RNA helicase Ddx5 and the noncoding RNA SRA act as co-activators in the Notch signaling pathway:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2013
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | VII, 100 Bl. Ill., graph. Darst. 30 cm 1 CD-ROM |
Internformat
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| adam_text | IMAGE 1
TABLE OF CONTENTS
TABLE OF CONTENTS
PUBLICATIONS I
ABBREVIATIONS II
SUMMARY 1
ZUSAMMENFASSUNG 2
1 INTRODUCTION 3
1.1 THE NOTCH SIGNALING PATHWAY 3
1.1.1 DISCOVERY OF NOTCH 3
1.1.2 THE NOTCH RECEPTORS AND LIGANDS 4
1.1.3 THE CANONICAL NOTCH SIGNALING PATHWAY 5
1.1.4 THE 1 NOTCH PATHWAY CO-REPRESSOR AND CO-ACTIVATOR COMPLEXES 6
1.1.5 THE NOTCH TARGET GENES 9
1.1.6 NOTCH SIGNALING IN T CELL DEVELOPMENT 10
1.1.7 NOTCH SIGNALING IN TUMORIGENESIS 11
1.2 THE RNA HELICASES 12
1.2.1 STRUCTURE AND FUNCTION OF THE DEAD-BOX RNA HELICASES 12
1.2.2 THE DEAD-BOX PROTEIN DDX5 14
1.2.3 THE TRANSCRIPTIONAL CO-REGULATIVE FUNCTIONS OF DDX5 16
1.3 THE STEROID RECEPTOR RNA ACTIVATOR: AN ATYPICAL CO-ACTIVATOR 17
1.3.1 SRA INTERACTING PROTEINS 19
1.4 DDX5, DDXL7 AND SRA ACT TOGETHER AS TRANSCRIPTIONAL CO-ACTIVATORS 19
1.5 DDX5, DDXL7 AND SRA IN CANCER DEVELOPMENT 21
AIM OF THE THESIS 23
2 MATERIALS 24
2.1 MOUSE STRAINS 24
2.2 CELL LINES, CELL CULTURE MEDIA AND REAGENTS 24
2.3 BACTERIA STRAINS 2 5
2.4 PLASMIDS 25
2.4.1 EXPRESSION PLASMIDS 25
2.4.2 RETROVIRAL PLASMIDS 26
2.4.3 BACTERIAL EXPRESSION PLASMIDS 26
2.5 OLIGONUCLEOTIDES 26
2.5.1 GENOTYPING OLIGONUCLEOT IDES 26
2.5.2 RT-PCR OLIGONUCLEOTIDES 27
2.5.3 CHIP OLIGONUCLEOTIDES 27
2.5.4 PROBES 27
2.5.5 SEQUENCES OF SHRNA HAIRPINS 28
2.6 ENZYMES AND STANDARDS 28
2.7 ANTIBODIES 28
2.8 COMMON BUFFERS AND SOLUTIONS 29
2.8.1 ELECTROPHORESIS AND GENERAL BUFFERS 29
2.8.2 NUCLEAR EXTRACT BUFFERS 29
2.8.3 CHIP BUFFERS 29
2.8.4 PULLDOWN BUFFERS 29
2.8.5 WESTERN BLOT BUFFERS 30
2.8.6 GST-PULLDOWN BUFFERS 30
2.8.7 NATIVE RNA-IMMUNOPRECIPITATION BUFFERS 30
2.8.8 MASS-SPECTROMETIY BUFFERS 30
V
HTTP://D-NB.INFO/1038684595
IMAGE 2
TABLE OF CONTENTS
2.9 CHEMICALS 30
2.9.1 RADIOCHEMICALS 31
2.9.2 KITS AND RESINS 32
2.10 CONSUMABLES 32
2.11 TECHNICAL EQUIPMENT 32
3 METHODS 34
3.1 DEVELOPMENT OF NEW MODEL SYSTEMS TO STUDY NOTCH SIGNALING IN VIVO 34
3.1.1 ESTABLISHMENT OF AN INDUCIBLE RBP-] KNOCKOUT PRO-B CELL LINE 34
3.1.2 ESTABLISHMENT OF AN INDUCIBLE SWITCH-ON SYSTEM IN MATURE T CELLS
35 3.2 CELL CULTURE METHODS 36
3.2.1 CULTURE CONDITIONS 36
3.2.2 ISOLATION OF BONE MARROW CELLS AND RED BLOOD CELL LYSIS 36
3.2.3 CRYOPRESERVATION 36
3.2.4 PHOENIX RETROVIRAL EXPRESSION SYSTEM 36
3.2.5 SHRNA KNOCKDOWN 37
3.2.6 LUCIFERASE ASSAY 37
3.3 WORKING WITH DNA 38
3.3.1 GENOTYPING 38
3.3.2 DETERMINATION OF DNA CONCENTRATIONS 38
3.3.3 RESTRICTION DIGEST OF DNA 38
3.3.4 DEPHOSPHORYLATION OF DNA 5 - ENDS 39
3.3.5 AGAROSE GEL ELECTROPHORESIS 39
3.3.6 PURIFICATION OF DNA FROM AN AGAROSE GEL 40
3.3.7 LIGATION OF DNA-FRAGMENTS INTO VECTOR-DNA 40
3.3.8 ELECTRO-TRANSFORMATION OF DNA IN BACTERIA 40
3.3.9 ANALYTIC ISOLATION OF PLASMID-DNA IN BACTERIA 41
3.3.10 PHENOL-CHLOROFORM EXTRACTION 41
3.3.11 AMPLIFICATION OF DNA VIA POLYMERASE CHAIN REACTION 42
3.3.12 DNA-SEQUENCING AND SEQUENCE ANALYSIS 43
3.4 WORKING WITH RNA 44
3.4.1 ISOLATION OF RNA 44
3.4.2 CDNA-SYNTHESIS 44
3.4.3 NATIVE RNA-IMMUNOPRECIPITATION 45
3.4.4 QUANTITATIVE REAL-TIME RT-PCR 46
3.5 PROTEIN BIOCHEMICAL METHODS 46
3.5.1 NUCLEAR EXTRACT PREPARATION 46
3.5.2 NUCLEAR EXTRACT PREPARATION FOR MASS-SPECTROMETRY 47
3.5.3 PROTEIN CONCENTRATION MEASUREMENT 47
3.5.4 SDS POLYACRYLAMID GEL ELECTROPHORESIS 47
3.5.5 WESTERN BLOT 48
3.5.6 PULLDOWN OF BIOTINYLATED PROTEINS 49
3.5.7 LIQUID CHROMATOGRAPHY MASS-SPECTROMETRIC ANALYSIS 50
3.5.8 COUPLED IN VITRO TRANSCRIPTION/TRANSLATION AND GST-PULLDOWN 52
3.5.9 CHROMATIN IMMUNOPRECIPITATION 53
4 RESULTS 54
4.1 IDENTIFICATION OF THE RNA HELICASE DDX5 AS A NEW NOTCH PATHWAY
PROTEIN 54
4.1.1 STUDYING NOTCH COMPLEXES USING IN VIVO BIOTINYLATION APPROACH
FOLLOWED BY MASS-SPECTROMETRY 54
4.1.2 PURIFICATION OF BIOTIN-TAGGED-NOTCHL-INTRACELLULAR DOMAIN IN BEKO
PRE-T CELLS 54
4.1.3 MASS-SPECTROMETRIC ANALYSIS OF BIOTIN-TAGGED NOTCHL INTRACELLULAR
DOMAIN 55
VI
IMAGE 3
TABLE OF CONTENTS
4.1.4 IDENTIFICATION OF NEW COMPLEX PROTEINS OF THE NOTCH SIGNALING
PATHWAY
USING SILAC 57
4.2 RNA HELICASE DDX5 PHYSICALLY INTERACTS WITH THE RBP-J/NOTCH
COMPLEX.. 64 4.2.1 DDX5 CO-IMMUNPRECIPITATES WITH NOTCH IN VIVO 64
4.2.2 THE RNA HELICASE DDX5 CO-PURIFIES WITH RBP-J/NICD CO-ACTIVATOR
COMPLEX 64
4.2.3 THE RNA HELICASE DDX5 DIRECTLY INTERACTS WITH RBP-) IN VITRO 66
4.3 THE RNA HELICASE DDX5 DIRECTLY REGULATES NOTCH TARGET GENES IN VIVO
69 4.3.1 NOTCH DEPENDENT RECRUITMENT OF DDX5 TO NOTCH TARGET GENES 69
4.3.2 RBP-J DEPENDENT RECRUITMENT OF DDX5 TO NOTCH TARGET GENES 70 4.4
FUNCTIONAL CHARACTERISATION OF DDX5 IN NOTCH SIGNALING 72
4.4.1 RNA HELICASE DDX5 ACTS AS A CO-ACTIVATOR IN NOTCH/RBP-J DEPENDENT
TRANSCRIPTION 72
4.4.2 DOWNREGULATION OF NOTCH TARGET GENES UPON DDX5 KNOCKDOWN IN VIVO
72 4.5 FUNCTIONAL CHARACTERIZATION OF THE LONG NON-CODING RNA SUA IN
NOTCH SIGNALING.- ............ ............ 73
4.5.1 SRA ASSOCIATES WITH DDX5 AND N1CD IN T CELLS 73
4.5.2 SRA ACTS TOGETHER WITH DDX5 AS A TRANSCRIPTIONAL CO-ACTIVATOR IN
VITRO 74 4.5.3 SRA ACTS AS A TRANSCRIPTIONAL CO-ACTIVATOR IN NOTCH
SIGNALING IN VIVO 75 4.6 OCCUPANCY OF P300 IS LOST AT NOTCH TARGET GENES
UPON SRA KNOCKDOWN AND OVEREXPRESSION 76
4.7 HISTONE ACETYLATION IS LOST UPON SRA KNOCKDOWN AND OVEREXPRESSION AT
NOTCH TARGET GENES 79
5 DISCUSSION 81
5.1 IDENTIFICATION OF THE RNA HELICASE DDX5 AS NOVEL COMPONENT OF THE
RBP-J/NOTCH COMPLEX 81
5.2 FUNCTIONAL CHARACTERISATION OF THE RNA HELICASE DDX5 AND THE LONG
NONCODING RNA SRA IN NOTCH SIGNALING 82
6 ATTACHMENTS 87
6.1 DDX5 KNOCKDOWN WITH 2 ND INDEPENDENT HAIRPIN 87
6.2 MASS-SPECTROMETRY LISTS 87
7 REFERENCES 88
VII
|
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| author | Jung, Claudia |
| author_GND | (DE-588)1038684269 |
| author_facet | Jung, Claudia |
| author_role | aut |
| author_sort | Jung, Claudia |
| author_variant | c j cj |
| building | Verbundindex |
| bvnumber | BV041283846 |
| ctrlnum | (OCoLC)858929182 (DE-599)DNB1038684595 |
| dewey-full | 572.864 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 572 - Biochemistry |
| dewey-raw | 572.864 |
| dewey-search | 572.864 |
| dewey-sort | 3572.864 |
| dewey-tens | 570 - Biology |
| discipline | Biologie Medizin |
| format | Thesis Book |
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| spelling | Jung, Claudia Verfasser (DE-588)1038684269 aut RNA helicase Ddx5 and the noncoding RNA SRA act as co-activators in the Notch signaling pathway vorgelegt von Claudia Jung 2013 VII, 100 Bl. Ill., graph. Darst. 30 cm 1 CD-ROM txt rdacontent n rdamedia nc rdacarrier Freiburg (Breisgau), Univ., Diss., 2013 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026732978&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Jung, Claudia RNA helicase Ddx5 and the noncoding RNA SRA act as co-activators in the Notch signaling pathway |
| subject_GND | (DE-588)4113937-9 |
| title | RNA helicase Ddx5 and the noncoding RNA SRA act as co-activators in the Notch signaling pathway |
| title_auth | RNA helicase Ddx5 and the noncoding RNA SRA act as co-activators in the Notch signaling pathway |
| title_exact_search | RNA helicase Ddx5 and the noncoding RNA SRA act as co-activators in the Notch signaling pathway |
| title_full | RNA helicase Ddx5 and the noncoding RNA SRA act as co-activators in the Notch signaling pathway vorgelegt von Claudia Jung |
| title_fullStr | RNA helicase Ddx5 and the noncoding RNA SRA act as co-activators in the Notch signaling pathway vorgelegt von Claudia Jung |
| title_full_unstemmed | RNA helicase Ddx5 and the noncoding RNA SRA act as co-activators in the Notch signaling pathway vorgelegt von Claudia Jung |
| title_short | RNA helicase Ddx5 and the noncoding RNA SRA act as co-activators in the Notch signaling pathway |
| title_sort | rna helicase ddx5 and the noncoding rna sra act as co activators in the notch signaling pathway |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026732978&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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