An expanded tool kit for the auxin‐inducible degron system in budding yeast

doi:10.1002/YEA.2967

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An expanded tool kit for the auxin‐inducible degron system in budding yeast

Journal Article•DOI•

An expanded tool kit for the auxin‐inducible degron system in budding yeast

Magdalena E. Morawska¹, Helle D. Ulrich¹•Institutions (1)

London Research Institute¹

01 Sep 2013-Yeast (Wiley-Blackwell)-Vol. 30, Iss: 9, pp 341-351

TL;DR: The construction of a series of vectors is reported that significantly enhance the versatility of this auxin‐inducible degron (AID) system in Saccharomyces cerevisiae and provides evidence for a general usefulness of the system.

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Abstract: Fusion of inducible degradation signals, so-called degrons, to cellular proteins is an elegant method of controlling protein levels in vivo. Recently, a degron system relying on the plant hormone auxin has been described for use in yeast and vertebrate cells. We now report the construction of a series of vectors that significantly enhance the versatility of this auxin-inducible degron (AID) system in Saccharomyces cerevisiae. We have minimized the size of the degron and appended a series of additional epitope tags, allowing detection by commercial antibodies or fluorescence microscopy. The vectors are compatible with PCR-based genomic tagging strategies, allow for C- or N-terminal fusion of the degron, and provide a range of selection markers. Application to a series of yeast proteins, including essential replication factors, provides evidence for a general usefulness of the system.

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Journal Article•DOI•

Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization.

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Elphège P. Nora, Anton Goloborodko¹, Anne-Laure Valton², Johan H. Gibcus², Alec Uebersohn, Nezar Abdennur¹, Job Dekker², Leonid A. Mirny¹, Benoit G. Bruneau - Show less +5 more•Institutions (2)

Massachusetts Institute of Technology¹, University of Massachusetts Medical School²

18 May 2017-Cell

TL;DR: The data support that CTCF mediates transcriptional insulator function through enhancer blocking but not as a direct barrier to heterochromatin spreading, and provides new fundamental insights into the rules governing mammalian genome organization.

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1,259 citations

Cites methods or result from "An expanded tool kit for the auxin‐..."

...The CTCF-AID-EGFP targeting vector (pEN84) was assembled by serial modification of the base vector pFNF (Addgene #22687) using Gibson assembly with the following templates: the minimal functional AID tag (aa 71-114) described by (Morawska and Ulrich, 2013) was PCR amplified from pAID (Nishimura et al....
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...Plasmid Construction We used the smallest functional truncation of the AID tag (AID*, 44 amino-acids), initially developed in yeast (Morawska and Ulrich, 2013), shorter than the mini-AID (67 amino-acids (Kubota et al....
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...To deplete endogenous CTCF in mESCs, we targeted the stop codon of both Ctcf alleles to introduce a 44-amino-acid version of the AID tag (residues 71–114) (Morawska and Ulrich, 2013; Nishimura et al., 2009) with an eGFP cassette (Figure 1A, Table S1)....
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...Acute CTCF Depletion with the AID System To deplete endogenous CTCF in mESCs, we targeted the stop codon of both Ctcf alleles to introduce a 44-amino-acid version of the AID tag (residues 71–114) (Morawska and Ulrich, 2013; Nishimura et al., 2009) with an eGFP cassette (Figure 1A, Table S1)....
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...As reported previously, the AID fusion led to slight constitutive destabilization (Morawska and Ulrich, 2013), so that basal CTCF levels were about 2- to 3-fold less in the AID-eGFP fusion line as compared to the untagged parental line (Figures 1B and 1C)....
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Journal Article•DOI•

Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins

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Gordana Wutz¹, Csilla Várnai², Kota Nagasaka¹, David A. Cisneros¹, Roman R. Stocsits¹, Wen Tang¹, Stefan Schoenfelder², Gregor Jessberger¹, Matthias Muhar¹, M. Julius Hossain, Nike Walther, Birgit Koch, Moritz Kueblbeck, Jan Ellenberg, Johannes Zuber¹, Peter Fraser², Peter Fraser³, Jan-Michael Peters¹ - Show less +14 more•Institutions (3)

Research Institute of Molecular Pathology¹, Babraham Institute², Florida State University³

15 Dec 2017-The EMBO Journal

TL;DR: It is shown that cohesin suppresses compartments but is required for TADs and loops, that CTCF defines their boundaries, and that the cohes in unloading factor WAPL and its PDS5 binding partners control the length of loops.

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Abstract: Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compartments, TAD ...

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586 citations

Cites methods from "An expanded tool kit for the auxin‐..."

...The homologous recombination template used introduced the sequences coding for monomeric EGFP (L221K) and the Arabidopsis thaliana IAA1771114 (AID*) mini-degron (Morawska & Ulrich, 2013)....
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Journal Article•DOI•

The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans.

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Liangyu Zhang¹, Jordan D. Ward², Ze Cheng³, Abby F. Dernburg¹•Institutions (3)

California Institute for Quantitative Biosciences¹, University of California, San Francisco², University of California, Berkeley³

15 Dec 2015-Development

TL;DR: The auxin-inducible degradation (AID) system is adapted to C. elegans to enable conditional depletion of degron-tagged protein targets in as little as twenty minutes, and provides a powerful new tool for spatiotemporal regulation and analysis of protein function in a metazoan model organism.

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Abstract: Experimental manipulation of protein abundance in living cells or organisms is an essential strategy for investigation of biological regulatory mechanisms. Whereas powerful techniques for protein expression have been developed in Caenorhabditis elegans, existing tools for conditional disruption of protein function are far more limited. To address this, we have adapted the auxin-inducible degradation (AID) system discovered in plants to enable conditional protein depletion in C. elegans. We report that expression of a modified Arabidopsis TIR1 F-box protein mediates robust auxin-dependent depletion of degron-tagged targets. We document the effectiveness of this system for depletion of nuclear and cytoplasmic proteins in diverse somatic and germline tissues throughout development. Target proteins were depleted in as little as 20-30 min, and their expression could be re-established upon auxin removal. We have engineered strains expressing TIR1 under the control of various promoter and 3′ UTR sequences to drive tissue-specific or temporally regulated expression. The degron tag can be efficiently introduced by CRISPR/Cas9-based genome editing. We have harnessed this system to explore the roles of dynamically expressed nuclear hormone receptors in molting, and to analyze meiosis-specific roles for proteins required for germ line proliferation. Together, our results demonstrate that the AID system provides a powerful new tool for spatiotemporal regulation and analysis of protein function in a metazoan model organism.

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450 citations

Cites methods from "An expanded tool kit for the auxin‐..."

...We fused a 44-amino acid minimal degron sequence (Morawska and Ulrich, 2013) (Fig....
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Journal Article•DOI•

Chemically induced proximity in biology and medicine

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Benjamin Z. Stanton¹, Benjamin Z. Stanton², Emma J. Chory², Gerald R. Crabtree²•Institutions (2)

National Institutes of Health¹, Stanford University²

09 Mar 2018-Science

TL;DR: Basic conceptual advances enabled by synthetic proximity as well as emerging CIP-based therapeutic approaches are reviewed, anticipating that the translation of CIP methodology through both humanized gene therapies and degradation-by-dimerization approaches will have far-reaching clinical impact.

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Abstract: Proximity, or the physical closeness of molecules, is a pervasive regulatory mechanism in biology. For example, most posttranslational modifications such as phosphorylation, methylation, and acetylation promote proximity of molecules to play deterministic roles in cellular processes. To understand the role of proximity in biologic mechanisms, chemical inducers of proximity (CIPs) were developed to synthetically model biologically regulated recruitment. Chemically induced proximity allows for precise temporal control of transcription, signaling cascades, chromatin regulation, protein folding, localization, and degradation, as well as a host of other biologic processes. A systematic analysis of CIPs in basic research, coupled with recent technological advances utilizing CRISPR, distinguishes roles of causality from coincidence and allows for mathematical modeling in synthetic biology. Recently, induced proximity has provided new avenues of gene therapy and emerging advances in cancer treatment.

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221 citations

Cites background from "An expanded tool kit for the auxin‐..."

...Later, a short 44–amino acid tag referred to as IAA17 (AID*) was developed to expand the utility of the auxin system (52)....
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Journal Article•DOI•

Current challenges of research on filamentous fungi in relation to human welfare and a sustainable bio-economy: a white paper

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Vera Meyer¹, Mikael Rørdam Andersen², Axel A. Brakhage³, Axel A. Brakhage⁴, Gerhard H. Braus⁵, Mark X. Caddick⁶, Timothy C. Cairns¹, Ronald P. de Vries⁷, Thomas Haarmann, Kim Hansen⁸, Christiane Hertz-Fowler⁶, Sven Krappmann⁹, Uffe Hasbro Mortensen², Miguel A. Peñalva¹⁰, Arthur F. J. Ram¹¹, Ritchie M. Head - Show less +12 more•Institutions (11)

Technical University of Berlin¹, Technical University of Denmark², Leibniz Association³, University of Jena⁴, University of Göttingen⁵, University of Liverpool⁶, Utrecht University⁷, Novozymes⁸, University of Erlangen-Nuremberg⁹, Spanish National Research Council¹⁰, Leiden University¹¹

31 Aug 2016

TL;DR: This white paper reports on the discussions of the first academic-industry Think Tank meeting and the suggestions made for moving fungal bio(techno)logy forward.

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Abstract: The EUROFUNG network is a virtual centre of multidisciplinary expertise in the field of fungal biotechnology. The first academic-industry Think Tank was hosted by EUROFUNG to summarise the state of the art and future challenges in fungal biology and biotechnology in the coming decade. Currently, fungal cell factories are important for bulk manufacturing of organic acids, proteins, enzymes, secondary metabolites and active pharmaceutical ingredients in white and red biotechnology. In contrast, fungal pathogens of humans kill more people than malaria or tuberculosis. Fungi are significantly impacting on global food security, damaging global crop production, causing disease in domesticated animals, and spoiling an estimated 10 % of harvested crops. A number of challenges now need to be addressed to improve our strategies to control fungal pathogenicity and to optimise the use of fungi as sources for novel compounds and as cell factories for large scale manufacture of bio-based products. This white paper reports on the discussions of the Think Tank meeting and the suggestions made for moving fungal bio(techno)logy forward.

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193 citations

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References

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Open Access

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Journal Article•DOI•

Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae

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Mark S. Longtine¹, Amos Mckenzie¹, Douglas J. Demarini¹, Nirav Shah¹, Achim Wach², Arndt Brachat², Peter Philippsen², John R. Pringle¹ - Show less +4 more•Institutions (2)

University of North Carolina at Chapel Hill¹, University of Basel²

01 Jul 1998-Yeast

TL;DR: A new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications that should further facilitate the rapid analysis of gene function in S. cerevisiae.

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Abstract: An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications Using as selectable marker the S cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5 + or Escherichia coli kan r gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging) Because of the modular nature of the plasmids, they allow eYcient and economical use of a small number of PCR primers for a wide variety of gene manipulations Thus, these plasmids should further facilitate the rapid analysis of gene function in S cerevisiae ? 1998 John Wiley & Sons, Ltd

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5,301 citations

"An expanded tool kit for the auxin‐..." refers methods in this paper

...Gene deletions and tags were introduced by means of PCR-generated cassettes (Longtine et al., 1998), using the primers listed in Table S1 (see Supporting information)....
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Book Chapter•DOI•

Getting started with yeast.

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Fred Sherman¹•Institutions (1)

University of Rochester¹

01 Jan 2002-Methods in Enzymology

TL;DR: The yeast Saccharomyces cerevisiae is now recognized as a model system representing a simple eukaryote whose genome can be easily manipulated and made particularly accessible to gene cloning and genetic engineering techniques.

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Abstract: Publisher Summary The yeast Saccharomyces cerevisiae is now recognized as a model system representing a simple eukaryote whose genome can be easily manipulated. Yeast has only a slightly greater genetic complexity than bacteria and shares many of the technical advantages that permitted rapid progress in the molecular genetics of prokaryotes and their viruses. Some of the properties that make yeast particularly suitable for biological studies include rapid growth, dispersed cells, the ease of replica plating and mutant isolation, a well-defined genetic system, and most important, a highly versatile DNA transformation system. Being nonpathogenic, yeast can be handled with little precautions. Large quantities of normal baker's yeast are commercially available and can provide a cheap source for biochemical studies. The development of DNA transformation has made yeast particularly accessible to gene cloning and genetic engineering techniques. Structural genes corresponding to virtually any genetic trait can be identified by complementation from plasmid libraries. Plasmids can be introduced into yeast cells either as replicating molecules or by integration into the genome. In contrast to most other organisms, integrative recombination of transforming DNA in yeast proceeds exclusively via homologous recombination. Cloned yeast sequences, accompanied by foreign sequences on plasmids, can therefore be directed at will to specific locations in the genome.

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3,547 citations

Journal Article•DOI•

An auxin-based degron system for the rapid depletion of proteins in nonplant cells

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Kohei Nishimura¹, Tatsuo Fukagawa², Haruhiko Takisawa¹, Tatsuo Kakimoto¹, Masato T. Kanemaki¹ - Show less +1 more•Institutions (2)

Osaka University¹, National Institute of Genetics²

15 Nov 2009-Nature Methods

TL;DR: The auxin-inducible degron (AID) system allowed rapid and reversible degradation of target proteins in response to auxin and enabled us to generate efficient conditional mutants of essential proteins in yeast as well as cell lines derived from chicken, mouse, hamster, monkey and human cells, thus offering a powerful tool to control protein expression and study protein function.

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Abstract: Plants have evolved a unique system in which the plant hormone auxin directly induces rapid degradation of the AUX/IAA family of transcription repressors by a specific form of the SCF E3 ubiquitin ligase Other eukaryotes lack the auxin response but share the SCF degradation pathway, allowing us to transplant the auxin-inducible degron (AID) system into nonplant cells and use a small molecule to conditionally control protein stability The AID system allowed rapid and reversible degradation of target proteins in response to auxin and enabled us to generate efficient conditional mutants of essential proteins in yeast as well as cell lines derived from chicken, mouse, hamster, monkey and human cells, thus offering a powerful tool to control protein expression and study protein function

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1,335 citations

"An expanded tool kit for the auxin‐..." refers background or methods in this paper

...The recently described auxin-inducible degron (AID) system uses a plant hormone-induced degradation signal to control protein levels (Nishimura et al., 2009)....
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...TIR1 strains were created by integration of pNHK53 (encoding OsTIR1 under control of the ADH1 promoter) into theURA3 locus (Nishimura et al., 2009)....
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...…due to the lack of an auxin-responsive system in animals or yeast, the hormone as well as the F-box protein are otherwise biologically silent and cause no measurable physiological changes in the absence of a target, thus minimizing possible side-effects of the treatment (Nishimura et al., 2009)....
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...In budding yeast, the AID system has been successfully applied to several proteins resident in different cellular compartments (Nishimura et al., 2009; Watase et al., 2012)....
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...As reported, the full-length AID-tag is responsive to even low auxin concentrations (Nishimura et al., 2009)....
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Journal Article•DOI•

Epitope tagging of yeast genes using a PCR‐based strategy: more tags and improved practical routines

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Michael Knop, Katja Siegers, Gislene Pereira, Wolfgang Zachariae¹, Barbara Winsor, Kim Nasmyth¹, Elmar Schiebel - Show less +3 more•Institutions (1)

Research Institute of Molecular Pathology¹

01 Jul 1999-Yeast

TL;DR: In the yeast Saccharomyces cerevisiae, molecular biological techniques have been developed that use a simple PCR‐based strategy to introduce epitope tags to chromosomal loci to produce PCR amplificable modules.

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Abstract: Epitope tagging of proteins as a strategy for the analysis of function, interactions and the subcellular distribution of proteins has become widely used. In the yeast Saccharomyces cerevisiae, molecular biological techniques have been developed that use a simple PCR-based strategy to introduce epitope tags to chromosomal loci (Wach et al., 1994). To further employ the power of this strategy, a variety of novel tags was constructed. These tags were combined with different selectable marker genes, resulting in PCR amplificable modules. Only one set of primers is required for the amplification of any module. Furthermore, convenient laboratory techniques are described that facilitate the genetic manipulations of yeast strains, as well as the analysis of the epitope-tagged proteins.

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1,070 citations

"An expanded tool kit for the auxin‐..." refers background or methods in this paper

...Plasmids pNHK53 (OsTIR1), pSM409 (IAA17–hphNT1), pMK43 (IAA17–KanMX4), pSM410 (IAA17–HIS3MX6) and pMK38 (PCUP1– IAA17–KanMX4) were obtained from the National BioResource Project–Yeast (Japan). pFA–Nat–NT2 and pAW8–6xFLAG were from EUROSCARF, and pYM3 from M. Knop (Knop et al., 1999)....
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...In higher eukaryotes, downregulation of gene expression by RNAimediated depletion of the mRNA is most commonly used today, but this technology exhibits a rather slow response, is still prone to off-target effects and is unavailable in simple model organisms such as budding yeast (Carthew, 2001)....
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Journal Article•DOI•

Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation

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Philipp Stelter¹, Helle D. Ulrich¹•Institutions (1)

Max Planck Society¹

11 Sep 2003-Nature

TL;DR: These findings assign a function to SUMO during S phase and demonstrate how ubiquitin and SUMO, by regulating the accuracy of replication and repair, contribute to overall genomic stability.

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Abstract: Protein modification by ubiquitin is emerging as a signal for various biological processes in eukaryotes, including regulated proteolysis, but also for non-degradative functions such as protein localization, DNA repair and regulation of chromatin structure. A small ubiquitin-related modifier (SUMO) uses a similar conjugation system that sometimes counteracts the effects of ubiquitination. Ubiquitin and SUMO compete for modification of proliferating cell nuclear antigen (PCNA), an essential processivity factor for DNA replication and repair. Whereas multi-ubiquitination is mediated by components of the RAD6 pathway and promotes error-free repair, SUMO modification is associated with replication. Here we show that RAD6-mediated mono-ubiquitination of PCNA activates translesion DNA synthesis by the damage-tolerant polymerases eta and zeta in yeast. Moreover, polymerase zeta is differentially affected by mono-ubiquitin and SUMO modification of PCNA. Whereas ubiquitination is required for damage-induced mutagenesis, both SUMO and mono-ubiquitin contribute to spontaneous mutagenesis in the absence of DNA damage. Our findings assign a function to SUMO during S phase and demonstrate how ubiquitin and SUMO, by regulating the accuracy of replication and repair, contribute to overall genomic stability.

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840 citations

"An expanded tool kit for the auxin‐..." refers methods in this paper

...DOI: 10.1002/yea concentrations of auxin and/or hydroxyurea and treatment by UV irradiation using a Stratalinker 2400 (Stratagene) where indicated, as described previously (Stelter and Ulrich, 2003)....
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