRCA Exon Duplication Screening breast and/or ovarian English-speaking countries

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Prediction of complete gene structures in human genomic DNA

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JOURNAL OF MOLECULAR BIOLOGY 1997年第1期268卷 78-94页

作者： Burge, C Karlin, S STANFORD UNIV DEPT MATH STANFORD CA 94305 USA

We introduce a general probabilistic model of the gene structure of human genomic sequences which incorporates descriptions of the basic transcriptional, translational and splicing signals, as well. as length distributions and compositional features of exons, introns and intergenic regions. Distinct sets of model parameters are derived to account for the many substantial differences in gene density and structure observed in distinct C + G compositional regions of the human genome. Lu addition, new models of the donor and acceptor splice signals are described which capture potentially important dependencies between signal positions. The model is applied to the problem of gene identification in a computer program, GENSCAN, which identifies complete exon/intron structures of genes in genomic DNA. Novel features of the program include the capacity to predict multiple genes in a sequence, to deal with partial as well as complete genes, and to predict consistent sets of genes occurring on either or both DNA strands. GENSCAN is shown to have substantially higher accuracy than existing methods when tested on standardized sets of human and vertebrate genes, with 75 to 80% of exons identified exactly. The program is also capable of indicating fairly accurately the reliability of each predicted exon. Consistent lv high levels of accuracy are observed for sequences of differing C + G content and for distinct groups of vertebrates. (C) 1997 Academic Press Limited.

关键词： exon prediction gene identification coding sequence probabilistic model splice signal

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sequence AND HETEROGENEITY IN THE 5S RNA GENE-CLUSTER OF DROSOPHILA-MELANOGASTER

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NUCLEIC ACIDS RESEARCH 1980年第3期8卷 441-451页

作者： TSCHUDI, C PIRROTTA, V European Molecular Biology Laboratory Postfach 10.2209 Heidelberg GFR

The sequence of the entire 5S RNA gene of Drosophila melanogaster was determined by sequencing collectively 23 copies contained in a cloned fragment of Drosophila DNA and by sequencing individually four subcloned gene copies. A repetitive heptamer (GCTG CCT) present in variable numbers immediately following the coding sequence, is responsible for the length heterogeneity in the spacer region. Some of the gene copies contain a nucleotide change in the coding region which results in a new site for the restriction enzyme Mn1 I. The variant 5S RNA produced by these gene copies has not been detected in vivo. Two other single nucleotide variations were identified in the spacer region.

关键词： .coding region melanogaster variant fragment spacer variations restriction coding sequence repeats RNA gene gene copies cuts repeat unit

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A coding-independent function of an alternative Ube3a transcript during neuronal development

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NATURE NEUROSCIENCE 2015年第5期18卷 666-+页

作者： Valluy, Jeremy Bicker, Silvia Aksoy-Aksel, Ayla Lackinger, Martin Sumer, Simon Fiore, Roberto Wuest, Tatjana Seffer, Dominik Metge, Franziska Dieterich, Christoph Woehr, Markus Schwarting, Rainer Schratt, Gerhard Univ Marburg Inst Physiol Chem Biochem Pharmacol Ctr Marburg D-35032 Marburg Germany Heidelberg Univ Interdisciplinary Ctr Neurosci SFB Jr Grp 488 Heidelberg Germany Univ Marburg Behav Neurosci Expt & Biol Psychol D-35032 Marburg Germany Max Planck Inst Biol Ageing Computat RNA Biol Lab Cologne Germany

The E3 ubiquitin ligase Ube3a is an important regulator of activity-dependent synapse development and plasticity. Ube3a mutations cause Angelman syndrome and have been associated with autism spectrum disorders (ASD). However, the biological significance of alternative Ube3a transcripts generated in mammalian neurons remains unknown. We report here that Ube3a1 RNA, a transcript that encodes a truncated Ube3a protein lacking catalytic activity, prevents exuberant dendrite growth and promotes spine maturation in rat hippocampal neurons. Surprisingly, Ube3a1 RNA function was independent of its coding sequence but instead required a unique 3' untranslated region and an intact microRNA pathway. Ube3a1 RNA knockdown increased activity of the plasticity-regulating miR-134, suggesting that Ube3a1 RNA acts as a dendritic competing endogenous RNA. Accordingly, the dendrite-growth-promoting effect of Ube3a1 RNA knockdown in vivo is abolished in mice lacking miR-134. Taken together, our results define a noncoding function of an alternative Ube3a transcript in dendritic protein synthesis, with potential implications for Angelman syndrome and ASD.

关键词： heart atrium septum defect Angelman Syndrome transcript RNA coding sequence protein synthesis dendrite growth Catalyst activity

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A eubacterial gene conferring spectinomycin resistance on Chlamydomonas reinhardtii: Integration into the nuclear genome and gene expression

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GENETICS 1997年第1期145卷 97-110页

作者： Cerutti, H Johnson, AM Gillham, NW Boynton, JE DUKE UNIV DEPT BOTDEV CELL & MOL BIOL GRPDURHAMNC 27708 DUKE UNIV DEPT ZOOLDEV CELL & MOL BIOL GRPDURHAMNC 27708

We have constructed a dominant selectable marker for nuclear transformation of C. reinhardtii, composed of the coding sequence of the eubacterial aadA gene (conferring spectinomycin resistance) fused to the 5' and 3' untranslated regions of tile endogenous RbcS2 gene. Spectinomycin-resistant transformants isolated by direct selection (1) contain the chimeric gene(s) stably integrated into the nuclear genome, (2) show cosegregation of the resistance phenotype with the introduced DNA, and (3) synthesize the expected mRNA and protein. Small linearized plasmids appeared to be inserted into the nuclear genome preferentially through their ends, with relatively few large deletions and/or rearrangements. Multiple copy transformants often integrated concatemers of transforming DNA. Our detailed analysis of the complex integration patterns of plasmid DNA in C. reinhardtii nuclear transformants should be useful for improving the technique of insertional mutagenesis. We also found that the spectinomycin-resistance phenotype was unstable in about half of the transformants. When maintained under nonselective conditions, neither the aadA mRNA nor the AadA protein were detected in these subclones. Moreover, since the integrated transforming DNA was not altered or lost, expression of the RbcS2::aadA::RbcS2 gene(s) appears to be repressed. Measurements of transcriptional activity, mRNA accumulation, and mRNA stability suggest that expression of this chimeric gene(s) may also be affected by rapid RNA degradation, presumably due to defects in mRNA processing and/or nuclear export. Thus, both gene silencing and transcript instability, rather than biased codon usage, may explain the difficulties Encountered in the expression of foreign genes in the nuclear genome of Chlamydomonas.

关键词： .transformants inserted linearized transforming plasmids coding sequence chimeric gene eubacterial concatemers spectinomycin

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Superfolder GFP reporters validate diverse new mRNA targets of the classic porin regulator, MicF RNA

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MOLECULAR MICROBIOLOGY 2012年第3期84卷 428-445页

作者： Corcoran, Colin P. Podkaminski, Dimitri Papenfort, Kai Urban, Johannes H. Hinton, Jay C. D. Vogel, Joerg Univ Wurzburg Inst Mol Infect Biol D-97080 Wurzburg Germany Duke Univ Med Ctr Dept Surg Durham NC 27710 USA Trinity Coll Dublin Sch Genet & Microbiol Dept Microbiol Moyne Inst Prevent Med Dublin 2 Ireland

MicF is a textbook example of a small regulatory RNA (sRNA) that acts on a trans-encoded target mRNA through imperfect base pairing. Discovery of MicF as a post-transcriptional repressor of the major Escherichia coli porin OmpF established the paradigm for a meanwhile common mechanism of translational inhibition, through antisense sequestration of a ribosome binding site. However, whether MicF regulates additional genes has remained unknown for almost three decades. Here, we have harnessed the new superfolder variant of GFP for reportergene fusions to validate newly predicted targets of MicF in Salmonella. We show that the conserved 5' end of MicF acts by seed pairing to repress the mRNAs of global transcriptional regulator Lrp, and periplasmic protein YahO, while a second targeting region is also required to regulate the mRNA of the lipid A-modifying enzyme LpxR. Interestingly, MicF targets lpxR at both the ribosome binding site and deep within the coding sequence. MicF binding in the coding sequence of lpxR decreases mRNA stability through exacerbating the use of a native RNase E site proximal to the short MicF-lpxR duplex. Altogether, this study assigns the classic MicF sRNA to the growing class of Hfq-associated regulators that use diverse mechanisms to impact multiple loci.

关键词： Binding Sites mRNA Stability Ribosomes MICF gene Porins coding sequence Regulators Messenger RNA Green Fluorescent Proteins Translational Repression Journalists

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An upstream ORF with non-AUG start codon is translated in vivo but dispensable for translational control of GCN4 mRNA

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NUCLEIC ACIDS RESEARCH 2011年第8期39卷 3128-3140页

作者： Zhang, Fan Hinnebusch, Alan G. Eunice K Shriver Natl Inst Child Hlth & Human Dev Lab Gene Regulat & Dev NIH Bethesda MD 20892 USA

Genome-wide analysis of ribosome locations in mRNAs of Saccharomyces cerevisiae has revealed the translation of upstream open reading frames that initiate with near-cognate start codons in many transcripts. Two such non-translation initiation codon (AUG)-initiated upstream open reading frames (uORFs) (nAuORFs 1 and 2) occur in GCN4 mRNA upstream of the four AUG-initiated uORFs (uORFs 1-4) that regulate GCN4 translation. We verified that nAuORF2 is translated in vivo by demonstrating beta-galactosidase production from lacZ coding sequences fused to nAuORF2, in a manner abolished by replacing its non-AUG initiation codon (AUA) start codon with the non-cognate triplet AAA, whereas translation of nAuORF1 was not detected. Importantly, replacing the near-cognate start codons of both nAuORFs with non-cognate triplets had little or no effect on the repression of GCN4 translation in non-starved cells, nor on its derepression in response to histidine limitation, nutritional shift-down or treatment with rapamycin, hydrogen peroxide or methyl methanesulfonate. Additionally, we found no evidence that initiation from the AUA codon of nAuORF2 is substantially elevated, or dependent on Gcn2, the sole eIF2 alpha kinase of yeast, in histidine-deprived cells. Thus, although nAuORF2 is translated in vivo, it appears that this event is not stimulated by eIF2 alpha phosphorylation nor significantly influences GCN4 translational induction under various starvation or stress conditions.

关键词： hydrogen peroxide: 7722-84-1 rapamycin: 53123-88-9 lacZ methyl methanesulfonate: 66-27-3 eIF2-alpha: phosphorylation beta-galactosidase: 9031-11-2 EC 3.2.1.23 production Gcn2 GCN4 mRNA histidine: 4998-57-6 limitation non-translation initiation codon-initiated upstream open reading frame 1: translation non-translation initiation codon-initiated upstream open reading frame 2: translation non-AUG initiation codon start codon stress condition translational control starvation condition coding sequence nutritional shift-down translational control Initiator Codon In vivo stress conditions Upstream coding sequence EIF2AK4 gene

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Flow-cytometric visualization of C>U mRNA editing reveals the dynamics of the process in live cells

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RNA BIOLOGY 2015年第4期12卷 389-397页

作者： Severi, Francesco Conticello, Silvestro G. Ist Toscano Tumori Core Res Lab Florence Italy Univ Careggi Azienda Osped Dept Oncol Florence Italy

APOBEC1 is the catalytic subunit of the complex that edits ApolipoproteinB (ApoB) mRNA, which specifically deaminates cytidine 6666 to uracil in the human transcript. The editing leads to the generation of a stop codon, resulting in the synthesis of a truncated form of ApoB. We have developed a method to quantitatively assay ApoB RNA editing in live cells by using a double fluorescent mCherry-EGFP chimera containing a approximate to 300bp fragment encompassing the region of ApoB subject to RNA editing. Coexpression of APOBEC1 together with this chimera causes specific RNA editing of the ApoB fragment. The insertion of a stop codon between the mCherry and EGFP thus induces the loss of EGFP fluorescence. Using this method we analyze the dynamics of APOBEC1-dependent RNA editing under various conditions. Namely we show the interplay of APOBEC1 with known interactors (ACF, hnRNP-C1, GRY-RBP) in cells that are RNA editing-proficient (HuH-7) or -deficient (HEK-293T), and the effects of restricted cellular localization of APOBEC1 on the efficiency of the editing. Furthermore, our approach is effective in assaying the induction of RNA editing in Caco-2, a cellular model physiologically capable of ApoB RNA editing.

关键词： AID APOBECs cytosine deaminase lipid metabolism mRNA post-transcriptional modification RNA editing APOBEC1 Apolipoprotein B mRNA editing enzyme catalytic polypeptide 1 ApoB Apolipoprotein B ACF APOBEC1 Complementation Factor ADAR Adenosine Deaminase RNA-specific ADAT Adenosine Deaminase tRNA-specific cds coding sequence hnRNP-C1 heterogeneous nuclear ribonucleoprotein C1 GRY-RBP Glycine-Arginine-Tyrosine-rich RNA-binding protein EGFP Enhanced Green Fluorescent Protein RBM47 RNA binding motif protein 47 FBS Fetal bovine serum FACS Fluorescence activated cell sorting

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Characterisation of two genes encoding cytochrome P450 mono-oxygenases involved in gibberellin biosynthesis in apple (Malus X domestica Borkh.)

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JOURNAL OF HORTICULTURAL SCIENCE & BIOTECHNOLOGY 2014年第3期89卷 329-337页

作者： Wang, C. H. Bai, M. D. Tian, Y. K. Tian, W. Qingdao Agr Univ Coll Hort Qingdao 266109 Shandong Peoples R China

Gibberellins are important phytohormones that regulate plant stem elongation. This study reports the isolation of the MdKO and MdKAO genes from apical stem tissue of the apple (Malus x domestica Borkh.) cultivar 'Fuji'. The genes encode the CytP450 mono-oxygenases, ent-kaurene oxidase (KO) and ent-kaurenoic acid oxidase (KAO), which play key roles in the central steps of the gibberellin biosynthesis pathway. The genomic DNA sequences of both MdKO and MdKAO contained eight exons and seven introns. The coding sequence of MdKO consisted of 1,545 bp and encoded a predicted polypeptide of 514 amino acids, whereas the coding sequence of MdKAO was 1,512 bp and encoded a putative polypeptide of 503 amino acids. The transcripts MDP0000201930 and MDP0000326359 that spanned chromosome 8 from locations 3,980,774 - 3,983,650 and chromosome 2 from locations 26,788,993 - 26,793,838 in the 'Golden Delicious' apple genome, corresponded to the products of MdKO and MdKAO, respectively. Homology studies on the deduced amino acid sequences of MdKO and MdKAO indicated that both shared a high level of similarity (66 - 94%) with KO and KAO proteins from other plant species. Quantitative real-time PCR analyses showed that the level of transcription of MdKAO was significantly lower in the columnar parent, and in F-1 progeny with the columnar phenotype, than in the standard parent and F-1 progeny with the standard phenotype. This was confirmed in the columnar wild-type and in corresponding standard phenotype mutants. It is conceivable that lower expression of the MdKAO gene was responsible for the reduced concentration of bio-active gibberellins in the apical tissues of columnar apple stems.

关键词： Genes biosynthesis reduced concentration Chromosomes, Human, Pair 2 coding sequence Malus domestica coding Cytochrome P-450 Enzyme System Crab Apple Gibberellins

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