Analysis of the nuclear egress complex of mouse cytomegalovirus [Elektronische Ressource] / vorgelegt von Mark Lötzerich

Mark Loetzerich

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• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 24
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Analysis of the nuclear egress complex of
mouse cytomegalovirus



















Dissertation zur Erlangung des Doktorgrades der
Naturwissenschaften
der Fakultät für Biologie
der Ludwig-Maximilians-Universität München



vorgelegt von
Mark Lötzerich
München, 2007




































Dissertation eingereicht am: 28.03.2007

Erstgutachterin: PD Dr. Bettina Kempkes
Zweitgutachter: Prof. Dr. Heinrich Leonhardt
Sondergutachter: Prof. Dr. Ulrich H. Koszinowski


Tag der mündlichen Prüfung: 19.12.2007
Table of contents


A. Summary ...................................................................................................... 1


B. Introduction.................................................................................................. 2

1. The cytomegaloviruses as family members of the herpesviruses .................. 2
1.1 Biology of CMV infection and host immune defense .................................... 3
1.2 Transmission and clinical relevance of human cytomegalovirus (HCMV) .... 4
1.3 Mouse cytomegalovirus (MCMV) as animal model for HCMV infection ....... 5
2. Structure of cytomegaloviruses...................................................................... 5
3. Replication of cytomegaloviruses................................................................... 7
4. The nuclear envelope..................................................................................... 9
4.1 The lamin proteins...................................................................................... 11
4.2 Proteins and protein complexes interacting with lamins............................. 12
4.2.1 The lamin B receptor-complex (LBR or p58) ....................................... 12
4.2.2 LEM-domain proteins and BAF complexes.......................................... 14
5. The egress of herpesvirus capsids from the nucleus ................................... 15
5.1 The nuclear egress complex (NEC) ........................................................... 17
6. Aims and concepts....................................................................................... 20


C. Material and methods................................................................................ 22

1. Material ........................................................................................................ 22
1.1 Devices ...................................................................................................... 22
1.2 Consumables ............................................................................................. 23
1.3 Reagents.................................................................................................... 23
1.4 Commercial kits.......................................................................................... 25
1.5 Oligonucleotide-peptides............................................................................ 26
1.6 Plasmids..................................................................................................... 26
1.6.1 Commercially available and published plasmids ................................. 26
1.6.2 Plasmids constructed over the project................................................. 27
1.6.2.1 M53/p38 expression plasmids....................................................... 27
1.6.2.2 pOriR6k-zeo-ie derived plasmids .................................................. 27
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Table of contents

1.6.2.3 Plasmid with temperature sensitive origin of replication (T )......... 29 s
1.6.2.4 MCMV-BACs................................................................................. 29
1.7 Bacterial strains.......................................................................................... 31
1.8 Cells ........................................................................................................... 31
1.8.1 Cell culture reagents............................................................................ 31
1.8.1.1 Basal media .................................................................................. 31
1.8.1.2 Supplements and sera .................................................................. 31
1.9 Viruses ....................................................................................................... 32
1.10 Antibodies ................................................................................................ 32
1.10.1 Primary antibodies............................................................................. 32
1.10.1.1 Rabbit polyclonal antisera........................................................... 32
1.10.1.2 Rabbit monoclonal antibodies ..................................................... 32
1.10.1.3 Mouse m 32
1.10.1.4 Rat polyclonal antisera................................................................ 32
1.10.1.5 Goat polyclonal antisera.............................................................. 33
1.10.2 Secondary antibodies ........................................................................ 33
1.10.2.1 FITC-conjugates ......................................................................... 33
1.10.2.2 Texas-Red-conjugates 33
1.10.2.3 Alexa-488-conjugates ................................................................. 33
1.10.2.4 Alexa-633-conjugates 33
1.10.2.5 Peroxidase (Pox)-conjugates ...................................................... 34
2. Methods ....................................................................................................... 34
2.1 Isolation and purification of nucleic acids ................................................... 34
2.1.1 Small scale isolation of plasmid DNA .................................................. 34
2.1.2 Large scale isolation of plasmid DNA 36
2.1.3 Small scale isolation of BAC-DNA ....................................................... 37
2.1.4 Large scale isolation of BAC-DNA 38
2.1.5 Determination of DNA concentration and purity of the isolated DNA... 39
2.2 Analysis and cloning of DNA...................................................................... 40
2.2.1 Restriction digest of DNA..................................................................... 40
2.2.2 Dephosphorylation of DNA .................................................................. 40
2.2.3 Amplification of DNA by Polymerase Chain Reaction (PCR)............... 41
2.2.3 Agarose gel electrophoresis ................................................................ 43

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Table of contents

2.2.4 Isolation of DNA fragments from agarose-gels .................................... 44
2.2.5 Annealing of synthetic oligonucleotides............................................... 44
2.2.6 Ligation of DNA fragments................................................................... 44
2.2.7 Transformation of recombinant DNA ................................................... 45
2.2.7.1 Preparation of electro-competent bacteria .................................... 45
2.2.7.2 Transformation of electrocompetent bacteria................................ 45
2.2.8 Linker scanning mutagenesis of the M53 ORF 46
2.3 Cells and viruses........................................................................................ 48
2.3.1 Tissue culture ...................................................................................... 48
2.3.1.1 Cultivation of cells ......................................................................... 48
2.3.1.2 Freezing and thawing of eukaryotic cells ...................................... 48
2.3.2 Working with MCMV ............................................................................ 49
2.3.2.1 Generation of recombinant MCMV-BACs ..................................... 49
2.3.2.2 Reconstitution of recombinant MCMV-BACs to virus.................... 50
2.3.2.3 MCMV virus stock preparation ...................................................... 51
2.3.2.4 Growth curves............................................................................... 52
2.3.2.5 MCMV titer determination by plaque assay................................... 52
2.4 Analysis of proteins .................................................................................... 53
2.4.1 Transfection of eukaryotic cells using calciumphosphate-precipitation 53
®2.4.2 Transfection of eukaryotic cells using Superfect transfection reagent 54
2.4.3 Protein extraction from eukaryotic cells ............................................... 54
2.4.3.1 Protein extraction from eukaryotic cells using total lysis buffer ..... 54
2.4.3.2 Protein extraction from eukaryotic cells using IP lysis buffer......... 55
2.4.3.3 Protein extraction from eukaryotic cells using high salt lysis buffer55
2.4.3.4 Preparation of nuclear extracts ..................................................... 56
2.4.4 Metabolic labeling of proteins