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Dokument 1.Pdf N ENTDECKUNG UND CHARAKTERISIERUNG E I R E EINER NEUEN GENFAMILIE, SBFDCP7, BEI T K A VERTEBRATEN UND BAKTERIEN B D N U N E T A R B JOSÉ RODRIGO GODOY BERTHET E T R E V I E B 7 P C D F B S N O V G N U K C E D T N E T E H INAUGURAL-DISSERTATION T R zur Erlangung des Grades eines E B Dr. med. vet. Y beim Fachbereich Veterinärmedizin édition scientifique O VVB LAUFERSWEILER VERLAG D der Justus-Liebig-Universität Gießen O VVB LAUFERSWEILER VERLAG ISBN 3-8359-5122-X G STAUFENBERGRING 15 . R D-35396 GIESSEN . J Tel: 0641-5599888 Fax: -5599890 [email protected] www.doktorverlag.de 9 7 8 3 8 3 5 9 5 1 2 2 8 édition scientifique VVB VVB LAUFERSWEILER VERLAG Das Werk ist in allen seinen Teilen urheberrechtlich geschützt. Jede Verwertung ist ohne schriftliche Zustimmung des Autors oder des Verlages unzulässig. Das gilt insbesondere für Vervielfältigungen, Übersetzungen, Mikroverfilmungen und die Einspeicherung in und Verarbeitung durch elektronische Systeme. 1. Auflage 2007 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the Author or the Publishers. 1st Edition 2007 © 2007 by VVB LAUFERSWEILER VERLAG, Giessen Printed in Germany VVB LAUFERSWEILER VERLAG édition scientifique STAUFENBERGRING 15, D-35396 GIESSEN Tel: 0641-5599888 Fax: 0641-5599890 email: [email protected] www.doktorverlag.de Aus dem Institut für Pharmakologie und Toxikologie, Fachbereich Veterinärmedizin der Justus-Liebig-Universität Gießen Betreuer: Prof. Dr. Ernst Petzinger Entdeckung und Charakterisierung einer neuen Genfamilie, SBFDCP7, bei Vertebraten und Bakterien INAUGURAL-DISSERTATION zur Erlangung des Grades eines Dr. med. vet. beim Fachbereich Veterinärmedizin der Justus-Liebig-Universität Gießen eingereicht von José Rodrigo Godoy Berthet Tierarzt aus Traiguén, Chile Gießen 2006 Mit Genehmigung des Fachbereiches Veterinärmedizin der Justus-Liebig-Universität Gießen Dekan: Prof. Dr. M. Reinacher Gutachter: Prof. Dr. Ernst Petzinger Prof. Dr. Till Rümenapf Tag der Disputation: 09. Januar 2007 „Bajo el cielo nacido tras la lluvia escucho un leve deslizarse de remos en el agua...” Jorge Teillier Marcela und unserem Sohn Vicente Jesús in Liebe und Bewunderung gewidmet INHALTSVERZEICHNIS Inhaltsverzeichnis Verzeichnis der Abbildungen ___________________________________________________ iv Verzeichnis der Tabellen _______________________________________________________v Abkürzungen________________________________________________________________ vi 1. Einleitung __________________________________________________________________ 1 2. Literaturübersicht ____________________________________________________________ 3 2.1 Grundlagen des Membrantransportes _________________________________________ 3 2.2 Funktionelle Unterschiede von Membrantransportsystemen ________________________ 3 2.3 Die Superfamilie der Solute Carrier (SLC) ______________________________________ 5 2.4 Die Familie der Sodium Bile Acid Symporter (SBF) _______________________________ 5 2.5 Die Solute Carrier-Familie 10 (SLC10) _________________________________________ 7 2.5.1 Der natriumabhängige Gallensäuretransporter der Leber NTCP__________________ 7 2.5.2 Der natriumabhängige Gallensäuretransporter des Dünndarms ASBT _____________ 8 2.5.3 Der natriumabhängige Steroidsulfattransporter SOAT _________________________ 8 2.6 Die Familie der ACR3-Carrier ________________________________________________ 9 2.7 Zielsetzung der Arbeit _____________________________________________________ 11 3. Material ___________________________________________________________________ 13 3.1 Verwendete Primer _______________________________________________________ 13 3.2 Klonierung, cRNA-Synthese und Insertion des FLAG-Motivs_______________________ 15 3.2.1 Bakterienstämme _____________________________________________________ 15 3.2.2 Vektoren____________________________________________________________ 15 3.2.3 Kulturmedien ________________________________________________________ 18 3.2.4 Agarose-Gelelektrophorese _____________________________________________ 18 3.2.5 Enzyme ____________________________________________________________ 19 3.2.6 Kommerziell erhältliche Kits und Material __________________________________ 20 3.2.7 cDNA-Panels und RNA ________________________________________________ 20 3.3 Expression in Xenopus laevis-Oozyten________________________________________ 20 3.3.1 Versuchstiere ________________________________________________________ 20 3.3.2 Verwendete Lösungen und Puffer für die Oozyten ___________________________ 20 3.3.3 Radioaktiv markierte Substanzen ________________________________________ 21 3.3.4 Materialien __________________________________________________________ 21 3.4 Immunfluoreszenz in Xenopus laevis-Oozyten__________________________________ 21 3.4.1 Verwendete Lösungen und Puffer ________________________________________ 21 3.4.2 Antikörper___________________________________________________________ 22 3.5 Agarose/Formaldehyd-Gelelektrophorese und Northern Blot_______________________ 22 3.5.1 Verwendete Lösungen und Puffer ________________________________________ 22 3.5.2 Gelelektrophorese ____________________________________________________ 23 3.5.3 Blotten _____________________________________________________________ 23 3.6 Zellkultur _______________________________________________________________ 24 3.6.1 Eukaryontische Zelllinie ________________________________________________ 24 3.6.2 Zellkulturbedarf ______________________________________________________ 24 3.6.3 Zellkulturmedien und Zusätze ___________________________________________ 25 3.6.4 Transiente Transfektion in HEK293-Zellen _________________________________ 25 3.7 Immunfluoreszenz________________________________________________________ 25 3.7.1 Primäre und sekundäre Antikörper _______________________________________ 25 i INHALTSVERZEICHNIS 3.7.2 Reagenzien und Puffer ________________________________________________ 26 3.7.3 Verwendete Lösungen und Puffer ________________________________________ 26 3.8 Reagenzien_____________________________________________________________ 27 3.9 Geräte _________________________________________________________________ 27 3.10 Bioinformatik ___________________________________________________________ 28 4. Methoden _________________________________________________________________ 30 4.1 Allgemeine molekularbiologische Methoden____________________________________ 30 4.2 Total-RNA Isolierung aus tierischem Gewebe __________________________________ 31 4.3 RNA-Gele zur Qualitätskontrolle_____________________________________________ 32 4.4 Gewinnung von polyA+-RNA aus Total-RNA ___________________________________ 32 4.5 cDNA-Synthese _________________________________________________________ 33 4.6 Polymerase-Kettenreaktion (PCR) ___________________________________________ 34 4.6.1 Klonierungs-PCR _____________________________________________________ 34 4.6.2 Mutagenese-PCR mit DpnI-Restriktion ____________________________________ 35 4.7 Klonierung______________________________________________________________ 38 4.8 Heterologe Expression in Xenopus laevis-Oozyten ______________________________ 41 4.8.1 Linearisierung der Plasmid-DNA und Abschneiden überhängender 3’-Enden ______ 41 4.8.2 cRNA-Synthese ______________________________________________________ 42 4.8.3 Froschoperation zur Entnahme der Oozyten ________________________________ 43 4.8.4 Aufbereitung der Oozyten ______________________________________________ 43 4.8.5 Mikroinjektion der Oozyten______________________________________________ 44 4.8.6 Transportmessung an Oozyten __________________________________________ 44 4.9 Nachweis der Expression mittels FLAG-Tag und Immunfluoreszenz an Oozyten _______ 45 4.9.1 Präparation und Permeabilisierung der Oozyten, primäre Antikörperreaktion_______ 45 4.9.2 Sekundäre Antikörperreaktion ___________________________________________ 45 4.9.3 Fixierung und Einbettung der Oozyten_____________________________________ 46 4.9.4 Einbettung der Oozyten und Schneiden der Präparate ________________________ 46 4.10 Northern Blot___________________________________________________________ 47 4.10.1 Agarose/Formaldehyd-Gelelektrophorese _________________________________ 47 4.10.2 Übertragung der RNAs auf eine Nylonmembran (Transfering) _________________ 47 4.10.3 Erstellung radioaktiv markierter DNA-Sonden ______________________________ 48 4.10.4 Hybridisierung ______________________________________________________ 50 4.11 Subklonierung in den Vektor pcDNA9________________________________________ 50 4.12 Transiente Transfektion in HEK293-Zellen ____________________________________ 51 4.13 Indirekte Immunfluoreszenz in HEK293-Zellen_________________________________ 51 5. Ergebnisse ________________________________________________________________ 53 5.1 Klonierung des P7 von Mensch, Ratte, Maus und Frosch _________________________ 53 5.1.1 Die in silico Identifizierung der P7-Proteine _________________________________ 53 5.1.2 Klonierung der Leserahmen und genomische Organisation der P7-Gene _________ 55 5.1.3 Alternatives Spleißen der P7-Transkripte __________________________________ 57 5.1.4 Im Genom von Xenopus laevis existieren zwei P7-Gene ______________________ 59 5.1.5 Proteinsequenz und SBF-Domäne-Architektur der P7-Proteine _________________ 60 5.1.6 Membrantopologie der P7-Isoformen______________________________________
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