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Funktionelle Charaktersierung Der Arginin Methyltransferase PRMT6

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Funktionelle Charaktersierung Der Arginin Methyltransferase PRMT6 Funktionelle Charaktersierung der Arginin Methyltransferase PRMT6 Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Chemie der Philipps-Universität Marburg vorgelegt von Dawin Michael Hyllus aus Frankenthal/Pfalz Marburg/Lahn, 2009 Vom Fachbereich Chemie der Philipps-Universität Marburg als Dissertation am 27.März 2009 angenommen. Erstgutachter: Prof. Dr. Lars-Oliver Essen Zweitgutachter: Prof. Dr. Uta-Maria Bauer Tag der mündlichen Prüfung am 01. April 2009 Inhaltsverzeichnis Inhaltsverzeichnis 1 Einleitung ............................................................................... 1 1.1 Protein Arginin Methyltransferasen (PRMT) ..................................................... 1 1.1.1 Posttranslationale Modifikation von Proteinen ......................................................................... 1 1.1.2 Struktur der Protein Arginin Methyltransferasen ...................................................................... 2 1.1.3 Mechanismus der Arginin Methylierung .................................................................................. 5 1.1.4 Substrate und Funktionen der Protein Arginin Methyltransferasen .......................................... 7 1.1.5 Protein Arginin Methyltransferase 6 (PRMT6) ........................................................................ 9 1.2 Der Histoncode ...................................................................................................... 11 1.2.1 Die Struktur des Chromatins ................................................................................................... 11 1.2.2 Posttranslationale Modifikationen an Histon N-Termini ........................................................ 12 1.2.3 Erkennung von Histonmodifikationen .................................................................................... 14 1.2.4 Lysin 4 Methylierung und MLL-Komplexe ........................................................................... 16 1.2.5 Lysin 27 Methylierung und Polycomb-Komplexe .................................................................. 19 1.3 Zielsetzung ............................................................................................................. 22 2 Material ................................................................................ 23 2.1 Zelllinien ................................................................................................................ 23 2.2 Bakterienstämme .................................................................................................. 23 2.3 Plasmide ................................................................................................................. 24 2.4 Peptide .................................................................................................................... 24 2.5 Antikörper ............................................................................................................. 25 2.6 Oligonukleotide ..................................................................................................... 26 2.6.1 qPCR-Primer .......................................................................................................................... 26 2.6.2 Chromatin-IP Primer .............................................................................................................. 27 2.6.3 siRNA ..................................................................................................................................... 27 2.7 Radioaktive Substanzen ....................................................................................... 27 2.8 Substrate für Methyltransferase-Reaktionen .................................................... 28 2.9 Standards ............................................................................................................... 28 3 Methoden .............................................................................. 29 3.1 Zellkultur Methoden ............................................................................................. 29 3.1.1 Kultivierung von eukaryotischen Zellen ................................................................................. 29 3.1.2 Transfektion von Plasmid-DNA mit Calciumphosphat .......................................................... 29 3.1.3 Transfektion von Plasmid-DNA mit Polyethylenimin ............................................................ 30 3.1.4 Transfektion von siRNA mit Dharmafect ............................................................................... 31 Inhaltsverzeichnis 3.1.5 Induktion von HT29-Zellen mit Zinkchlorid .......................................................................... 32 3.1.6 Differenzierung von NT2/D1 Zellen ....................................................................................... 32 3.2 Molekularbiologische Methoden .......................................................................... 33 3.2.1 Herstellung kompetenter Bakterien ......................................................................................... 33 3.2.2 Bestimmung der Nukleinsäurekonzentration .......................................................................... 33 3.2.3 Transformation kompetenter Bakterien .................................................................................. 34 3.2.4 Agarose-Gelelektrophorese ..................................................................................................... 34 3.2.5 Präparative Isolierung von Plasmid-DNA aus E. coli ............................................................. 35 3.2.6 Präparation von Gesamt-RNA aus eukaryotischen Zellen ...................................................... 36 3.2.7 Reverse Transkriptase Reaktion .............................................................................................. 36 3.2.8 Quantitative Polymerasekettenreaktion (qPCR) ..................................................................... 37 3.3 Biochemische Methoden ....................................................................................... 40 3.3.1 Bestimmung der Proteinkonzentration (Bradford) .................................................................. 40 3.3.2 Konzentrierung von Proteinen ................................................................................................ 41 3.3.3 SDS-Polyacrylamid Gelelektrophorese (PAGE) .................................................................... 41 3.3.4 Färbung von Proteingelen mittels Coomassie ......................................................................... 42 3.3.5 Färbung von Proteingelen mittels colloidalem Coomassie ..................................................... 42 3.3.6 Silberfärbung von Proteingelen............................................................................................... 43 3.3.7 Proteintransfer / Western Blot (nass) ...................................................................................... 44 3.3.8 Proteintransfer / Western Blot (halbtrocken) .......................................................................... 45 3.3.9 Ponceau-Färbung von Membranen ......................................................................................... 45 3.3.10 Immundetektion ...................................................................................................................... 46 3.3.11 Entfernen von Antikörpern von einer Blotmembran ............................................................... 47 3.3.12 Präparation von GST-Fusionsproteinen aus Bakterien ........................................................... 48 3.3.13 Präparation von His-Fusionsproteinen aus Bakterien ............................................................. 49 3.3.14 Präparation von unlöslichen His-Fusionsproteinen aus Bakterien .......................................... 50 3.3.15 Herstellung von Gesamtzellextrakt aus eukaryotischen Zellen (IPH-Extrakt) ........................ 51 3.3.16 Herstellung von Gesamtzellextrakt aus eukaryotischen Zellen (IPH-Extrakt) ........................... mit Benzonase-Behandlung .................................................................................................... 52 3.3.17 Herstellung von cytoplasmatischem Extrakt / Kernextrakt aus eukaryotischen Zellen .......... 53 3.3.18 Aufreinigung von Strep-Tag Fusionsproteinen aus Gesamtzellextrakt ................................... 54 3.3.19 Immunpräzipitation (IP) .......................................................................................................... 55 3.3.20 in vitro Methyltransferase-Reaktion ....................................................................................... 56 3.3.21 Fluorographie .......................................................................................................................... 57 3.3.22 Szintillationsmessung von radioaktiv markierten Peptiden .................................................... 57 3.3.23 Szintillationsmessung von Edman-Sequenzanalysefraktionen ............................................... 58 3.3.24 Flüssig-Methyltransferase Test ............................................................................................... 58 3.3.25 Kopplung von Peptiden an Sulfolink-Gel ............................................................................... 59 3.3.26 Peptid-Pulldown ...................................................................................................................... 60 3.3.27 Chromatin-Immunpräzipitation (ChIP) ..................................................................................
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