Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universtität München Characterization of two novel myelin proteins Karin Schaefer aus Nordenham 2009 Erklärung Diese Dissertation wurde im Sinne von §13 Abs. 3 der Promotionsordnung vom 29. Januar 1998 von PD Dr. Winklhofer betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet. München, am 15.12.2009 …………………………………….. (Karin Schaefer) Dissertation eingereicht am 15.12.2009 1. Gutachter PD Dr. Konstanze Winklhofer 2. Gutachter Prof. Dr. Christian Haass Mündliche Prüfung am 08.03.2010 Contents 1 SUMMARY ........................................................................................................................................... 1 2 INTRODUCTION ................................................................................................................................... 4 2.1 GLIAL CELLS ................................................................................................................................................... 4 2.2 EVOLUTION OF MYELIN .................................................................................................................................... 4 2.3 MYELINATING GLIA ......................................................................................................................................... 6 2.3.1 Myelin composition and structure ................................................................................................... 8 2.3.2 Axo-glia support ............................................................................................................................. 21 2.3.3 Metabolism of myelinating glia as risk factor for disease ............................................................. 21 2.3.4 Myelination in zebrafish ................................................................................................................. 23 2.4 MICROARRAY EXPRESSION SCREEN TO IDENTIFY NOVEL MYELIN SPECIFIC GENES ......................................................... 26 2.5 PROMOTERS DRIVING EXPRESSION IN MYELINATING GLIA ...................................................................................... 28 3 RESULTS ............................................................................................................................................. 31 3.1 ZWILLING .................................................................................................................................................... 31 3.1.1 Gene structure ............................................................................................................................... 31 3.1.2 Phylogenetic conservation of Zwilling-A and -B proteins and the zwilling locus............................ 32 3.1.3 mRNA expression ........................................................................................................................... 34 3.1.4 Zwilling protein .............................................................................................................................. 36 3.2 CLAUDIN K .................................................................................................................................................. 39 3.2.1 Claudin k gene ................................................................................................................................ 39 3.2.2 Claudin k protein structure ............................................................................................................ 39 3.2.3 Phylogeny ....................................................................................................................................... 41 3.2.4 mRNA expression ........................................................................................................................... 44 3.2.5 Claudin k antibodies ....................................................................................................................... 46 3.2.6 Claudin k protein expression and localization in cells and tissues during development ................ 48 3.2.7 Claudin k protein localization in cells ............................................................................................. 52 3.2.8 Morpholino knock down ................................................................................................................ 54 3.2.9 Other Claudins related to zebrafish myelin .................................................................................... 57 3.3 CLAUDIN K PROMOTER .................................................................................................................................. 60 3.3.1 Conservation of claudin promoter in teleost .................................................................................. 60 3.3.2 Tol2 mediated Gal4-UAS expression system .................................................................................. 61 3.3.3 mb eGFP under the control of claudin k promoter specifically and strongly labels the processes of myelinating glia. ........................................................................................................................................... 62 3.3.4 Claudin k promoter driving fluorescently tagged Claudin k ........................................................... 64 4 DISCUSSION ....................................................................................................................................... 68 4.1 ZWILLING ................................................................................................................................................... 68 4.2 CLAUDIN K .................................................................................................................................................. 71 4.3 CLAUDIN K PROMOTER .................................................................................................................................. 76 5 METHODS .......................................................................................................................................... 80 5.1 SEQUENCE DATA ANALYSIS ............................................................................................................................. 80 5.1.1 Database searches ......................................................................................................................... 80 5.1.2 Motive Searches............................................................................................................................. 80 5.1.3 Alignments ..................................................................................................................................... 80 5.1.4 Construction of phylogenetic trees ................................................................................................ 80 5.1.5 Synteny .......................................................................................................................................... 80 5.1.6 Percent identity plot ...................................................................................................................... 81 5.1.7 Ks/Ka value .................................................................................................................................... 81 5.2 MOLECULARBIOLOGAL METHODS .................................................................................................................... 81 5.2.1 5’-RACE .......................................................................................................................................... 81 5.2.2 RT-PCR ........................................................................................................................................... 82 5.2.3 Claudin k promoter ........................................................................................................................ 83 5.2.4 Fluorescently tagged Claudin k ...................................................................................................... 84 5.2.5 Agarose gel electrophoresis ........................................................................................................... 86 5.2.6 Restriction digests .......................................................................................................................... 86 5.2.7 Gateway cloning ............................................................................................................................ 86 5.2.8 Transformation of competent E.coli .............................................................................................. 87 5.2.9 Analyzing transformants by PCR.................................................................................................... 87 5.2.10 Amplification and preparation of plasmids ............................................................................... 87 5.2.11 Amplification and preparation of BAC ...................................................................................... 87 5.2.12 Measuring DNA and RNA absorbance ...................................................................................... 88 5.2.13 Sequencing ................................................................................................................................ 88 5.3 FISH HUSBANDRY AND CARE TAKING ................................................................................................................