O-linked N-Acetylglucosaminylation of Sp1 inhibits the human immunodeficiency virus type-1 promoter [Elektronische Ressource] / vorgelegt von Ramona Jochmann

Moni Grego

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2009
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

O-linked N-Acetylglucosaminylation of Sp1 Inhibits
the Human Immunodeficiency Virus Type-1
Promoter



Den Naturwissenschaftlichen Fakultäten der
Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades



vorgelegt von
Ramona Jochmann
aus Reschitza (Rumänien)


Als Dissertation genehmigt von den Naturwissenschaftlichen
Fakultäten der Friedrich-Alexander-Universität Erlangen-Nürnberg











Tag der mündlichen Prüfung: 8. Mai 2009
Vorsitzender der Promotionskommission: Prof. Dr. Eberhard Bänsch
Erstberichterstatter: Prof. Dr. Michael Stürzl
Zweitberichterstatter: Prof. Dr. Thomas Winkler Table of Contents
Table of Contents

1. SUMMARY............................................................................................................................. 1
ZUSAMMENFASSUNG............................................................................................................. 2
2. INTRODUCTION...................................................................................................................... 3
2.1 The Human Immunodeficiency Virus Type-1 ................................................................. 3
2.1.1 The Structure of HIV-1........................................................................................ 3
2.1.2 The HIV-1 Proteins ............................................................................................. 4
2.1.3 The Regulation of HIV-1 Gene Expression......................................................... 5
2.1.4 The HIV-1 Replication Cycle .............................................................................. 6
2.1.5 The Clinical Course of HIV-1 Infection .............................................................. 6
2.1.6 The Highly Active Antiretroviral Therapy .......................................................... 8
2.1.7 Residual Viremia and HIV-1 Latency 9
2.2 The Transcription Factor Sp1...................................................................................... 13
2.2.1 Characteristics of Sp1 ....................................................................................... 13
2.2.2 Transcriptional Activation by Sp1..................................................................... 15
2.2.3 Regulation of the Transcriptional Activity of Sp1............................................. 17
2.2.3.1 Regulation of Sp1 Expression Level ..................................................... 17
2.2.3.2 Regulation by Posttranslational Modifications.................................... 17
2.2.3.3 Regulation by Interaction Partners ...................................................... 19
2.3 Posttranslational Modification of Proteins with O-GlcNAc ........................................ 21
2.3.1 The Hexosamine Biosynthesis Pathway ............................................................ 21
2.3.2 The Cycling Enzymes Involved in O-GlcNAc Modification of Proteins ........... 23
2.3.2.1 O-GlcNAc Transferase ......................................................................... 23
2.3.2.2 O-GlcNAc Hexosaminidase.................................................................. 24
2.3.2.3 Interplay Between NCOAT and OGT at the Chromatin....................... 25
2.3.3 Diverse Regulation of Protein Function by O-GlcNAc..................................... 26
2.3.4 The Yin-Yang Hypothesis .................................................................................. 29
2.4 Aims of the Study.......................................................................................................... 31
3. MATERIAL AND METHODS .................................................................................................. 32
3.1 Material........................................................................................................................ 32
3.1.1 Chemicals, Media and Reagents ....................................................................... 32
3.1.2 Consumables ..................................................................................................... 34
3.1.3 Equipment 34
3.1.4 Kits .................................................................................................................... 35
3.1.5 Enzymes............................................................................................................. 35
3.1.6 Standards........................................................................................................... 36
3.1.7 Software 36
3.1.8 Buffer and Solutions.......................................................................................... 36
3.1.9 Primary Antibodies ........................................................................................... 38
3.1.10 Secondary Antibodies...................................................................................... 39
3.1.11 Oligonucleotides.............................................................................................. 39
3.1.11.1 siRNA-Oligonucleotides ..................................................................... 39
3.1.11.2 Cloning Oligonucleotides ................................................................... 39
3.1.11.3 Gel Shift Oligonucleotides.................................................................. 40
3.1.11.4 Oligonucleotides for RT-PCR............................................................. 40
3.1.11.5 Sequencing Oligonucleotides 40
3.1.12 Plasmids .......................................................................................................... 41
3.1.13 Biological Material ......................................................................................... 45
ITable of Contents
3.1.13.1 Bacterial Strains ................................................................................. 45
3.1.13.2 Eukaryontic Cell Lines ....................................................................... 45
3.1.13.3 Primary Cells...................................................................................... 45
3.1.13.4 Recombinant Viruses .......................................................................... 46
3.2 Methods ........................................................................................................................ 46
3.2.1 RNA-Techniques................................................................................................ 46
3.2.1.1 Isolation of Cellular RNA from Eukaryotic Cells................................. 46
3.2.1.2 Agarose Gel Electrophoresis of RNA ................................................... 46
3.2.1.3 Quantitative Measurement of RNA....................................................... 46
3.2.1.4 In vitro Transcription of mRNA............................................................ 47
3.2.2 cDNA Synthesis ................................................................................................. 48
3.2.3 DNA-Techniques ............................................................................................... 49
3.2.3.1 Polymerase Chain Reaction (PCR) ...................................................... 49
a) Reverse Transcription (RT)-PCR ..................................................... 49
b) Cloning-PCR .................................................................................... 50
c) Mutagenesis-PCR ............................................................................. 51
3.2.3.2 Agarose Gel Electrophoresis of DNA................................................... 52
3.2.3.3 Isolation of DNA Fragments from Gels................................................ 52
3.2.3.4 Restriction Digest of DNA Molecules 52
3.2.3.5 Purification of Linearized DNA with Phenol/Chloroform/Ethanol...... 53
3.2.3.6 Dephosphorylation of Vector DNA....................................................... 54
3.2.3.7 Klenow Reaction................................................................................... 54
3.2.3.8 Ligation of DNA 54
3.2.3.9 Heat Shock Transformation of Bacteria............................................... 54
3.2.3.10 Isolation of Plasmid DNA................................................................... 55
3.2.3.11 DNA Sequencing................................................................................. 55
3.2.3.12 Quantitative Measurement of DNA .................................................... 56
3.2.4 Cell Biological Methods.................................................................................... 56
3.2.4.1 Cultivation of E.Coli............................................................................. 56
3.2.4.2 Cultivation of Mammalian Cells........................................................... 56
3.2.4.3 HIV-1 Pseudovirus Production ............................................................ 57
3.2.4.4 Infection of Lymphocytes with HIV-1 and Stimulation with GlcN ....... 57
3.2.4.5 Cell Transfection .................................................................................. 58
a) Transfection of HEK 293T Cells ...................................................... 58
b) Transfection of Jurkat Cells ............................................................. 58
+c) Transfe