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Identifikation Und Charakterisierung Von Zellulären Zielproteinen Zur Antiviralen Therapie Der SARS-Coronavirus Infektion

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Identifikation Und Charakterisierung Von Zellulären Zielproteinen Zur Antiviralen Therapie Der SARS-Coronavirus Infektion Aus dem Max von Pettenkofer-Institut für Hygiene und Medizinische Mikrobiologie der Ludwig-Maximilians-Universität München Vorstand: Prof. Dr. med. Dr. h.c. Ulrich Koszinowski Identifikation und Charakterisierung von zellulären Zielproteinen zur antiviralen Therapie der SARS-Coronavirus Infektion Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften an der Medizinischen Fakultät der Ludwig-Maximilians-Universität München vorgelegt von Julia Schöpf aus Schrobenhausen - 2011 - Gedruckt mit Genehmigung der Medizinischen Fakultät der Ludwig-Maximilians-Universität München Betreuer: Prof. Dr. med. Dr. rer. nat. Jürgen Haas Zweitgutachter: Priv. Doz. Dr. Ursula Zimber-Strobl Dekan: Prof. Dr. med Dr. h.c. M. Reiser, FACR, FRCR Tag der mündlichen Prüfung: 29. November 2011 Ehrenwörtliche Versicherung Ich versichere hiermit ehrenwörtlich, dass die vorgelegte Dissertation von mir selbständig und ohne unerlaubte Hilfe angefertigt worden ist. München, den 03.03.2011 (Julia Schöpf) Erklärung Hiermit erkläre ich, - dass die Dissertation nicht ganz oder in wesentlichen Teilen einer anderen Prüfungskommission vorgelegt worden ist. - dass ich mich nicht anderweitig einer Doktorprüfung ohne Erfolg unterzogen habe. München, den 03.03.2011 (Julia Schöpf) für meine Eltern So eine Arbeit wird eigentlich nie fertig, man muss sie für fertig erklären, wenn man nach Zeit und Umständen das Möglichste getan hat. J. W. Goethe, Italienische Reise, 16. März 1787 Inhalte dieser Arbeit wurden wie folgt veröffentlicht: THE SARS-CORONAVIRUS-HOST INTERACTOME: IDENTIFICATION OF CYCLOPHILINS AS TARGET FOR PAN-CORONAVIRUS INHIBITORS S. Pfefferle*, J. Schöpf *, M. Kögl*, C. C. Friedel, M. A. Müller, J. Carbajo-Lozoya, T. Stellberger, E. von Dall’armi, P. Herzog, S. Kallies, D. Niemeyer, V. Ditt, T. Kuri, R. Züst, K. Pumpor, R. Hilgenfeld, F. Schwarz, R. Zimmer, I. Steffen, F. Weber, V. Thiel, G. Herrler, H.-J. Thiel, C. Schwegmann-Weßels, S. Pöhlmann, J. Haas, C. Drosten, A. von Brunn. * = equal contribution PLoS Pathogens 7 (10): e1002331.doi:10.1371/journal.ppat:100233 Beiträge zu wissenschaftlichen Tagungen: Jahrestagung der Gesellschaft für Virologie Heidelberg, 05. bis 07. März 2008 ANALYSIS OF INTRAVIRAL PROTEIN-PROTEIN INTERACTIONS OF THE SARS CORONAVIRUS ORFEOME BY PEPTIDE SCANNING von Brunn A. , Ester C., Schöpf J. , Uetz P. and Haas J. Art der Präsentation: Vortrag Jahrestreffen SARS-BMBF Zoonoseverbund Bonn, 11. April 2008 GENOMWEITE ANALYSE VON PROTEIN-PROTEIN INTERAKTIONEN ZWISCHEN DEM SARS- CORONAVIRUS MIT DER HUMANEN WIRTSZELLE Schöpf J. , Kögl M., von Dall’armi E., Endesfelder M., Haas J., von Brunn A. Art der Präsentation: Vortrag Jahrestagung der Gesellschaft für Virologie Leipzig, 18. bis 21. März 2009 GENOME-WIDE ANALYSIS OF PROTEIN-PROTEIN INTERACTIONS BETWEEN THE SARS- CORONAVIRUS AND THE HUMAN HOST Schöpf J. , Kögl M., von Dall’armi E., Endesfelder M., Weber F., Kuri T., Glowaka I., Bertram S., Pöhlmann S., Pfefferle S., Drosten C., Haas J., von Brunn A. Art der Präsentation: Poster Jahrestreffen SARS-BMBF Zoonoseverbund Frankfurt, 14. Januar 2010 IDENTIFICATION OF CYCLOSPORIN A AS AN INHIBITOR OF VIRAL REPLICATION OF SEVERAL CORONAVIRUSES Schöpf J. , Pfefferle S., Kögl M., Herzog P., Müller M.A., Muth D., Kuri T., Ebel F., Friedel C., Zimmer R., Weber F., Haas J., Thiel H.-J., Herrler G., Thiel V., Drosten C., von Brunn A. Art der Präsentation: Vortrag 4th European Congress of Virology Cernobbio, Comer See 07. bis 11. April 2010 APPLYING SYSTEMS BIOLOGY TO SEVERE ACUTE RESPIRATORY CORONAVIRUS AND ITS HUMAN HOST: IDENTIFICATION OF A CELLULAR TARGET AND ITS INHIBITOR FOR ANTIVIRAL INTERVENTION Schöpf J. , Pfefferle S., Kögl M., Herzog P., Müller M.A., Muth D., Kuri T., Ebel F., Friedel C., Zimmer R., Weber F., Haas J., Thiel H.-J., Herrler G., Thiel V., Drosten C., von Brunn A. Art der Präsentation: Vortrag Inhaltsverzeichnis 1 Einleitung 1 1.1 Die Familie der Coronaviridae ........................................................................................ 1 1.1.1 Struktureller Aufbau eines Coronavirus........................................................................... 2 1.1.2 Genomorganisation und Replikationszyklus ................................................................... 3 1.2 Das SARS-assoziierte Coronavirus............................................................................... 7 1.2.1 Der zoonotische Ursprung des SARS-CoV..................................................................... 7 1.2.2 Pathogenese des SARS-CoV.......................................................................................... 8 1.2.3 Das SARS-CoV und die Zytokin-Antwort ........................................................................ 9 1.2.4 Antivirale Strategien gegen das SARS-CoV ................................................................. 11 1.3 Der Calcineurin / NFAT-Signalweg .............................................................................. 12 1.3.1 Proteine der NFAT-Familie............................................................................................ 12 1.3.2 Die Calcineurin / NFAT - Signaltransduktionskaskade ................................................. 14 1.3.3 Inhibitoren des Calcineurin/NFAT-Signalwegs.............................................................. 16 1.3.4 Das antivirale Potential von Cyclosporin A.................................................................... 17 1.4 Zielsetzung der Arbeit ................................................................................................... 19 2 Material und Methoden 20 2.1 Materialien ...................................................................................................................... 20 2.1.1 Laborgeräte.................................................................................................................... 20 2.1.2 Chemikalien ................................................................................................................... 20 2.1.3 Verbrauchsmaterialien................................................................................................... 22 2.1.4 Enzyme und Größenstandards...................................................................................... 23 2.1.5 Antikörper....................................................................................................................... 23 2.1.6 Fertigsätze (Kits)............................................................................................................ 23 2.1.7 Zelllinien......................................................................................................................... 24 2.1.8 Viren............................................................................................................................... 24 2.1.9 Bakterienstämme........................................................................................................... 25 2.1.10 Plasmide ........................................................................................................................ 26 2.1.11 Oligonucleotide .............................................................................................................. 31 2.2 Mikrobiologische Methoden ......................................................................................... 34 2.2.1 Kultivierung von Bakterien............................................................................................. 34 2.2.2 Herstellung kompetenter Bakterien ............................................................................... 35 2.2.3 Transformation von Bakterien........................................................................................ 35 2.2.4 Automatisiertes Hefe-Zwei-Hybrid-Screening ............................................................... 35 2.3 DNA-Standardmethoden ............................................................................................... 36 2.3.1 Isolierung von Plasmid-DNA aus Bakterien .................................................................. 36 2.3.2 Polymerasekettenreaktion (PCR) .................................................................................. 36 2.3.3 Verdau mit Restriktionsenzymen................................................................................... 37 2.3.4 Dephosphorylierung des Vektor-Plasmids .................................................................... 37 2.3.5 Ligation........................................................................................................................... 37 2.3.6 Rekombinationsklonierung (Gateway®-Klonierung) ..................................................... 37 2.3.7 Agarose-Gelelektrophorese........................................................................................... 38 2.3.8 Aufreinigung von DNA aus Agarosegelen..................................................................... 38 2.3.9 Konzentrationsbestimmung von Nukleinsäuren............................................................ 39 2.3.10 Sequenzierung von DNA ............................................................................................... 39 2.4 RNA-Standardmethoden ............................................................................................... 39 2.4.1 RNA-Isolation aus Gewebe-Zellkulturen ....................................................................... 39 2.4.2 Synthese von cDNA....................................................................................................... 39 2.4.3 Echtzeit PCR-Quantifizierung
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  • Revisiting the Saccharomyces Cerevisiae Predicted Orfeome
    Downloaded from genome.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Letter Revisiting the Saccharomyces cerevisiae predicted ORFeome Qian-Ru Li,1,6 Anne-Ruxandra Carvunis,1,2,6 Haiyuan Yu,1,6 Jing-Dong J. Han,1,6,7 Quan Zhong,1 Nicolas Simonis,1 Stanley Tam,1 Tong Hao,1 Niels J. Klitgord,1 Denis Dupuy,1 Danny Mou,1 Ilan Wapinski,3,4 Aviv Regev,3,5 David E. Hill,1 Michael E. Cusick,1 and Marc Vidal1,8 1Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA; 2TIMC-IMAG, CNRS UMR5525, Faculte´deMe´decine, 38706 La Tronche Cedex, France; 3Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA; 4School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, USA; 5Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA Accurately defining the coding potential of an organism, i.e., all protein-encoding open reading frames (ORFs) or “ORFeome,” is a prerequisite to fully understand its biology. ORFeome annotation involves iterative computational predictions from genome sequences combined with experimental verifications. Here we reexamine a set of Saccharomyces cerevisiae “orphan” ORFs recently removed from the original ORFeome annotation due to lack of conservation across evolutionarily related yeast species. We show that many orphan ORFs produce detectable transcripts and/or translated products in various functional genomics and proteomics experiments. By combining a naïve Bayes model that predicts the likelihood of an ORF to encode a functional product with experimental verification of strand-specific transcripts, we argue that orphan ORFs should still remain candidates for functional ORFs.
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