Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Mamatha Sauermann, M.Sc. in Biotechnology Born in Srikakulam, India Date of oral-examination: 27th September, 2006 Development and application of a high throughput cell based assay to identify apoptosis inducing proteins, and functional characterization of the candidate Vacuole Membrane Protein 1 (Vmp1) Referees: PD. Dr. Stefan Wiemann PD. Dr. Frank Breitling To my husband and parents Contents SUMMARY ..................................................................................................................... 1 ZUSAMMENFASSUNG ................................................................................................ 2 1 INTRODUCTION ................................................................................................... 3 1.1 Types of cell death........................................................................................................ 4 1.1.1 Necrosis.................................................................................................................................. 5 1.1.2 Apoptosis ............................................................................................................................... 5 1.2 Organelle specific initiation of apoptosis pathways .................................................. 6 1.2.1 Death Receptor pathway from the plasma membrane (Extrinsic pathway)........................... 7 1.2.2 Mitochondrial pathway of apoptosis (Intrinsic pathway) ...................................................... 8 1.2.3 The nuclear death pathway .................................................................................................... 9 1.2.4 Endoplasmic reticulum (ER) mediated apoptosis................................................................ 10 1.2.4.1 Unfolded protein response and apoptosis................................................................................... 10 1.2.4.2 Role of Ca2+ in ER stress-induced apoptosis.............................................................................. 11 1.2.5 Golgi apparatus and apoptosis ............................................................................................. 12 1.2.6 Lysosome-mediated apoptosis ............................................................................................. 12 1.2.7 Role of cytoskeleton and cell adhesion in apoptosis............................................................ 13 1.2.8 Integration of different apoptosis pathways......................................................................... 14 1.3 Apoptosis dysregulation and its clinical implications ............................................. 14 1.3.1 Apoptosis and cancer ........................................................................................................... 15 1.3.2 Apoptosis and autoimmunity ............................................................................................... 15 1.3.3 Apoptosis and AIDS ............................................................................................................ 16 1.3.4 Apoptosis and Neurodegeneration....................................................................................... 16 1.4 Detection of apoptosis ................................................................................................ 16 1.4.1 Analysis of cell morphology................................................................................................ 17 1.4.2 Plasma membrane changes .................................................................................................. 17 1.4.3 Mitochondrial changes......................................................................................................... 18 1.4.4 DNA and nuclear changes.................................................................................................... 18 1.4.5 Biochemical changes ........................................................................................................... 20 1.5 cDNA resources for identification of novel apoptosis activators ........................... 21 1.5.1 Selection of novel full-length cDNAs for screening............................................................ 21 1.6 Aim of the project....................................................................................................... 22 2 MATERIALS AND METHODS.......................................................................... 23 2.1 Materials ..................................................................................................................... 23 2.1.1 Equipment............................................................................................................................ 23 2.1.2 Chemicals............................................................................................................................. 24 2.1.3 Kits....................................................................................................................................... 26 2.1.4 Plastic and glassware ...........................................................................................................26 2.1.5 Oligonucleotides .................................................................................................................. 27 2.1.6 siRNAs................................................................................................................................. 28 2.1.7 Peptides................................................................................................................................ 29 2.1.8 Antibiotics............................................................................................................................ 29 2.1.9 Restriction enzymes and buffers.......................................................................................... 29 Contents 2.1.10 Bacterial Strains...............................................................................................................30 2.1.11 Cell Lines......................................................................................................................... 30 2.1.12 Antibodies........................................................................................................................ 31 2.1.13 Plasmids........................................................................................................................... 32 2.2 Methods....................................................................................................................... 35 2.2.1 Molecular Biology methods................................................................................................. 35 2.2.1.1 Polymerase Chain Reaction (PCR)............................................................................................. 35 2.2.1.2 Purification of PCR products ...................................................................................................... 38 2.2.1.3 Ligation........................................................................................................................................ 39 2.2.1.4 Gateway Reactions...................................................................................................................... 40 2.2.1.5 Transformation of bacteria.......................................................................................................... 41 2.2.1.6 Small – scale preparation of plasmid DNA (mini prep)............................................................. 42 2.2.1.7 Large – scale preparation of plasmid DNA (maxi prep)............................................................ 43 2.2.1.8 Measurement of DNA concentration.......................................................................................... 44 2.2.1.9 Restriction digest......................................................................................................................... 44 2.2.1.10 Gel electrophoresis...................................................................................................................... 45 2.2.1.11 Extraction of total RNA from cells............................................................................................. 45 2.2.1.12 Quantification of the RNA using Ribogreen .............................................................................. 46 2.2.1.13 Reverse transcription................................................................................................................... 46 2.2.1.14 Quantitative Real - time PCR (TaqMan).................................................................................... 47 2.2.2 Cell biology methods ........................................................................................................... 48 2.2.2.1 Subculturing of cells.................................................................................................................... 48 2.2.2.2 Freezing and thawing the cells.................................................................................................... 49 2.2.2.3 Counting of cells ......................................................................................................................... 49 2.2.2.4 Transfection with plasmid DNA and siRNA.............................................................................. 50 2.2.2.5 Generation of Stable cell