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Identification and Characterization of Zebrafish 17Beta-HSD Type 1 and Type 3: a Comparative Analysis of Androgen/Estrogen Activity Regulators

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Identification and Characterization of Zebrafish 17Beta-HSD Type 1 and Type 3: a Comparative Analysis of Androgen/Estrogen Activity Regulators Institut für Experimentelle Genetik GSF-Forschungzentrum für Umwelt und Gesundheit, Neuherberg Identification and characterization of zebrafish 17beta-HSD type 1 and type 3: A comparative analysis of androgen/estrogen activity regulators Rebekka Mindnich Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.- Prof. Dr. Bertold Hock Prüfer der Dissertation: 1. Priv.-Doz. Dr. Jerzy Adamski 2. Univ.-Prof. Dr. Johannes Buchner 3. Univ.-Prof. Dr. Wolfgang Wurst Die Dissertation wurde am 30.06.2004 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 07.10. 2004 angenommen. Table of contents Table of contents ABSTRACT................................................................................................................................... 7 ZUSAMMENFASSUNG................................................................................................................ 9 ABBREVIATIONS....................................................................................................................... 11 1 INTRODUCTION ................................................................................................................ 13 1.1 THE AIM OF THIS STUDY ................................................................................................ 14 1.2 FUNCTION AND SYNTHESIS OF ANDROGENS AND ESTROGENS......................................... 14 1.2.1 MEMBERS OF THE STEROID HORMONE CLASS ............................................................ 14 1.2.1.1 The synthesis pathway of the steroid hormone classes ............................................. 14 1.2.1.2 Tissues and compartments of steroid hormone synthesis ......................................... 16 1.2.2 ANDROGENS AND THEIR FUNCTIONS ......................................................................... 16 1.2.3 ESTROGENS AND THEIR FUNCTIONS .......................................................................... 17 1.2.4 EVOLUTIONARY CONSIDERATIONS............................................................................. 18 1.2.4.1 The receptors............................................................................................................. 18 1.2.4.2 The metabolising enzymes......................................................................................... 19 1.3 CHARACTERISTICS AND FUNCTIONS OF 17BETA-HSDS.................................................. 19 1.3.1 CLASSIFICATION AND FUNCTIONS.............................................................................. 19 1.3.2 MEMBERS OF THE SHORT-CHAIN DEHYDROGENASE/ REDUCTASE (SDR) SUPERFAMILY 21 1.3.3 17BETA-HSD TYPE 1 ............................................................................................... 23 1.3.4 17BETA-HSD TYPE 3 ............................................................................................... 23 1.4 THE (ZEBRA) FISH MODEL ORGANISM ............................................................................ 24 1.4.1 FUNCTIONS OF ANDROGENS AND ESTROGENS ........................................................... 24 1.4.2 ENZYMES AND RECEPTORS OF STEROIDOGENESIS ..................................................... 25 1.4.3 ZEBRAFISH BIOLOGY ................................................................................................ 25 2 METHODS.......................................................................................................................... 29 2.1 DNA ........................................................................................................................... 29 2.1.1 ISOLATION AND PURIFICATION PROCEDURES.............................................................. 29 2.1.1.1 Isolation of genomic DNA from zebrafish tissue......................................................... 29 2.1.1.2 Isolation of plasmid DNA from bacteria ...................................................................... 30 2.1.1.3 Purification of linear dsDNA from solutions ................................................................ 30 2.1.1.4 Purification of dsDNA from gels ................................................................................. 30 2.1.2 MEASUREMENT........................................................................................................ 30 2.1.2.1 Separation and monitoring by agarose gel electrophoresis ....................................... 30 2.1.2.2 Measurement by optical density................................................................................. 31 2.1.3 CLONING STRATEGIES .............................................................................................. 31 2.1.3.1 TOPO-TA cloning....................................................................................................... 31 2.1.3.2 Cloning via restriction sites......................................................................................... 31 2.1.3.3 Restriction site independent cloning........................................................................... 32 2.1.4 PCR-BASED METHODS............................................................................................. 33 2.1.4.1 PCR (polymerase chain reaction) .............................................................................. 33 2.1.4.2 Fusion-PCR................................................................................................................ 33 2.1.4.3 Secondary PCR/nested PCR ..................................................................................... 34 2.1.4.4 Sequencing ................................................................................................................ 34 2.1.4.5 Site-directed mutagenesis.......................................................................................... 34 2.1.5 LIBRARY SCREEN ..................................................................................................... 34 2.1.5.1 Probe labeling ............................................................................................................ 35 2.1.5.2 Filter-screen and signal-detection .............................................................................. 35 2.2 RNA ........................................................................................................................... 36 2.2.1 ISOLATION FROM TISSUES......................................................................................... 36 2.2.2 HANDLING AND MEASUREMENT ................................................................................. 36 2.2.3 REVERSE TRANSCRIPTION INTO CDNA...................................................................... 36 2.3 PROTEIN CHEMISTRY .................................................................................................... 37 2.3.1 PRODUCTION OF RECOMBINANT PROTEIN IN E. COLI................................................... 37 2.3.2 SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) ................................ 38 2.3.3 WESTERN BLOT ....................................................................................................... 39 3 Table of contents 2.3.4 MEASUREMENT OF SUBSTRATE SPECIFICITY .............................................................. 39 2.4 BIOINFORMATIC METHODS ............................................................................................ 40 2.4.1 IN SILICO SCREEN..................................................................................................... 40 2.4.2 IN SILICO NORTHERN BLOT ....................................................................................... 41 2.4.3 ASSEMBLIES AND GENE STRUCTURE ......................................................................... 41 2.4.4 ALIGNMENTS AND PHYLOGENY.................................................................................. 42 2.4.5 CALCULATION OF IDENTITY PLOTS ............................................................................ 42 2.4.6 3D-MODELING OF PROTEIN STRUCTURE .................................................................... 42 2.4.7 IDENTIFICATION OF TERMINAL SIGNALLING MOTIFS AND GLOBULAR DOMAINS................ 43 2.5 WORK WITH ORGANISMS .............................................................................................. 43 2.5.1 WORKING WITH E. COLI ............................................................................................ 43 2.5.2 WORKING WITH ZEBRAFISH....................................................................................... 44 3 MATERIALS, EQUIPMENT, PROGRAMS........................................................................ 45 3.1 MATERIALS.................................................................................................................. 45 3.1.1 ANTIBODIES............................................................................................................. 45 3.1.2 BACTERIA................................................................................................................ 45 3.1.3 CDNA-LIBRARIES (COLONY MACROARRAYS FROM RZPD)......................................... 46 3.1.4 CHEMICALS (SPECIAL).............................................................................................. 46 3.1.5 ENZYMES ...............................................................................................................
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