Cloning and characterization of the HIPK2 protein kinase:
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| Main Author: | |
|---|---|
| Format: | Book |
| Language: | English |
| Published: |
2000
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Item Description: | Ulm, Univ., Diss., 2000 |
| Physical Description: | 111 Bl. Ill., graph. Darst. : 30 cm |
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MARC
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| 100 | 1 | |a Wang, Ying |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Cloning and characterization of the HIPK2 protein kinase |c submitted by Ying Wang |
| 264 | 1 | |c 2000 | |
| 300 | |a 111 Bl. |b Ill., graph. Darst. : 30 cm | ||
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| 500 | |a Ulm, Univ., Diss., 2000 | ||
| 650 | 7 | |a Hochschulschrift |2 gtt | |
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| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-009268930 | |
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TABLE OF CONTENTS
1. ABBREVIATIONS 1
2. INTRODUCTION 3
2.1. THE CD95 SYSTEM AND APOPTOSIS 3
2.1.1. THE DEATH RECEPTOR CD95 3
2.1.2. CD95 ASSOCIATED SIGNALING MOLECULES AND PATHWAY 6
2.2. THE P53 PATHWAY AND APOPTOSIS 7
2.2.1. STRUCTURE AND FUNCTION OF P53 AND ITS FAMILY 7
2.2.2. STABILITY AND ACTIVITY OF P53 9
2.2.2. STABILITY AND ACTIVITY OF P53 11
2.3. AIMS AND PLANNING OF WORK 14
3. MATERIALS AND METHODS 16
3.1. MATERIALS 16
3.1.1 BIOCHEMICAL REGENTS 16
3.1.2. EXPRESSION VECTORS 16
3.1.3. ENZYMES 16
3.1.4. ANTIBODIES 17
3.1.5. CDNA LIBRARIES 17
3.2. METHODS 17
3.2.1. GENERAL PROCESS OF CONSTRUCTING AND SEQUENCING PLASMIDS 17
3.2.2. ISOLATION OF HUMAN AND MURINE HIPK2 CDNAS 18
3.2.3 NORTHERN AND DOT BLOT ANALYSIS 20
3.2.3.1. PROBE 20
3.2.3.2. HYBRIDIZATION 21
3.2.4. CONSTRUCTION OF EXPRESSION PLASMIDS AND MUTAGENESIS 21
3.2.5. CELL LINES AND CELL CULTURE 22
3.2.6. TRANSIENT TRANSFECTION AND STABLE TRANSFECTION 22
3.2.7. RT-PCR ANALYSIS 23
3.2.8. MEASUREMENT OF APOPTOSIS 24
3.2.9. SIGNAL TRANSDUCTION PATHWAY REPORTING SYSTEMS 25
3.2.9.1. ELKL ACTIVATION ASSAY 26
3.2.9.2. P53 AND
NF-KB ACTIVATION ASSAY 26
3.2.10. IMMUNOPRECIPITATION 27
3.2.11. WESTERN BLOT 27
3.2.12. SUBCELLULAR FRACTIONATION 28
3.2.13. QUANTITATIVE FLUOROMETRIC ASSAY 29
3.2.14. YEAST TWO-HYBRID ASSAY 29
3.2.14.1. YEAST HOST STRAINS 29
3.2.14.2. CONSTRUCTING THE BAIT PLASMIDS 30
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/960380949
3.2.14.3. TESTING CELL TOXICITY EFFECT AND TRANSCRIPTIONAL ACTIVATION OF
THE BAITS 30
3.2.14.4. LIBRARY SCREENING 31
3.2.14.5. REPROOFING TWO-HYBRID POSITIVE INTERACTIONS 32
3.2.14.6. SEQUENCING AND ANALYZING INSERTS IN THE POSITIVE AD/LIBRARY
PLASMIDS 33
3.2.15. INTERACTING ANALYSIS IN YEAST 3 3
4. RESULTS 35
4.1. CDNA CLONING OF HIPK2 35
4.1.1. CDNA CLONING AND CONSTRUCTION ANALYSIS 35
4.1.2. COMPARISON HIPK2 WITH THE CDNAS WE FOUND 37
4.1.3. HIPK2 MUTAGENESIS 37
4.2. MRNA EXPRESSION PATTERN OF HIPK2 41
4.2.1. NORTHERN BLOT ANALYSIS 41
4.2.2. DOT BLOT ANALYSIS _ 43
4.3. OVEREXPRESSION OF HIPK2 INCREASES CELL DEATH 46
4.4. HIPK2 AFFECTS THE EXPRESSION OF TUMOR SUPPRESSOR PROTEIN P53 51
4.4.1. HIPK2 ACTIVATES P53-MEDIATED TRANSCRIPTION 51
4.4.2. WILDTYPE HIPK2 LEADS TO AN INCREASE OF P53 EXPRESSION 53
4.4.3. HIPK2 DOES NOT LEAD TO PHOSPHORYLATION OF SERINE 15, SERINE 20
AND
SERINE 392 OF P53 55
4.4.4. HIPK2 AFFECTS THE EXPRESSION OF MDM2 AND THE OTHER TARGET GENE 57
4.4.5. HIPK2 CAN NOT INTERACT WITH P53 AND MDM2 DIRECTLY IN VIVO 59
4.5. HIPK2 INDUCES ELKL- BUT NOT NF-KB-MEDIATED TRANSCRIPTION 61
4.6. YEAST TWO-HYBRID SCREENING ANALYSIS 63
4.6.1. PHENOTYPES OF YEAST HOST STRAINS 63
4.6.2. THE EFFECT ON CELL TOXICITY AND TRANSCRIPTIONAL ACTIVATION OF THE
BAITS 63
4.6.3. LIBRARY SCREENING RESULT ^ 64
4.6.4. RANBPM BINDS TO HIPK2 68
5. DISCUSSION 72
5.1. THE IDENTIFICATION OF THE NEW GENE HIPK2 72
5.1.1. THE FULL LENGTH CDNA 72
5.1.2. THE EXPRESSION AND LOCALIZATION OF HIPK2 72
5.2. HIPK2 ENHANCES CD95-INDUCED APOPTOSIS SLIGHTLY AND DOES NOT
BIND TO CD95 74
5.3. OVEREXPRESSION HIPK2 DIRECTLY LEAD TO CELL DEATH 75
5.4. HIPK2 IS INVOLVED IN P53 PATHWAY 76
5.4.1. OVEREXPRESSION HIPK2 ENHANCES THE TRANSCRIPTIONAL ACTIVATION OF
P53 76
5.4.2. OVEREXPRESSION OF HIPK2 LEADS TO ACCUMULATION OF P53 77
5.4.3. HIPK2IS INVOLVED IN THE BALANCE BETWEEN MDM2 AND P53 80
5.4.4. HIPK2 AND P53 TARGET GENES AND PATHWAY 82
5.5. HIPK2 AND MAP KINASE PATHWAY 84
5.6. INVESTIGATING THE FUNCTION OF HIPK2 AND SEARCHING FOR HIPK2
BINDING PROTEINS 85
5.6.1. THE NOVEL PROTEIN HIPK2 AND ITS FUNCTION DESCRIBED BY OTHER
GROUPS 85
5.6.2. INTERACTING INVESTIGATION VIA YEAST TWO-HYBRID SYSTEM 87
6. SUMMARY 90
7. REFFERENCES 92
8. ACKNOWLEDGEMENTS 111 |
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| author_facet | Wang, Ying |
| author_role | aut |
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| indexdate | 2024-10-09T18:10:34Z |
| institution | BVB |
| language | English |
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| physical | 111 Bl. Ill., graph. Darst. : 30 cm |
| publishDate | 2000 |
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| spelling | Wang, Ying Verfasser aut Cloning and characterization of the HIPK2 protein kinase submitted by Ying Wang 2000 111 Bl. Ill., graph. Darst. : 30 cm txt rdacontent n rdamedia nc rdacarrier Ulm, Univ., Diss., 2000 Hochschulschrift gtt (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=009268930&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Wang, Ying Cloning and characterization of the HIPK2 protein kinase Hochschulschrift gtt |
| subject_GND | (DE-588)4113937-9 |
| title | Cloning and characterization of the HIPK2 protein kinase |
| title_auth | Cloning and characterization of the HIPK2 protein kinase |
| title_exact_search | Cloning and characterization of the HIPK2 protein kinase |
| title_full | Cloning and characterization of the HIPK2 protein kinase submitted by Ying Wang |
| title_fullStr | Cloning and characterization of the HIPK2 protein kinase submitted by Ying Wang |
| title_full_unstemmed | Cloning and characterization of the HIPK2 protein kinase submitted by Ying Wang |
| title_short | Cloning and characterization of the HIPK2 protein kinase |
| title_sort | cloning and characterization of the hipk2 protein kinase |
| topic | Hochschulschrift gtt |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=009268930&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT wangying cloningandcharacterizationofthehipk2proteinkinase |