Characterization of the {β4GalNAc [beta-4GalNAc] transferases and the {β4GalNAcTB [beta-4GalNAcTB] pilot protein (GABPI) from Drosophila melanogaster [Elektronische Ressource] / von Anita Stolz

Anita Stolz

Sujets

• Biologie

Informations

• Publié par: gottfried_wilhelm_leibniz_universitat_hannover
• Publié le: 01 janvier 2008
• Nombre de lectures: 31
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Characterization of the β4GalNAc Transferases
and the β4GalNAcTB Pilot Protein (GABPI) from
Drosophila melanogaster




Der Naturwissenschaftlichen Fakultät
der Gottfried Wilhelm Leibniz Universität Hannover
zur Erlangung des Grades

Doktorin der Naturwissenschaften
Dr. rer. nat.

genehmigte Dissertation







von
Dipl.-Biochem. Anita Stolz
geboren am 17. August 1979 in Essen

Hannover 2008




























Referentin : Prof. Dr. Rita Gerardy-Schahn
Korreferent : Prof. Dr. Jürgen Alves
Tag der Promotion : Montag, den 03.03.08Content i
ABSTRACT ..................................................................................................... 1
ZUSAMMENFASSUNG ................................................................................ 2
1 INTRODUCTION.................................................................................... 4
1.1 Glycosylation............................................................................................................4
1.1.1 General overview...............................................................................................4
1.1.2 N-linked glycosylation......................................................................................4
1.1.3 O-linked 5
1.1.4 Glycosphingolipids............................................................................................5
1.2 The secretory pathway – from the ER to the Golgi.............................................. 6
1.2.1 General overview...............................................................................................6
1.2.2 Quality control system in the ER....................................................................... 8
1.2.3 Transport between ER and Golgi .................................................................... 10
1.3 Glycosyltransferases..............................................................................................10
1.3.1 The β1,4-galactosaminyltransferase family .................................................... 12
1.4 The DHHC protein family .................................................................................... 14
1.5 Glycosylation in Drosophila melanogaster........................................................... 15
1.6 Aim of this study.................................................................................................... 16
2 MATERIALS AND METHODS........................................................... 17
2.1 Material..................................................................................................................17
2.1.1 Chemicals........................................................................................................17
2.1.2 Standard buffer and media............................................................................... 19
2.1.3 Culture media and additives ............................................................................ 20
2.1.4 Kits and further materials ................................................................................ 20
2.1.5 Laboratory Equipment.....................................................................................21
2.1.5 Enzymes..........................................................................................................22
2.1.6 Molecular weight markers 22
2.1.7 Antibodies........................................................................................................22
2.1.7.1 Primary Antibodies......................................................................................22 Content ii
2.1.7.2 Secondary Antibodies..................................................................................23
2.1.8 Oligonucleotides..............................................................................................23
2.1.9 Plasmids...........................................................................................................25
2.1.9.1 Plasmids with site-directed mutagenesis .................................................... 27
2.1.10 Laboratory animals..........................................................................................28
2.1.11 Eukaryotic cell lines........................................................................................28
2.1.12 Bacterial strains...............................................................................................29
2.2 Methods..................................................................................................................30
2.2.1 Cell biological techniques ............................................................................... 30
2.2.1.1 Transient transfection of HEK293 cells ........................................................ 30
2.2.1.2 Transient transfection of S2 cells .................................................................. 31
2.2.1.3 Generation of semi stable HEK293 cells....................................................... 31
2.2.1.3 RNAi treatment of Drosophila Schneider cells ............................................. 31
2.2.1.4 Immunocytochemistry................................................................................... 32
2.2.1.5 Immunofluorescence ..................................................................................... 32
2.2.2 Molecular biological techniques......................................................................33
2.2.2.1 Plasmid preparation33
2.2.2.2 Polymerase chain reaction (PCR)................................................................ 33
2.2.2.3 Determination of DNA and RNA concentrations........................................ 35
2.2.2.4 Agarose gel electrophoresis of DNA........................................................... 35
2.2.2.5 Isolation of DNA fragments from agarose gels........................................... 35
2.2.2.6 Restriction digest of DNA ........................................................................... 35
2.2.2.7 Ligation of DNA..........................................................................................36
2.2.2.8 Precipitation of nucleic acids....................................................................... 36
2.2.2.9 Transformation of chemically competent E.coli ......................................... 36
2.2.2.10 ation of electro competent E.coli YZ2000.................................. 36
2.2.2.11 Preparation of chemically competent E.coli................................................ 36
2.2.2.12 Preparation of E.coli DMSO-Stocks ........................................................... 37
2.2.2.13 Synthesis of dsRNA .................................................................................... 37
2.2.2.14 Agarose gel electrophoresis of RNA 37
2.2.3 Biochemical techniques...................................................................................38
2.2.3.1 Immunoprecipitation38
2.2.3.2 Analyses of proteins from transfected HEK293 cells ................................. 38
2.2.3.3 Polyacrylamide gelelectrophoresis (SDS-PAGE).......................................39 Content iii
2.2.3.4 Western blot.................................................................................................39
2.2.3.5 Immunostaining of Western blots ............................................................... 39
2.2.3.6 Protein estimation........................................................................................40
2.2.3.7 Golgi preparation.........................................................................................40
2.2.3.8 In vitro assay for β4GalNAc transferases.................................................... 40
2.2.3.9 Reverse-phase chromatography (in vitro assay).......................................... 41
2.2.3.10 Glycosphingolipid preparation from HEK293 cells.................................... 41
2.2.3.11 D. melanogaster................................ 42
2.2.3.12 High-performance thin-layer chromatography (HPTLC)............................ 42
2.2.3.13 Immunostaining of HPTLC......................................................................... 42
2.2.3.14 Matrix assistance laser desorption (MALDI) mass spectrometry ............... 43
3 RESULTS................................................................................................ 44
3.1 Function of β4GalNAcTA and β4GalNAcTB in vivo ......................................... 44
3.1.1 High-performance thin-layer chromatography analysis.................................. 44
3.1.2 Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF-MS) ......................................................................................... 45
3.2 In vitro characterization of β4GalNAcTA and β4GalNA