A genome wide analysis of protein-protein interactions in Kaposi's sarcoma associated herpesvirus (KSHV) [Elektronische Ressource] / vorgelegt von Christine Zeretzke

Christine Zeretzke

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 33
• Langue: English
• Poids de l'ouvrage: 8 Mo

Extrait

Aus dem Max von Pettenkofer-Institut für Hygiene und
Medizinische Mikrobiologie der Ludwig-Maximilians-Universität
München






A genome-wide analysis
of protein-protein interactions in
Kaposi’s sarcoma-associated herpesvirus (KSHV)







Dissertation
zum Erwerb des Doktorgrades der Medizin
an der Medizinischen Fakultät der
Ludwig-Maximilians-Universität zu München









vorgelegt von

Christine Zeretzke

aus Münster


2006






Mit Genehmigung der Medizinischen Fakultät
der Universität München



Berichterstatter: Priv. Doz. Dr. Dr. J. Haas

Mitberichterstatter: Prof. Dr. M. Volkenandt
Prof. Dr. H.-G. Klobeck Th. Kirchner



Mitbetreuung durch
den promovierten Mitarbeiter:


Dekan: Prof. Dr. med. D. Reinhardt





Tag der mündlichen Prüfung: 19.10.2006








































In Liebe und Dankbarkeit,
meinen Eltern gewidmet

Inhaltsverzeichnis

1 ZUSAMMENFASSUNG...................................................................................... 1
1 SUMMARY ......................................................................................................... 2
2 INTRODUCTION................................................................................................ 3
2.1 Herpesviridae .............................................................................................. 3
2.2 The replication cycle of herpesviridae ......................................................... 6
2.3 The Kaposi’s sarcoma-associated herpesvirus (KSHV).............................. 7
2.3.1 The Kaposi’s sarcoma (KS).................................................................. 7
2.3.2 The primary effusion lymphoma (PEL)................................................. 9
2.3.3 The multicentric Castleman’s disease (MCD) ...................................... 9
2.3.4 The virus particle................................................................................ 10
2.3.5 The KSHV genome ............................................................................ 11
2.3.6 The life cycle of KSHV ....................................................................... 13
2.4 The KSHV Y2H screen.............................................................................. 14
2.4.1 From sequencing to understanding.................................................... 14
2.4.2 The yeast two-hybrid system.............................................................. 15
2.4.3 Libraires versus arrays 16
2.4.4 Genome-wide yeast two-hybrid screens ............................................ 17
2.4.5 Yeast two-hybrid screens for viral protein interactions ....................... 18
2.5 Topology of protein interaction networks................................................... 20
2.5.1 Scale-free networks ........................................................................... 20
2.5.2 The small world phenomenon ............................................................ 21
2.6 Aims of this project.................................................................................... 22
3 MATERIAL AND METHODS ............................................................................ 23
3.1 Materials.................................................................................................... 23
3.1.1 Equipment.......................................................................................... 23
3.1.2 Chemicals 24
3.1.3 Additional materials 27
3.1.4 Cell lines ............................................................................................ 27
3.1.5 Recombinant vaccinia viruses............................................................ 27
3.1.6 Bacterial strains.................................................................................. 28
3.1.7 Yeast strains ...................................................................................... 28
3.1.8 Plasmids 28
3.1.9 Oligonucleotides................................................................................. 30
3.1.10 Molecular weight markers .................................................................. 36
i Inhaltsverzeichnis


3.1.11 Kits ..................................................................................................... 37
3.1.12 Antibodies .......................................................................................... 37
3.1.12.1 Primary antibodies ....................................................................... 37
3.1.12.2 Secondary antibodies .................................................................. 37
3.1.13 Enzymes ............................................................................................ 37
3.2 Methods .................................................................................................... 38
3.2.1 Bacterial cell culture ........................................................................... 38
3.2.1.1 Cultivation of bacteria .................................................................. 38
3.2.1.2 Preparation of competent bacteria ............................................... 38
3.2.1.3 Transformation............................................................................. 39
3.2.2 DNA techniques ................................................................................. 39
3.2.2.1 Purification of plasmid DNA ......................................................... 39
3.2.2.2 Determination of DNA concentration............................................ 39
3.2.2.3 Restriction endonuclease digestion ............................................. 39
3.2.2.4 5’-Dephosphorylation reaction ..................................................... 40
3.2.2.5 Nested polymerase chain reaction (PCR) for recombinatorial
cloning.......................................................................................... 40
3.2.2.6 Isolation of DNA fragments .......................................................... 42
3.2.2.7 Phenol/chloroform extraction and ethanol precipitation ............... 42
3.2.2.8 Ligation ........................................................................................ 43
3.2.2.9 Recombinatorial cloning (RC) ...................................................... 43
3.2.2.9.1 The BP reaction ........................................................................ 43
3.2.2.9.2 The LR reaction 43
3.2.2.10 Agarose gel electrophoresis......................................................... 44
3.2.2.11 Plasmid construction.................................................................... 44
3.2.2.11.1 Inserting the recombination cassette into bait and prey vectors 44
3.2.2.11.2 Cloning the PCR products into the donor vector..................... 45
3.2.2.11.3 Subcloning of the array into bait and prey vector.................... 45
3.2.3 Tissue culture..................................................................................... 45
3.2.3.1 Cultivation and cryoconservation ................................................. 45
3.2.3.2 Calcium phosphate transfection................................................... 46
3.2.4 Protein techniques ............................................................................. 46
3.2.4.1 Co-immunoprecipitation ............................................................... 46
ii Inhaltsverzeichnis


3.2.4.2 SDS-PAGE .................................................................................. 47
3.2.4.3 Western blot................................................................................. 48
3.2.5 Yeast cell culture................................................................................ 49
3.2.5.1 Competent yeast cells.................................................................. 49
3.2.5.2 Transformation into yeast............................................................. 50
3.2.5.3 Mating and selection by a robot device........................................ 50
4 RESULTS......................................................................................................... 53
4.1. Generation of KSHV arrays in Y2H bait and prey vectors......................... 53
4.1.1 Amplification of KSHV genes 53
4.1.2 Adding attB sites to both ends of the PCR-product............................ 55
4.1.3 Cloning the PCR-products into the entry vector ................................. 55
4.1.4 Subcloning into the bait and prey vector ............................................ 55
4.1.5 Transformation into yeast and arrangement of the plates .................. 56
4.1.6 The robot-assisted steps.................................................................... 58
4.2 Identification of KSHV protein-protein