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Inaugural-Dissertation INAUGURAL-DISSERTATION zur Erlangung der Doktorwürde der Naturwissenschaftlich-Mathematischen Gesamtfakultät der Ruprecht-Karls-Universität Heidelberg vorgelegt von Bärbel Großmann aus Bad Kreuznach Tag der mündlichen Prüfung: Strukturelle und funktionale Analyse des genomischen DAZL1-Locus in Mammalia Gutachter: Prof. Dr. Werner Buselmaier Prof. Dr. Michael Wink Die vorliegende Arbeit wurde von Januar 1998 bis März 2002 unter Anleitung von Herrn PD Dr. rer. nat. P. H. Vogt in der Fachgruppe „Reproduktionsgenetik“ am Institut für Humangenetik der Universität Heidelberg angefertigt. Danksagung Herrn PD Dr. P.H. Vogt danke ich für die Überlassung des Themas und die fachliche Betreuung. Mein besonderer Dank gilt Herrn Prof. Dr. W. Buselmaier für die Übernahme des Erstgutachtens. Ebenso gilt mein besonderer Dank Herrn Prof. Dr. Michael Wink für die Übernahme der Funktion des Zweitgutachters. Herrn Dr. Hager und seinem Team danke ich für die Durchführung der cytogenetischen Studien. Bei Herrn Prof. Strowitzki von der Abteilung Gynäkologische Endokrinologie und Reproduktionsmedizin der Universität Heidelberg bedanke ich mich für die Kooperation bei der DAZL1-Mutataionsanalyse bei POF-Patientinnen und bei Herrn Prof. Fryns aus der Abteilung Humangenetik des Fachbereichs Humanbiologie der Universität Leuven für die Bereitstellung von Patientenmaterial. Ganz besonders möchte ich mich bei den Mitarbeitern der „Arbeitsgruppe Vogt“ bedanken, die mir fachlich zur Seite gestanden haben und für eine nette und angenehme Atmosphäre gesorgt haben. Den Mitarbeitern der anderen Arbeitsgruppen des Instituts für Humangenetik danke ich für ihre Hilfsbereitschaft und die gute Zusammenarbeit. Bei Mostafa Kabiri und meinem Sohn Manusch bedanke ich mich für die Geduld, die sie aufbringen mussten. Zuletzt möchte ich mich bei meiner Familie bedanken, die mich immer bedingungslos unterstützt hat. In jeder Antwort kann man eine neue Frage finden. Jiddische Spruchweisheit Inhaltsverzeichnis _______________________________________________________________ Inhalt Seite 1. Einleitung 1 1.1 Der Gametogeneselocus DAZL1 1 1.1.1 DAZL1-Homologe beim Menschen 2 1.1.1.1 Die DAZ-Genfamilie auf dem humanen Y-Chromosom 2 1.1.1.2 Das humane BOULE-Gen auf Chromosom 2 4 1.1.2 Das Dazl1-Gen der Maus 5 1.1.3 DAZL1-Homologe in Xenopus und Zebrafisch 6 1.1.4 Evolution der DAZL1-Genfamilie 7 1.2 Keimbahnspezifische Genregulation 9 1.3 Genomische Transpositionsmechanismen in Primaten: die THE- Sequenzfamilie 11 1.4 DAZL1 und POF-Syndrom 12 1.5 Zielsetzung 14 2. Material 15 2.1 Untersuchungsmaterial 15 2.1.1 Patientenmaterial 15 2.1.2 Genomische DNA-Klone 15 2.1.2.1 Cosmid-Library von Callithrix jacchus 15 2.1.2.2 Cosmid-Library vom Gibbon (Hylobates klossi) 16 2.1.2.3 BAC-Klone vom Rind (Bos taurus) 16 2.1.2.4 BAC-Klone von der Maus (Mus musculus) 16 2.1.2.5 PAC-Klone von der Maus (Mus musculus) 17 2.1.2.6 Humane PAC-Klone 17 2.1.2.7 Humane BAC-Klone 18 2.2 Bakterienstämme 18 2.3 Vektoren 19 2.3.1 Plasmide 19 2.3.1.1 pBluescript SK (+) von Stratagene: 19 2.3.1.2 pCR®2.1-TOPO (Invitrogen, TOPOTM TA Cloning) 20 2.3.2 Cosmid-Vektoren 20 2.3.2.1 Lawrist 7 20 I Inhaltsverzeichnis _______________________________________________________________ 2.3.3 BAC-Vektor 21 2.3.4 PAC-Vektoren 21 2.3.4.1 PAC-Vektor pCYPAC2 21 2.3.4.2 PAC-Vektor pPAC4 22 2.4 Enzyme 22 2.4.1 Restriktionsendonukleasen 22 2.4.2 Sonstige Enzyme 22 2.5 Nukleinsäuren 23 2.5.1 Längenmarker für die Agarosegelelektrophprese 23 2.5.2 Nukleotide 24 2.5.3 Oligonukleotide 24 2.5.4 Sonstige Nukleinsäuren 26 2.6 Antibiotika 26 2.7 Antikörper, Farbstoffe und Antifading 26 2.8 Software und Datenbanken zur „in silico“-Sequenzanalyse 26 2.9 Filmmaterial 27 2.9.1 Fotographie 27 2.9.2 Autoradiographie 27 2.10 Kits 27 2.11 Geräte 28 2.12 Nährmedien und Zusätze 28 2.12.1 Medien für die Bakterienkultivierung 28 2.13 Puffer und Lösungen 29 2.13.1 Lösungen für die Agarose-Gelelektrophosese 29 2.13.2 Puffer und Lösungen für Southern-Blots 29 2.13.3 Puffer und Lösungen für RNA-Gele 30 2.13.4 Puffer und Lösungen für die radioaktive Hybridisierung 30 2.13.5 Puffer und Lösungen für die nicht radioaktive Hybridisierung mit Digoxigenin 31 2.13.6 Puffer und Lösungen für Sequenziergele 31 2.13.7 Puffer und Lösungen für Plasmidpräparationen 31 2.13.8 Puffer und Lösungen für die Präparation genomischer DNA aus Blut 32 2.13.9 Puffer und Lösungen für FISH 32 2.13.10 Puffer und Lösungen für die TMHA 33 II Inhaltsverzeichnis _______________________________________________________________ 3. Methoden 3.1 Mikrobiologische Techniken 34 3.1.1 Kultivierung von Bakterien auf Agarplatten 34 3.1.2 Kultivierung von Bakterien in Flüssigmedien 34 3.1.2.1 Photometrische Bestimmung der Bakteriendichte 34 3.1.3 Langzeitlagerung von Bakterien in Form von Glycerinstocks 34 3.2 Nukleinsäurepräparationen 35 3.2.1 DNA-Isolierung aus Blut 35 3.2.2 Isolierung von RNA aus Hodengewebe 35 3.2.3 Isolierung von RNA aus Blut 36 3.2.4 Isolierung von Plasmid-DNA 36 3.2.4.1 Plasmid-DNA-Aufreinigung über Silikagel-Säulen 37 3.2.4.1.1 Mini-Präparation 37 3.2.4.1.2 Maxi-Präparation 38 3.2.4.1.3 Präparation von BAC- und PAC-DNA 38 3.2.4.1.4 Alternatives Protokoll zur Isolierung von PAC-DNA 40 3.3 Agarose-Gelelektrophorese 41 3.3.1 Fragmentlängenbestimmung 42 3.3.2 Formaldehyd-Agarosegele für die RNA-Analyse 43 3.4 PCR (Polymerase-Kettenreaktion) 44 3.4.1 Berechnung der „Annealing-Temperatur“ 45 3.4.2 Standard-PCR-Ansatz: und Programm 45 3.5 Sequenzierung von DNA mit dem A.L.F. Express DNA Sequenzierer 46 3.6 Southern-Blot 48 3.6.1 Kapillar-DNA-Blot nach Southern 48 3.6.2 Bidirektionaler Blot (Sandwich-Blot) 50 3.6.3 Vakuum-Blot 50 3.7 Hybridisierungsverfahren 51 3.7.1 Radioaktive Markierung einer Sonde mit _-32-P-dCTP 51 3.7.1.1 Random-Priming 51 3.7.1.2 Markierung durch PCR 52 3.7.1.3 Aufreinigung radioaktiv markierter DNA-Sonden 52 3.7.1.4 Denaturierung der Sonde 53 3.7.1.5 Hybridisierung von Nukleinsäure-Blots 53 III Inhaltsverzeichnis _______________________________________________________________ 3.7.2 Nicht-radioaktive Hybridisierung mit dem Digoxigenin-System 53 3.7.2.1 Markierung der Sonde 53 3.7.2.1.1 DIG-Labelling durch Random Priming 54 3.7.2.1.2 DIG-Labelling mit Hilfe von PCR 55 3.7.2.2 DNA-Hybridisierung mit Digoxigenin-markierten DNA-Sonden 56 3.7.2.2.1 Hybridisierung in Hybridisierungslösung 56 3.7.2.2.2. Hybridisierung in DIG Easy Hyb 56 3.7.2.3 Stringenzwaschen 57 3.7.2.4 Detektion 57 3.7.3 Fluoreszenz-in-situ-Hybridisierung (FISH) auf Metaphase- Chromosomen 58 3.7.3.1 Herstellung von Chromosomenpräparaten aus Blut 59 3.7.3.2 Vorbehandlung der Chromosomenpräparate 60 3.7.3.3. Markierung der FISH-DNA-Sonden durch „Nick-Translation“ 61 3.7.3.4 Hybridisierung 62 3.7.3.5 Denaturierung der Chromosomenpräparate 62 3.7.3.6 Aufarbeitung der Proben 62 3.7.3.7 Detektion der FISH-Signale und Gegenfärbung der Chromosomen 63 3.7.3.8 Gegenfärbung der Chromosomen mit DAPI 64 3.7.3.9 Fluoreszenzmikroskopie und Bildbearbeitung 64 3.8 Mutationsscreening mit TMHA 65 3.8.1 Prinzip des WAVETM-Systems 66 3.8.2 Auswahl der Primer und PCR 67 3.8.3 Protokoll für die PCR 68 3.9 SNP-Analyse nach dem ARMS-Prinzip 69 3.9.1 Chi-Quadrat-Test 69 3.10 Screenen von auf Filter gespotteten Genbanken 70 3.11 Screening von BAC- bzw. PAC-Bibliotheken des RZPD mittels PCR 72 4. Ergebnisse 74 4.1 Strukturelle Analyse des DAZL1-Locus 74 4.1.1 Isolierung von genomischen DAZL1-Klonen 74 4.1.1.1 Humane DAZ- und DAZL1-Klone 74 4.1.1.1.1 Entstehung einer 2,2 kb-Insertion im DAZ-Locus von Primaten 83 4.1.1.1.2 Humane DAZL1 BAC-und PAC-Klone 85 IV Inhaltsverzeichnis _______________________________________________________________ 4.1.1.2 Genomische DAZL1-Klone von Mammalia 87 4.1.1.2.1 Hylobates klossi 87 4.1.1.2.2 Callithrix jacchus 87 4.1.1.2.3 Genomische Dazl1-Klone von der Maus 88 4.1.1.2.4 Genomische Dazl1-Klone vom Rind 89 4.2 Die genomische Struktur des DAZL1-Locus 89 4.2.1 Konservierte Elemente im genomischen DAZL1-Locus 96 4.3 In silico-Analyse der 5´-Upstream-Region von DAZL1 auf potentielle Promotorfunktion 100 4.4 Die 3´- untranslatierte Region des humanen DAZL1-Gens 106 4.5 Molekulare Analyse der DAZL1-Transposition 109 4.5.1 Identifizierung der ersten DAZ-Genkopie 114 4.5.2 Vergleich der Exon-Intron-Mutationsraten zwischen DAZL1 und DAZ2 115 4.6 Fluoreszenz-in-situ-Hybridisierung (FISH) von CosCaja-DAZL1 auf Metaphase-Chromosomen von Callithrix jacchus 118 4.7 DAZL1-Mutationsanalyse 120 4.7.1 DAZL1-Deletionsanalyse mittels Fluoreszenz in situ Hybridisierung (FISH) 120 4.7.2 DAZL1-Mutationsanalyse in POF-Patientinnen auf RNA-Ebene 124 4.7.3 DAZL1-Mutationsanalyse an POF-Patienninen auf DNA- Sequenzebene 127 4.7.3.1 Mutationsanalyse von Exon 3 127 4.7.3.1.1 Primer und Bedingungen für die Mutationsanalyse von Exon 3 127 4.7.3.1.2 Ergebnisse der TMHA für Exon 3 130 4.7.2.1.3 Ergebnis der Exon 3- Sequenzanalyse 131 4.7.3.2 Mutationsanalyse von Exon 5 und 6 132 4.7.3.2.1 Primer und Bedingungen für die Mutationsanalyse von Exon 5 und 6 132 4.7.3.2.2 Ergebnisse der TMHA für Exon 5 und 6 134 4.7.3.2.3 Ergebnis der Sequenzanalyse von Exon 5 und 6 136 4.7.3.3 Mutationsanalyse von Exon 9 137 4.7.3.3.1 Primer und Bedingungen für die Mutationsanalyse von Exon 9 137 4.7.3.3.2 Ergebnisse der TMHA für Exon 9 139 4.7.3.3.3 Ergebnis der Exon 9-Sequenzanalyse 140 4.8 SNP-Analyse 141 4.8.1 SNP Nr.
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