Aus Dem Institut Für Humangenetik Der Medizinischen Fakultät Charité – Universitätsmedizin Berlin

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Aus Dem Institut Für Humangenetik Der Medizinischen Fakultät Charité – Universitätsmedizin Berlin Aus dem Institut für Humangenetik der Medizinischen Fakultät Charité – Universitätsmedizin Berlin DISSERTATION Zelluläre Charakterisierung eines neuen Chromosomeninstabilitätssyndroms und Ausschluss von Kandidatengenen zur Erlangung des akademischen Grades Doctor medicinae (Dr. med.) vorgelegt der Medizinischen Fakultät Charité – Universitätsmedizin Berlin von Martin Markovski aus Berlin Gutachter: 1. Prof. Dr. M. Digweed 2. Prof. Dr. M. Stuhrmann-Spangenberg 3. Prof. Dr. F. Eckardt-Schupp Datum der Promotion: 26.11.2007 Für Ebru, Kian und Kiara Abkürzungsverzeichnis Abkürzungsverzeichnis A Adenin AS Aminosäure AT Ataxia telangiectasia ATM Ataxia telangiectasia, „mutated” BRCT „breast-cancer-carboxy-terminal” Domäne BLAST „Basic Local Alignment Search Tool” bp Basenpaare BSA Bovines Serumalbumin C Cytosin ca. circa CDK2 „Cyclin-dependent kinase 2” CDK4 „Cyclin-dependent kinase 4” cDNA „complementary Desoxyribonucleic acid” CT C-terminales Ende CHK2 Checkpoint Kinase 2 cM centi-Morgen DDIT3 „DNA-damage-inducible transcript 3” ddNTP 2´, 3´-Didesoxy-ribonukleosid-5`-triphosphat DDX23 „DEAD (Asp-Glu-Ala-Asp) box polypeptide 23” DNA „Desoxyribonucleic acid” (Desoxyribonukleinsäure) dNTP Desoxyribonukleosidtriphosphat DSB Doppelstrangbruch EBV Epstein-Barr-Virus EDTA Ethylendiamintetraessigsäure EST „Expressed sequence tag“ F „Forward primer“ (Vorwärts Primer) FA Fanconi-Anämie FAM 5'-Carboxy-Fluorescin FHA „fork-head-associated” Domäne FKS fetales Kälberserum G Guanin Gy Gray - Maßeinheit der Energiedosis HR homologe Rekombination HRP „Horse Radish Peroxidase” kb Kilobasen I Abkürzungsverzeichnis kDa Kilodalton KUB3 Ku70 binding protein LCL „Lymphoblastoide Cell Line” (Lymphblastoide Zelllinie) LOD „Logarithm of odds” M molar min. Minute mM millimolar NBS Nijmegen-Breakage-Syndrom NCBI „National Center of Biotechnology Information” NHEJ „Non homologous end-joining” NT N-terminales Ende OMIM „Online Mendelian Inheritance in Man (database)” ORF „Open reading frame” (Offener Leserahmen) PAA Polyacrylamid PA2G4 „Proliferation-associated 2G4” PCR „Polymerase chain reaction” (Polymerasekettenreaktion) PPM1H „Protein phosphatase 1H” R „Reverse primer” (Rückwärts Primer) RBMS2 „RNA binding motif, single stranded interacting protein 2” rpm „rotation per minute” (Umdrehungen pro Minute) RNA „Ribonucleic acid” (Ribonukleinsäure) RT Reverse Transkription SAP „Shrimp Alkaline Phosphatase” SDS Sodiumdodecylsulfat sek. Sekunden Ser Serin SMUG1 „Single-strand selective monofuncional uracil DNA glycosylase” SNP „Single nucleotid polymorphism” SSA „Single strand annealing” T Thymin Taq Thermus aquaticus U Unit U/min Umdrehungen pro Minute V Volt II Inhaltsverzeichnis Inhaltsverzeichnis 1 Einleitung________________________________________________________ 1 1.1 Chromosomeninstabilitätssyndrome____________________________________ 1 1.2 Nijmegen-Breakage-Syndrom (NBS) ____________________________________ 1 1.2.1 Klinisches Bild _________________________________________________________ 2 1.2.2 Molekulare Grundlagen __________________________________________________ 3 1.3 DNA-Reparatursysteme _______________________________________________ 6 1.3.1 Non homologous end joining (NHEJ)_______________________________________ 7 1.3.2 Homologe Rekombination (HR) / Single Strand Annealing (SSA) ________________ 8 1.4 Ein weiterer Gendefekt, der zum NBS-Phänotyp führt _____________________ 10 1.5 Zielsetzung ________________________________________________________ 11 1.5.1 Chromosomenbruchanalyse _____________________________________________ 11 1.5.2 Proteinbiochemische Analyse ___________________________________________ 11 1.5.3 Familienkopplungsanalyse ______________________________________________ 12 2 Materialien und Methoden _________________________________________ 13 2.1 Materialien_________________________________________________________ 13 2.1.1 Zelllinien _____________________________________________________________ 13 2.1.2 Zellkulturmedien_______________________________________________________ 13 2.1.3 Patienten-DNA ________________________________________________________ 13 2.1.4 Antikörper ____________________________________________________________ 14 2.1.4.1 Primäre Antikörper _________________________________________________ 14 2.1.4.2 Sekundäre Antikörper_______________________________________________ 14 2.1.5 Enzyme ______________________________________________________________ 14 2.1.6 Kits__________________________________________________________________ 15 2.1.7 Primer _______________________________________________________________ 15 2.1.7.1 Primer für Mikrosatelliten ____________________________________________ 15 2.1.7.2 Primer für cDNA-PCR _______________________________________________ 16 2.1.8 Lösungen und Puffer ___________________________________________________ 17 2.1.9 Chemikalien und Reagenzien ____________________________________________ 18 2.1.10 Verbrauchsmaterialien_________________________________________________ 19 2.1.11 Programme und Datenbanken___________________________________________ 19 2.1.12 Geräte und Zubehör ___________________________________________________ 20 2.2 Methoden _________________________________________________________ 21 2.2.1 Zellkultur _____________________________________________________________ 21 2.2.1.1 Allgemeine Bedingungen ____________________________________________ 21 2.2.1.2 Zellkultivierung ____________________________________________________ 21 III Inhaltsverzeichnis 2.2.1.3 Zellzahlbestimmung ________________________________________________ 21 2.2.2 Zellbiologische Methoden _______________________________________________ 21 2.2.2.1 Induzierte Chromosomenschädigung__________________________________ 21 2.2.2.2 Chromosomenaufarbeitung __________________________________________ 22 2.2.2.3 Chromosomenauswertung___________________________________________ 23 2.2.3 Proteinbiochemische Methoden __________________________________________ 23 2.2.3.1 Zellbestrahlung ____________________________________________________ 23 2.2.3.2 Proteinextraktion___________________________________________________ 24 2.2.3.3 Phosphatase-Behandlung ___________________________________________ 24 2.2.3.4 Immunpräzipitation _________________________________________________ 25 2.2.3.5 Western Blot ______________________________________________________ 26 2.2.3.6 Immundetektion____________________________________________________ 27 2.2.3.7 „Strippen" von Membranen __________________________________________ 28 2.2.4 Molekulargenetische Methoden __________________________________________ 29 2.2.4.1 Globale DNA-Amplifizierung _________________________________________ 29 2.2.4.2 Isolierung von RNA_________________________________________________ 29 2.2.4.3 Reverse Transkription ______________________________________________ 30 2.2.4.4 Polymerase-Kettenreaktion (PCR)_____________________________________ 31 2.2.4.5 Agarose-Gelelektrophorese __________________________________________ 33 2.2.4.6 PCR-Aufreinigung __________________________________________________ 33 2.2.4.7 Gel Extraktion _____________________________________________________ 34 2.2.4.8 Sequenzierung_____________________________________________________ 34 2.2.4.9 Fällung ___________________________________________________________ 35 3 Ergebnisse______________________________________________________ 36 3.1 Chromosomenbruchanalysen_________________________________________ 36 3.1.1 Auswertung der Chromosomenaberrationen des 1. Versuchsaufbaus __________ 37 3.1.2 Auswertung der Chromosomenaberrationen des 2.Versuchsaufbaus ___________ 42 3.1.3 Dizentrische Chromosomen _____________________________________________ 46 3.1.4 Endoreduplikationen ___________________________________________________ 47 3.2 Proteinanalysen ____________________________________________________ 48 3.2.1 Immunpräzipitation ____________________________________________________ 49 3.2.1.1 Nachweis von phospho-ATM (S1981) und ATM __________________________ 49 3.2.2 Western Blot __________________________________________________________ 50 3.2.2.1 Nachweis von Nibrin________________________________________________ 50 3.2.2.2 Nachweis von SMC1 und phospho-SMC1 (Ser957) _______________________ 51 3.2.2.3 Nachweis von p53 __________________________________________________ 52 3.2.2.4 Nachweis von phospo-p53 (Ser15) ____________________________________ 53 3.2.2.5 Nachweis von p21 __________________________________________________ 54 3.2.2.6 Nachweis von CHK2 ________________________________________________ 55 IV Inhaltsverzeichnis 3.2.2.7 Nachweis von FANCD2______________________________________________ 56 3.3 DNA-Analysen _____________________________________________________ 57 3.3.1 Homozygosity-Mapping _________________________________________________ 57 3.3.2 Feinkartierung_________________________________________________________ 59 3.3.3 Kandidatengenscreening________________________________________________ 60 3.3.4 RT-PCR-Kontrolle durch GAPDH-Amplifizierung ____________________________ 60 3.3.5 Auswertung der Kandidatengene _________________________________________ 61 3.3.5.1 DDX23 („DEAD (Asp-Glu-Ala-Asp) box polypeptide 23“) __________________ 62 3.3.5.2 SMUG1 („single-strand-selective monofunctional uracil-DNA glycosylase 1”) 63 3.3.5.3 CDK2 („cyclin-dependent kinase 2“)___________________________________ 63 3.3.5.4 PA2G4 („proliferation-associated 2G4“) ________________________________ 64 3.3.5.5 RBMS2 („RNA binding motif, single stranded interacting protein 2”) ________ 65 3.3.5.6 DDIT3
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