Cloning and characterization of the chick-neurotrophin-receptor trkA:
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| Main Author: | |
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| Format: | Thesis Book |
| Language: | English |
| Published: |
1995
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | 104 Bl. Ill., graph. Darst. |
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MARC
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| 100 | 1 | |a Schröpel, Andrea |d -2013 |e Verfasser |0 (DE-588)114150818 |4 aut | |
| 245 | 1 | 0 | |a Cloning and characterization of the chick-neurotrophin-receptor trkA |c vorgelegt von Andrea Schröpel |
| 264 | 1 | |c 1995 | |
| 300 | |a 104 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a Würzburg, Univ., Diss. | ||
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Record in the Search Index
| _version_ | 1812453616123904000 |
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| adam_text |
TABLE OF CONTENTS
1. ABBREVIATIONS 8
2. ZUSAMMENFASSUNG 10
3. INTRODUCTION 11
3.1. EARLY NEUROSCIENCE 11
3.2. CELL DEATH 11
3.3. NEUROTROPHIC THEORY 13
3.4. EFFECTS OF NGF ON NEURONAL SURVIVAL 15
3.5. THE NEUROTROPHIN FAMILY 15
3.6. ROLE OF NEUROTROPHINS 18
3.8. OBJECTIVES 21
4. MATERIALS AND METHODS
4.1. MATERIALS
4.1.1. SPECIAL CHEMICALS AND MATERIAL 22
4.1.2. SPECIAL APPARATUS 23
4.1.3. GENERALLY USED BUFFERS AND MEDIA 23
4.1.4. ENZYMES 29
4.1.5. BIOLOGICAL MATERIAL 29
4.1.5.1. CELL LINES AND ANIMALS 29
4.1.5.2. BACTERIAL STRAINS 29
4.1.5.3. PLASMIDS 29
4.1.5.4. PHAGES 29
4.2. METHODS
4.2.1.
TISSUE PREPARATION AND CELL CULTURE 29
4.2.1.1. PRODUCTION OF CULTURE MEDIUM AND CELL CULTURE 29
4.2.1.2. ISOLATION OF GANGLIA FROM EMBRYONIC CHICKEN 30
4.2.1.3. CELL CULTURE OF SYMPATHETIC NEURONS 30
4.2.1.4. SECONDARY CULTURE 30
4.2.2.
MOLECULAR BIOLOGY TECHNIQUES 30
4.2.2.1. ETHANOL PRECIPITATION 30
4.2.2.3. ISOPROPANOL PRECIPITATION 30
4.2.2.4. PHENOL/CHCL
3
EXTRACTION 31
4.2.2.5. AGAROSE GEL ELECTROPHORESIS 31
5
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/946587116
4.2.2.6. ACRYLAMIDE GEL ELECTROPHORESIS 31
4.2.2.7. FORMAMIDE GEL FOR SEPARATION OF RNA 31
4.2.2.9. HYBRIDIZATION PROTOCOLS 32
4.2.2.10. DETERMINATION OF DNA/RNA 32
4.2.2.11. RESTRICTION ENDONUCLEASE DIGESTION 33
4.2.2.12. ISOLATION OF DNA FRAGMENTS 33
4.2.2.13. LIGATION REACTION 33
4.2.2.14. PREPARATION OF COMPETENT CELLS FOR ELECTROPORATION 33
4.2.2.15. ELECTROPORATION OF E.COLI CELLS 33
4.2.2.16. RAPID, SMALL-SCALE ISOLATION OF PLASMIDS 33
4.2.2.17. LARGE-SCALE ISOLATION OF PLASMIDS 34
4.2.2.18. POLYMERASE CHAIN REACTION (PCR) 34
4.2.2.19. SYNTHESIS AND WORK-UP OF OLIGONUCLEOTIDES 35
4.2.2.22.
IN VITRO TRANSCRIPTION 35
4.2.2.23. EXTRACTION OF RNA FROM TISSUES OR CELLS 36
4.2.2.24. ISOLATION OFPOLY(A)
+RNA ^ 36
4.2.2.25. REVERSE TRANSCRIPTION (RT) AND RT-PCR 36
4.2.2.26. RAPID AMPLIFICATION OF CDNA ENDS (RACE) 37
4.2.2.27. DETERMINATION OF THE LIBRARY TITER 38
4.2.2.28. SCREENING 38
4.2.3.
PROTEIN BIOCHEMISTRY 39
4.2.3.1. IODINATION OF NGF 39
4.2.3.2. CROSSLINKING AND IMMUNOPRECIPITATION 40
4.2.3.3. SDS-POLYACRYLAMIDE GEL (PAGE) 40
4.2.3.4. BINDING STUDIES 41
5. RESULTS* 42
5
.1.
SCREENING FOR CHTRKA 42
5.1.2. SELECTION OF THE OLIGONUCLEOTIDES 42
5.1.3. AMPLIFICATION OF A TRKA LIKE FRAGMENT USING RT-PCR 46
5.1.5. SEQUENCE ANALYSIS 52
5.1.6. ALTERNATIVE TRANSCRIPT 53
5.1.7. CONCLUSION ABOUT THE SEQUENCE IDENTITY 53
5.2. ANALYSIS OF CHTRKA MRNA DURING DEVELOPMENT 53
5.2.1. NORTHERN BLOT ANALYSIS 53
5.2.2. LOCALIZATION OF CHTRKA EXPRESSION BY IN SITU STUDIES 5 5
5.3. ANALYSIS OF CHTRKA ON PROTEIN LEVEL 60
5.3.1. IMMUNOPRECIPITATION STUDIES ON SYMPATHETIC NEURONSQ 60
6
5.3.2. TRANSIENT EXPRESSION OF CHTRKA IN A293 CELLS AND BINDING 61
5.4. TRKA IN E7 SYMPATHETIC CULTURES 62
5.5. IN SITU STUDIES ON ANGF TREATED CHICK EMBRYOS 65
6. DISCUSSION 67
6.1. CLONING OF A FUNCTIONAL CHICK TRKA NGF RECEPTOR 67
6.1.1. CLONING OF CHTRKA USING RT-PCR AND SCREENING OF
SPECIFIC LIBRARIES 67
6.1.2. ANALYSIS OF THE 5'END 68
6.1.3. CONSERVATION OF THE SUBDOMAIN STRUCTURE IN CHTRKA 69
6.1.4. THE CHICK TRKA GENE CODES FOR ONE TRANSCRIPT 71
6.2. EXPRESSION OF CHTRKA
IN VIVO
72
6.3. THE CLONED CHTRKA RECEPTOR BINDS NGF WITH
INTERMEDIATE AFFINITY AND HIGH SPECIFICITY 76
6.4. IMMUNOPRECIPITATION OF TRKA USING ELL AND E7
SYMPATHETIC NEURONS _ 77
6.5. RETINOIC ACID DOES NOT INDUCE TRKA 79
6.6. TREATMENT OF EARLY CHICK EMBRYOS WITH ANGF
ABOLISHED TRKA POSITIVE NEURONS IN DRG 82
6.7. CONCLUSION 82
7. REFERENCES 84
8. APPENDIX 95
9. ACKNOWLEDGEMENTS 103
*
10. CURRICULUM VITAE 104
7 |
| any_adam_object | 1 |
| author | Schröpel, Andrea -2013 |
| author_GND | (DE-588)114150818 |
| author_facet | Schröpel, Andrea -2013 |
| author_role | aut |
| author_sort | Schröpel, Andrea -2013 |
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| building | Verbundindex |
| bvnumber | BV010406851 |
| ctrlnum | (OCoLC)613922465 (DE-599)BVBBV010406851 |
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| illustrated | Illustrated |
| indexdate | 2024-10-09T16:14:22Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-006930169 |
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| owner_facet | DE-20 DE-91 DE-BY-TUM DE-355 DE-BY-UBR DE-12 DE-11 DE-188 |
| physical | 104 Bl. Ill., graph. Darst. |
| publishDate | 1995 |
| publishDateSearch | 1995 |
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| spelling | Schröpel, Andrea -2013 Verfasser (DE-588)114150818 aut Cloning and characterization of the chick-neurotrophin-receptor trkA vorgelegt von Andrea Schröpel 1995 104 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Würzburg, Univ., Diss. Molekulargenetik (DE-588)4039987-4 gnd rswk-swf Huhn (DE-588)4026126-8 gnd rswk-swf Nervenwachstumsfaktor (DE-588)4171485-4 gnd rswk-swf Rezeptor (DE-588)4049722-7 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Nervenwachstumsfaktor (DE-588)4171485-4 s Rezeptor (DE-588)4049722-7 s Molekulargenetik (DE-588)4039987-4 s DE-604 Huhn (DE-588)4026126-8 s DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=006930169&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Schröpel, Andrea -2013 Cloning and characterization of the chick-neurotrophin-receptor trkA Molekulargenetik (DE-588)4039987-4 gnd Huhn (DE-588)4026126-8 gnd Nervenwachstumsfaktor (DE-588)4171485-4 gnd Rezeptor (DE-588)4049722-7 gnd |
| subject_GND | (DE-588)4039987-4 (DE-588)4026126-8 (DE-588)4171485-4 (DE-588)4049722-7 (DE-588)4113937-9 |
| title | Cloning and characterization of the chick-neurotrophin-receptor trkA |
| title_auth | Cloning and characterization of the chick-neurotrophin-receptor trkA |
| title_exact_search | Cloning and characterization of the chick-neurotrophin-receptor trkA |
| title_full | Cloning and characterization of the chick-neurotrophin-receptor trkA vorgelegt von Andrea Schröpel |
| title_fullStr | Cloning and characterization of the chick-neurotrophin-receptor trkA vorgelegt von Andrea Schröpel |
| title_full_unstemmed | Cloning and characterization of the chick-neurotrophin-receptor trkA vorgelegt von Andrea Schröpel |
| title_short | Cloning and characterization of the chick-neurotrophin-receptor trkA |
| title_sort | cloning and characterization of the chick neurotrophin receptor trka |
| topic | Molekulargenetik (DE-588)4039987-4 gnd Huhn (DE-588)4026126-8 gnd Nervenwachstumsfaktor (DE-588)4171485-4 gnd Rezeptor (DE-588)4049722-7 gnd |
| topic_facet | Molekulargenetik Huhn Nervenwachstumsfaktor Rezeptor Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=006930169&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT schropelandrea cloningandcharacterizationofthechickneurotrophinreceptortrka |