Identification and characterization of Eya1-interacting proteins [Elektronische Ressource] / von Kathrin Landgraf

Dekanat

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2008
• Nombre de lectures: 15
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

“Identification and characterization
of Eya1-interacting proteins”




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 Kathrin Landgraf


geboren am 12.10.1979
in Zwickau





Jena, im Juni 2008

























Gutachter:
1. Prof. Dr. Christoph Englert, FLI Jena
2. Prof. Dr. Frank Große, FLI Jena
3. Prof. Dr. Martin Blum, Universität Hohenheim

Tag der öffentlichen Disputation: 06.10.08
































Meinen Eltern gewidmet. Table of contents
TABLE OF CONTENTS
ZUSAMMENFASSUNG .......................................................................................... I
ABSTRACT............................................................................................................ II
ABBREVIATIONS................................................................................................. III
1 INTRODUCTION............................................................................................. 1
2 MATERIALS AND METHODS...................................................................... 13
2.1 MATERIALS............................................................................................................................ 13
2.1.1 Bacterial strains ........................................................................................................... 13
2.1.2 Yeast strains................................................................................................................. 13
2.1.3 cDNA library for yeast two-hybrid screening............................................................ 14
2.1.4 Eukaryotic cell lines .................................................................................................... 14
2.1.5 Antibodies..................................................................................................................... 14
2.1.6 Culture media ............................................................................................................... 15
2.1.6.1 Culture media for E.coli.......................................................................................... 15
2.1.6.2 a for S. cerevisiae .............................................................................. 15
2.1.7 Amino acids.................................................................................................................. 16
2.1.8 Plasmids ....................................................................................................................... 16
2.1.9 Oligonucleotides.......................................................................................................... 18
2.1.10 Morpholinos 18
2.1.11 siRNAs........................................................................................................................... 19
2.2 METHODS 20
2.2.1 Standard techniques ................................................................................................... 20
2.2.2 Yeast two-hybrid system............................................................................................. 20
2.2.2.1 Preparation of competent yeast cells (KFY1)......................................................... 20
2.2.2.2 Transformation of competent yeast cells (Klebe et al., 1983)................................ 20
2.2.2.3 Screening of the cDNA library................................................................................ 21
2.2.2.4 Qualitative colony-lift filter assay............................................................................ 22
2.2.2.5 Quantitative β-galactosidase liquid assay.............................................................. 22
2.2.2.6 Plasmid extraction from yeast cells........................................................................ 23
2.2.2.7 Protein extracs ......................................................................... 24
2.2.3 GST pulldown system ................................................................................................. 24
2.2.3.1 Purification of GST fusion proteins from E. coli BL21 ............................................ 24
2.2.3.2 In vitro interaction assay......................................................................................... 25
2.2.4 RNA isolation................................................................................................................ 25
2.2.5 RT-PCR analysis .......................................................................................................... 26
2.2.5.1 cDNA synthesis...................................................................................................... 26
2.2.5.2 Polymerase Chain Reaction (PCR)........................................................................ 26
Table of contents
2.2.5.3 Quantitative Real-Time RT-PCR (qRT-PCR)......................................................... 26
2.2.6 RNA in situ hybridization ............................................................................................ 27
2.2.6.1 Generation of digoxigenin-labeled riboprobes ....................................................... 27
2.2.6.2 RNA in situ hybridization on paraffin sections........................................................ 27
2.2.6.3 Whole mount in situ hybridization (WISH) of zebrafish embryos........................... 28
2.2.7 RNA interference.......................................................................................................... 29
2.2.8 Luciferase reporter assay ........................................................................................... 29
2.2.9 Lysis of eukaryotic cells for direct Immunoblotting ................................................ 29
2.2.10 Immunoprecipitation ................................................................................................... 30
2.2.11 In vivo ubiquitination assay ....................................................................................... 30
2.2.12 Fluorescence microscopy .......................................................................................... 31
3 RESULTS ..................................................................................................... 32
3.1 CHARACTERIZATION OF EYA1-SPECIFIC ANTIBODIES ............................................. 32
3.2 BIOCHEMICAL CHARACTERIZATION OF EYA1 ............................................................ 36
3.3 IDENTIFICATION OF EYA1-INTERACTING PROTEINS.................................................. 45
3.3.1 Yeast two-hybrid analysis........................................................................................... 45
3.3.1.1 Screening of a mouse embryo cDNA library.......................................................... 47
3.3.1.2 Verification of potential interaction partners of Eya1.............................................. 48
3.3.2 Characterization of the Eya1-Sipl1 interaction ......................................................... 49
3.3.2.1 Eya1 and Sipl1 interact in mammalian cells........................................................... 49
3.3.2.2 l1 bind directly to each other............................................................. 50
3.3.2.3 Interaction of Sipl1 with other Eya family members ............................................... 51
3.3.2.4 Localization of binding sites ................................................................................... 53
3.3.3 Interaction of Eya1 with Rbck1, a Sipl1-related protein........................................... 55
3.4 FUNCTIONAL RELEVANCE OF THE INTERACTIONS ................................................... 57
3.4.1 Cellular localization of Eya1, Sipl1, and Rbck1 ........................................................ 57
3.4.2 Co-expression of Eya1 and Sipl1 or Rbck1 in mouse.............................................. 59
3.4.3 Effect of Sipl1 and Rbck1 on transactivation function of Eya1 .............................. 61
3.4.4 Identification of orthologs of Sipl1 and Rbck1 in zebrafish .................................... 63
3.4.4.1 Co-expression of Eya1 and Sipl1/Rbck1 orthologs in zebrafish ............................ 64
3.4.4.2 Knockdown of Sipl1/Rbck1 orthologs in zebrafish ................................................. 67
3.5 ASSOCIATION OF SIPL1 AND RBCK1 WITH HUMAN DISEASE .................................. 70
3.5.1 Screening of BOR patients for mutations in SIPL1 or RBCK1................................ 71
R365C3.5.2 The BOR-associated mutation Sipl1 comprises the interaction with Eya1.... 71
4 DISCUSSION................................................................................................ 73
4.1 IMPORTANCE OF