CLONING AND FUNCTIONAL CHARACTERIZATION OF NOVEL GENES EXPRESSED PREFERENTIALLY IN THE HUMAN RETINA DISSERTATION ZUR ERLANGUNG DES NATURWISSENSCHAFTLICHEN DOKTORGRADES DER BAYERISCHEN JULIUS-MAXIMILIANS-UNIVERSITÄT WÜRZBURG VORGELEGT VON JELENA STOJIC AUS BELGRAD SERBIEN UND MONTENEGRO WÜRZBURG, 2005 Eingereicht am: 21.02.2005. Bei der Fakultät für Biologie Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Ulrich Scheer Gutachter: Prof. Dr. Bernhard Weber Gutachter: Prof. Dr. Roy Gross Tag des Promotionskolloquiums: 08.06.2005. Doktorurkunde ausgehändigt am: …………………. Erklärung gemäß §4, Absatz 3 der Promotionsordnung für die Fakultät für Biologie der Universität Würzburg: Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig durchgeführt und verfasst habe. Andere Quellen als die angegebenen Hilfsmittel und Quellen wurden nicht verwendet. Die Dissertation wurde weder in gleicher noch in ähnlicher Form in einem anderen Prüfungsverfahren vorgelegt. Es wurde zuvor kein anderer akademischer Grad erworben. Die vorliegende Arbeit wurde am Institut für Humangenetik der Universität Würzburg unter der Leitung von Prof. Bernhard H.F. Weber angefertigt. Würzburg, 2005 Jelena Stojic ACKNOWLEDGMENTS First of all, I would like to thank Prof. Dr. Bernhard Weber for giving me the opportunity to do my Ph.D. thesis in his group and for his excellent scientific guidance during my studies. I would also like to thank Prof. Dr. Roy Gross who accepted to be supervisor of this thesis as a member of the Faculty of Biology at the University of Würzburg. I gladly express my appreciation to Dr. Heidi Stöhr for her support at the beginning of my doctorate degree. In particular, I would like to thank to my lab colleagues, Heidi Schulz for non-selfishly sharing her knowledge and being there when necessary to answer all the questions I had, always with a smile, and for doing with me virtual Northern blot analysis, Christine Wiedemann for helping me with the quantitative real-time PCR analysis, Franziska Krämer for her enthusiasm and generous assistance, Vladimir Milenkovic for having a lot of patience to solve the computer problems over and beyond the call of duty, Andrea Rivera and Andrea Gehrig for having patience and kindness to teach me many technical approaches, Andrea Welcker for the proofreading and translation assistance. I would also like to express my thanks to Claudia, Susanne, Ines, Nicole, Birgit, Sandra, Peter, Andreas, Johanna and Jürgen for the many social and scientific events we have shared during the time I spent in the lab and for friendly atmosphere. And much thanks to everybody for being patient to overcome the language barriers at my shaky start and for teaching me German. The diversity of the group members, combined with all the new cultural experiences gained from living in Würzburg, have furnished me with many fond memories, and have certainly broadened my horizons. I am very grateful to Prof. Dr. med. Holger Höhn, for providing an international atmosphere at the Institute of Human Genetics. To the secretary, Ilse Neumann, I would like to thank for the smooth running of things. Very special thanks to my landlady and landlord, Chris and Uwe Berndt, who made me feel truly accepted as the member of the family and made my stay in Würzburg wonderful experience. The greatest acknowledgement I reserve for my family in Belgrade, for their support, and for giving me the best start in life I could ever have hoped for, to whom I dedicate this Dissertation. Jelena Stojic Table of contents TABLE OF CONTENTS 1 SUMMARY................................................................................................................................... 1 2 ZUSAMMENFASSUNG.............................................................................................................. 4 3 INTRODUCTION ........................................................................................................................ 8 3.1 Organization of the retina ...................................................................................................... 8 3.1.1 Photoreceptor cells ...................................................................................................... 10 3.1.2 Phototransduction cascade.......................................................................................... 12 3.2 Age related macular degeneration........................................................................................ 14 3.2.1 Disease phenotype....................................................................................................... 14 3.2.2 Risk factors in AMD ................................................................................................... 16 3.2.3 The genetics of age-related macular degeneration ...................................................... 17 3.2.4 Search for the complex disease genes ......................................................................... 20 3.2.4.1 Genotyping single nucleotide polymorphisms (SNPs) ........................................... 21 3.3 Goal of the thesis ................................................................................................................. 22 4 MATERIALS AND METHODS ............................................................................................... 24 4.1 Bioinformatics...................................................................................................................... 24 4.1.1 Computational analysis of nucleotide sequences.............................................................. 24 4.1.2 Computational analysis of amino acid sequences ....................................................... 24 4.2 RNA Methods...................................................................................................................... 25 4.2.1 RNA sources............................................................................................................... 25 4.2.2 RNA isolation.............................................................................................................. 25 4.2.2.1 Total RNA............................................................................................................... 25 4.2.2.2 Cytoplasmic RNA................................................................................................... 26 4.2.2.3 Poly(A)+ RNA......................................................................................................... 26 4.2.3 Quantification of RNA................................................................................................ 26 4.2.4 First-strand cDNA synthesis ....................................................................................... 27 4.2.5 Northern blot ............................................................................................................... 27 4.2.5.1 Agarose gel electrophoresis..................................................................................... 27 4.2.5.2 Transfer to Nylon membrane.................................................................................. 28 4.2.5.3 Radioactive random-prime probe labelling............................................................. 28 4.2.5.4 Membrane hybridization ......................................................................................... 28 4.3 DNA Methods...................................................................................................................... 29 4.3.1 Phenol / chlorophorm extraction ................................................................................. 29 4.3.2 Polymerase chain reaction (PCR) ............................................................................... 29 4.3.2.1 Standard PCR .......................................................................................................... 29 4.3.2.2 Quantitative Real-time PCR (qRT-PCR) ................................................................ 30 4.3.3 Agarose gel electrophoresis ........................................................................................ 31 4.3.4 DNA purification......................................................................................................... 32 4.3.4.1 Purification of PCR products.................................................................................. 32 4.3.4.2 Purification of plasmid DNA.................................................................................. 32 4.3.4.3 Restriction enzyme digestion.................................................................................. 33 4.3.4.4 Ligation and transformation.................................................................................... 33 4.3.5 Sequencing .................................................................................................................. 34 4.3.6 Virtual Northern blot analysis ..................................................................................... 34 4.4 cDNA libraries..................................................................................................................... 35 4.4.1 Generation of a suppression subtracted cDNA library from the human retina ........... 35 4.4.1.1 Sequencing of the clones from the library................................................................. 35 Table of contents 4.4.2 Full-length cDNA library ............................................................................................ 36 4.4.2.1 Construction ............................................................................................................