Identification and characterization of interacting partners of cytoplasmic domain of Xenopus paraxial protocadherin [Elektronische Ressource] / presented by Yingqun Wang

Qun

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2007
• Nombre de lectures: 20
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Identification and Characterization of
interacting partners of cytoplasmic domain of
Xenopus Paraxial Protocadherin























DISSERTATION

submitted to the
Combined Faculties of the Natural Sciences and Mathematics
of the Ruprechtr-Karls University of Heidelberg, Germany

for the degree of
Doctor of Natural Sciences







presented by
Master of Sciences Yingqun Wang
born in Wuhan, P. R. China



Oral examination: ……………………




Identification and Characterization of
interacting partners of cytoplasmic domain of
Xenopus Paraxial Protocadherin














Referees: Prof. Dr. Herbert Steinbeisser
Prof. Dr. Thomas Holstein





















DEDICATED TO THE MEMORY OF MY MOTHER

李竹生 ZHUSHENG LI









Table of Contents
Table of Contents

Table of Contents....................................................................................................................................i
1. SUMMARY ....................................................................................................................................... 1
2. INTRODUCTION............................................................................................................................. 3
2.1 Morphogenetic movements in gastrulation........................................................................... 4
2.1.1 CE movements.............................................................................................................. 6
2.1.2 Tissue seperation .......................................................................................................... 8
2.2 Molecular basis of gastrulation movements.......................................................................... 9
2.2.1 Wnt signaling pathway ................................................................................................ 9
2.2.1.1 Canonical Wnt/β-catenin pathway ................................................................ 11
2.2.1.2 Planar cell polarity pathway 11
2+
2.2.1.3 Wnt/Ca pathway........................................................................................... 12
2.2.1.4 Regulation of gastrulation by Wnt signaling ................................................ 13
2.2.2 FGF signaling ............................................................................................................. 14
2.2.3 BMP and Nodal signaling .......................................................................................... 16
2.2.4 Endocytosis ................................................................................................................. 17
2.2.5 Cytosketon remodeling .............................................................................................. 18
2.2.6 Extracelluar matrix.................................................................................................... 20
2.2.7 Cell adhesion molecules ............................................................................................. 21
2.2.7.1 Classic cadherins 22
2.2.7.1.1 Classical cadherins in morphogenesis ................................................ 22 2 Catenins in morphogenesis.................................................................. 22
2.2.7.2 Protocadherins................................................................................................. 23
2.2.7.2.1 Axial protocadherin ............................................................................. 23 2 NF-protocadherin................................................................................. 23
2.2.7.2.3 Paraxial protocadherin ........................................................................ 24 4 Protocadherin in Neural crest and Somites ....................................... 25
2.2.7.3 Atypical cadherins........................................................................................... 25
2.3 Aim of the study .................................................................................................................... 26
3. Materials and Methods................................................................................................................... 28
3.1 Materials ................................................................................................................................ 28
3.1.1 Chemicals 28
3.1.2 Enzyme and Kit systems............................................................................................ 28
3.1.3 Laboratory instruments and accessories.................................................................. 29
3.1.4 General buffers and media........................................................................................ 30
3.1.5 Stock solutions, media and buffers for yeast work.................................................. 31
3.1.6 Antibodies ................................................................................................................... 33
3.1.7 Bacterial, yeast strains and yeast two-hybrid cDNA library.................................. 33
3.1.8 Oligonucleotides ......................................................................................................... 33
3.1.9 Plasmids ...................................................................................................................... 35
3.1.9.1 Plasmids for yeast two-hybrid assay.............................................................. 35
3.1.9.2 Plasmids for GST pull-down assay ................................................................ 36
3.1.9.3 Plasmids for expression in Xenopus embryos ............................................... 36
3.2 Molecular biology methods .................................................................................................. 37
3.2.1 Maintenance of bacterial strains............................................................................... 37
3.2.2 Preparation of competent bacteria ........................................................................... 37
3.2.3 Transformation of E. coli .......................................................................................... 37
3.2.4 Plasmid Minipreparation 37
3.2.5 Plasmid Midipreparation 37
3.2.6 Enzymatic modification of DNA 38
i Table of Contents
3.2.6.1 Digestion of DNA by restriction endonucleases............................................ 38
3.2.6.2 Generation of blunt-end DNA fragments...................................................... 38
3.2.6.3 Dephosphorylation of plasmid DNA.............................................................. 38
3.2.6.4 Ligation of DNA fragments ............................................................................ 38
3.2.7 DNA electrophoresis .................................................................................................. 38
3.2.8 DNA purification........................................................................................................ 39
3.2.8.1 Purification of DNA fragments ...................................................................... 39
3.2.8.2 Extraction of DNA fragments from agarose gels.......................................... 39
3.2.9 In vitro transcription of CAP-mRNA ....................................................................... 39
3.2.10 Determination of DNA and RNA concentration.................................................... 39
3.2.11 Polymerase Chain Reaction..................................................................................... 39
3.2.12 Mutagenesis of plasmids .......................................................................................... 40
3.2.13 DNA Sequencing....................................................................................................... 40
3.3 Embryological methods ........................................................................................................ 41
3.3.1 Preparation of Xenopus laevis embryos.................................................................... 41
3.3.2 Microinjection and manipulation of Xenopus embryos.......................................... 41
3.3.2.1 Micorinjection ................................................................................................. 41
3.3.2.2 Explantation of animal caps........................................................................... 41
3.3.3 RT-PCR (Reverse transcription PCR)..................................................................... 42
3.3.3.1 Isolation of RNA from embryos or explants................................................. 42
3.3.3.2 Reverse Transcription (RT)............................................................................ 42
3.3.3.3 PCR .................................................................................................................. 42
3.4 Protein biochemical methods ............................