Click here to load reader

Sudheer Kumar Gara - COnnecting REpositories · PDF file REFEREES/ BERICHTERSTATTER 1. Berichterstatter: Prof. Dr. med. Mats Paulsson 2. Berichterstatter: Prof. Dr. med. Jens Brüning

  • View
    0

  • Download
    0

Embed Size (px)

Text of Sudheer Kumar Gara - COnnecting REpositories · PDF file REFEREES/ BERICHTERSTATTER 1....

  • IDENTIFICATION AND CHARACTERIZATION OF

    NOVEL COLLAGEN CHAINS

    Inaugural-Dissertation

    zur

    Erlangung des Doktorgrades

    der Mathematisch-Naturwissenschaftlichen Fakultät

    der Universität zu Köln

    vorgelegt von

    Sudheer Kumar Gara aus Visakhapatnam, Indien

    Köln, 2010

  • REFEREES/ BERICHTERSTATTER

    1. Berichterstatter: Prof. Dr. med. Mats Paulsson

    2. Berichterstatter: Prof. Dr. med. Jens Brüning

    Vorsitzender:

    Prof. Dr. rer. nat. Siegfried Roth


    Tag der mündlichen Prüfung: 20.04.2010

  • DEDICATED TO

    MY BELOVED PARENTS

  • I

    INDEX INDEX ……………………………………………………………………………… I ABBREVIATIONS………………………………………………………………….. IV 1. INTRODUCTION…………………………………………………………… 1 1.1 Collagen…………………………………………………………………… 1 1.2 Classification of collagens………………………………………………. 2 1.3 Von Willebrand factor A like domain (VWA).…………………………. 5 1.4 Collagen VI……………………………………………………………… 6 1.4.1 Alternative splicing of collagen VI………………………………… 7 1.4.2 Macromolecular assembly of collagen VI…………………………. 8 1.4.3 Interaction partners of collagen VI………………………………… 10 1.4.4 Human disorders associated with collagen VI……………………... 10 1.4.4.1 Bethlem Myopathy (BM)……………………………………….. 10 1.4.4.2 Ullrich Congenital Muscular Dystrophy (UCMD)……………… 12 1.4.4.3 Ossification of the Posterior Longitudinal Ligament (OPLL)....... 13 1.4.4.4 Diffuse Idiopathic Skeletal Hyperostosis (DISH)………………. 13 1.4.4.5 Osteoarthritis (OA)……………………………………………… 13 1.4.5 Animal models of collagen VI myopathies…………………………13 1.4.6 Cell attachment activity of collagen VI……………………………. 14 1.5 Collagen XXVIII…………………………………………………………15 1.6 Aims of the thesis……………………………………………………….. 16

    2 RESULTS………………………………………………………………….. 17 2.1 Novel collagen VI chains………………………………………………... 17 2.1.1 Cloning, gene and protein structure of new collagen VI chains…… 17 2.1.1.1 Cloning of cDNAs coding for new mouse collagen VI chains…. 17 2.1.1.2 Cloning of cDNAs coding for new human collagen VI chains…. 20 2.1.1.3 Domain architecture of the new chains…………………………. 20 2.1.1.4 Analysis of the collagenous domains…………………………… 24 2.1.1.5 Analysis of the VWA-domains………………………………….. 25 2.1.1.6 Analysis of the unique domains…………………………………. 25 2.1.1.7 Structure of the new collagen VI genes…………………………. 26 2.1.1.8 Human collagen VI α5 and α6 chains........................................... 29 2.1.1.9 Alternative splicing........................................................................ 29 2.1.2 Expression analysis of new collagen VI chains................................. 30 2.1.2.1 Analysis of mRNA of the new collagen VI genes in mouse......... 30 2.1.2.2 Preparation and evaluation of collagen VI specific antibodies...... 32 2.1.2.3 Analysis of antibody cross-reactivity between the collagen VI chains............................................................................................. 34 2.1.2.4 Collagen VI preparations from newborn mice contain the new chains............................................................................................. 37 2.1.2.5 Immunohistochemical analysis of the expression of the new chains in mouse.............................................................................. 38 2.1.2.5.1 Collagen VI chain expression in skeletal and cardiac muscle... 38 2.1.2.5.2 Collagen VI chain expression in smooth muscle....................... 41 2.1.2.5.3 Collagen VI chain expression in kidney ................................... 43 2.1.2.5.4 Collagen VI expression in mouse reproductive organs............. 44

  • II

    2.1.2.5.5 Collagen VI chain expression in skin........................................ 46 2.1.2.5.6 The new collagen VI chains are absent in cartilage.................. 48 2.1.3 Fate of the new collagen VI chains in the Col6a1 null mouse.......... 49 2.1.4 Expression of new collagen VI chains in man................................... 51 2.1.4.1 Expression in human skeletal muscle............................................ 52 2.1.4.2 Expression in human skin.............................................................. 52 2.1.5 The role of new collagen VI chains in human diseases..................... 54 2.1.5.1 Association to muscular dystrophies............................................. 54 2.1.5.2 Analysis of skin patients with COL6A1, -A2 and -A3 mutations... 55 2.1.5.3 Association to knee osteoarthritis.................................................. 57 2.2 Targeting of the collagen XXVIII gene in mouse..................................... 60 2.2.1 Strategy for the construction of the Col28a1 knockout targeting vector.................................................................................................. 61 2.2.2 ES-cell transfection and screening for positive clones...................... 62 2.2.3 Generation of chimeric mice.............................................................. 63 2.2.4 Examining the germ-line transmission of the targeted gene.............. 64

    3 DISCUSSION................................................................................... 64 3.1 Collagen VI ............................................................................................... 65 3.1.1 Gene structure and evolution of new chains.......................................... 65 3.1.2 Molecular assembly and interactions of new collagen VI chains.......... 67 3.1.3 Differential and restricted expression of the new chains....................... 72 3.1.4 Significance of the new chains in human diseases................................ 75 3.2 Collagen XXVIII....................................................................................... 81

    4 MATERIALS AND METHODS ............................................................. 83 4.1 Materials.................................................................................................... 83 4.1.1 General solutions and buffers................................................................ 83 4.1.2 Bacterial media and strains.................................................................... 84 4.1.3 Nucleic acid and protein standards........................................................ 84 4.1.4 Oligonucleotide primers........................................................................ 84 4.1.5 Restriction enzymes............................................................................... 86 4.1.6 Antibodies.............................................................................................. 87 4.2 Molecular biology methods....................................................................... 88 4.2.1 Culture and storage of bacteria.............................................................. 88 4.2.2 Preparation of competent cells............................................................... 88 4.2.3 Bacterial transformation……………………………………………… 88 4.2.4 Isolation of plasmid DNA…………………………………………….. 88 4.2.5 Determination of DNA concentration………………………………… 89 4.2.6 Agarose gel electrophoresis…………………………………………... 89 4.2.7 Gel-elution of DNA fragments……………………………………….. 89 4.2.8 Polymerase chain reaction (PCR)…………………………………….. 89 4.2.9 Restriction digestion of DNA………………………………………… 90 4.2.10 Ligation of DNA……………………………………………………… 90 4.2.11 DNA sequencing and analysis………………………………………... 91 4.2.12 DNA precipitation……………………………………………………..91 4.2.13 Isolation of total RNA………………………………………………… 91 4.2.14 Synthesis of cDNA…………………………………………………… 92 4.2.15 Northern blot………………………………………………………….. 92 4.2.15.1 DIG-labeling of RNA……………………………………………… 92

  • III

    4.2.15.2 Estimation of labeled RNA………………………………………… 93 4.2.15.3 Formaldehyde gels…………………………………………………. 93 4.2.15.4 Blotting……………………………………………………………..

Search related