Towards the architecture of the human inner kinetochore [Elektronische Ressource] / von Sandra Orthaus

Towards the architecture of the human inner kinetochore Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät der Friedrich-Schiller-Universität Jena von Diplom-Biologin Sandra Orthaus geboren am 02. Juli 1974 in Jena ITable of contents 1 1 Introduction 1.1. The centromere kinetochore complex 1 1.1.1. The centromere 1 1.1.2. Inner kinetochore proteins 2 1.1.2.1. CENP-A 3 1.1.2.2. CENP-B 4 1.1.2.3. CENP-C 5 1.1.2.4. CENP-H 5 1.1.2.5. CENP-I 6 1.1.3. Outer kinetochore proteins 6 1.2. The nucleosome 7 1.3. Objective 10 12 2 Materials and Methods 2.1. Materials 12 2.1.1. Chemicals 12 2.1.2. Standarts and Kits 12 2.2. Methods 12 2.2.1. Cell culture and transfection into HEp-2 cells 13 2.2.2. Analysis of the CENP-H genotype of HEp-2 cells 13 2.2.3. RNA interference 14 2.2.4. Cell viability assays 15 2.2.5. Cell cycle analysis and cell synchronisation 15 2.2.6. Antibodies and immuno-fluorescence 16 2.2.7. Confocal microscopy 16 2.2.8. Western Blots 17 2.2.9. Plasmids and cloning 17 2.2.10. Förster resonance energy transfer (FRET) 20 2.2.10.1. Acceptor Photobleaching based FRET measurements 24 2.2.10.2. FLIM (Fluorescence Lifetime Measurements) 25 29 3 Results 3.1. RNAi knock down of the human kinetochore protein CENP-H 29 3.1.1. The siRNA led to depletion of CENP-H in human HEp-2 cells 30 3.1.2.