Characterisation of the MID1/α4 multiprotein complex INAUGURAL DISSERTATION to obtain doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by Beatriz Aranda Orgillés Born on 07 November 1976 in Zaragoza, Spain Berlin, April 2006 1. Reviewer: Professor Dr. Susann Schweiger 2. Reviewer: Professor Dr. Volker A. Erdmann Date of defence: To Dirk, Gabriel, Mª Jesús and Ana Table of Contents Table of Contents TABLE OF CONTENTS............................................................................................................1 1 INTRODUCTION...............................................................................................................4 1.1 VENTRAL MIDLINE DEVELOPMENT .................................................................................4 1.1.1 Craniofacial development .....................................................................................7 1.1.2 Heart development ................................................................................................8 1.1.3 Ephrins as NCC pathfinders ................................................................................9 1.1.4 Midline development and disease .......................................................................10 1.2 X-LINKED OPITZ BBB/G SYNDROME (OS) AND MID1................................................11 1.2.1 The RBCC protein MID1.....................................................................................12 1.2.2 The MID1 function ..............................................................................................15 1.2.3 Regulation of MID1 function...............................................................................16 1.3 MTOR PATHWAY .........................................................................................................17 1.4 AIM OF THE STUDY .......................................................................................................19 2 MATERIALS AND METHODS......................................................................................20 2.1 MATERIALS ..................................................................................................................20 2.1.1 General reagents .................................................................................................20 2.1.2 Enzymes...............................................................................................................22 2.1.3 Kits ......................................................................................................................22 2.1.4 Instruments and Disposables ..............................................................................22 2.1.5 Antibodies............................................................................................................23 2.1.6 Vectors.................................................................................................................24 2.1.7 Buffers and Media ...............................................................................................24 2.1.8 Cell lines..............................................................................................................26 2.1.9 Bioinformatic tools and Databases.....................................................................26 2.1.10 Patients................................................................................................................27 2.2 NUCLEIC ACID METHODS ..............................................................................................27 2.2.1 Polymerase chain reaction (PCR).......................................................................27 2.2.2 Agarose gel electrophoresis................................................................................28 2.2.3 RNA preparation and cDNA synthesis................................................................28 2.2.4 Phenol:Chloroform extraction ............................................................................29 2.2.5 EtOH precipitation of nucleic acids....................................................................29 2.2.6 Cloning................................................................................................................29 2.2.6.1 Cloning of pEGFP constructs and mutagenesis ..............................................29 2.2.6.2 Preparation of the pCMVTag2C and pBMT116 constructs............................31 2.2.6.3 α4/44aa peptide clones....................................................................................31 2.2.7 Plasmid DNA isolation and transformation........................................................32 2.2.7.1 Plasmid DNA isolation....................................................................................32 2.2.7.2 Chemical transformation.................................................................................33 2.2.7.3 Electroporation ................................................................................................33 2.2.8 Transfection.........................................................................................................33 2.2.8.1 siRNA transfection..........................................................................................33 2.2.8.2 Polyfect transfection of plasmid DNA............................................................33 2.2.8.3 Lipofectamine transfection of plasmid DNA..................................................34 2.2.9 Sequencing ..........................................................................................................34 2.3 PROTEIN METHODS .......................................................................................................35 -1- Table of Contents 2.3.1 Bradford assay ....................................................................................................35 2.3.2 SDS-PAGE Gel....................................................................................................35 2.3.3 Western blot.........................................................................................................36 2.3.4 Silver staining......................................................................................................37 2.3.5 Coomassie/Colloidal coomassie staining............................................................37 2.3.6 Immunofluorescence............................................................................................37 2.3.7 Microtubule assembly experiment.......................................................................38 2.3.8 Immunoprecipitation ...........................................................................................38 2.3.9 Yeast two-hybrid experiments .............................................................................39 2.3.9.1 Yeast-transformation.......................................................................................39 2.3.9.2 β-Galactosidase activity ..................................................................................40 2.3.10 Cell culturing and trypisnation ...........................................................................41 2.4 PREPARATION OF THE 44AA PEPTIDES ..........................................................................41 2.4.1 Growth of E.Coli cultures ...................................................................................41 2.4.2 Preparation of cleared E.Coli lysates.................................................................42 2.4.3 Purification of 6xHis tagged proteins from E.Coli .............................................42 2.4.4 Thrombin digestion .............................................................................................42 2.5 CHROMATOGRAPHY COLUMN AND MASS SPECTROMETRY ...........................................43 2.6 SUCROSE GRADIENTS ...................................................................................................44 2.6.1 Discontinuous gradients......................................................................................44 2.6.2 Linear sucrose gradient ......................................................................................44 2.7 RNA-PROTEIN BINDING EXPERIMENTS.........................................................................45 2.7.1 Homopolymer binding assay...............................................................................45 2.7.2 Ephrin mRNAs-MID1 binding experiments ........................................................45 2.7.2.1 Amplification of G-quartets and EFNB1 mRNA............................................45 2.7.2.2 In vitro transcription of biotinylated RNA......................................................47 2.7.2.3 Biotinylation Efficiency Dot Blot ...................................................................47 2.7.2.4 RNA-PROTEIN Binding Assay......................................................................47 2.7.3 Biofinformatics....................................................................................................48 3 RESULTS...........................................................................................................................49 3.1 CHARACTERISATION OF THE BBOXES OF MID1 ...........................................................49 3.1.1 Mutations in Bbox1 disrupt MID1-α4 interaction..............................................49 3.1.2 Analysis of the structure of the Bbox2 domain....................................................52 3.1.3 C195F, mutated