XL2 - a novel neuroendocrine-specific luminal Golgi-resident protein derived from the XL alpha s/G alpha s gene:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | German |
| Veröffentlicht: |
1999
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | VI, 197 S. Ill., graph. Darst. |
Internformat
MARC
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| 100 | 1 | |a Wang, Yanzhuang |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a XL2 - a novel neuroendocrine-specific luminal Golgi-resident protein derived from the XL alpha s/G alpha s gene |c by Yanzhuang Wang |
| 264 | 1 | |c 1999 | |
| 300 | |a VI, 197 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a Heidelberg, Univ., Diss., 1999 | ||
| 650 | 0 | 7 | |a GTP-bindende Proteine |0 (DE-588)4248354-2 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Golgi-Apparat |0 (DE-588)4192795-3 |2 gnd |9 rswk-swf |
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 689 | 0 | 0 | |a Golgi-Apparat |0 (DE-588)4192795-3 |D s |
| 689 | 0 | 1 | |a GTP-bindende Proteine |0 (DE-588)4248354-2 |D s |
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| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-008668917 | |
Datensatz im Suchindex
| _version_ | 1812461177344622592 |
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| adam_text |
CONTENTS
CONTENTS
1. INTRODUCTION L
1.1. THE SECRETORY PATHWAY 1
1.1.1. THE CXOCYTIC PATHWAY 2
1.1.2. COAT PROTEINS AND ADAPTOR PROTEINS 9
1.1.3. VESICLE DOCKING MACHINERY AND THE "SNARE HYPOTHESIS" 11
1.1.4. MEMBRANE LIPIDS AND THE "RAFT HYPOTHESIS" 12
1.1.5. THE ENDOCYTIC PATHWAY 15
1.1.6. MEMBRANE TRAFFIC AND (SMALL) G-PROTEINS 15
1.2. HETEROTRIMERIC GUANINE-NUCLEOTIDE BINDING PROTEINS 18
1.2.1. THE DISCOVERY OF G PROTEINS 19
1.2.2. G PROTEIN ALPHA SUBUNITS 20
1.2.3. GPY SUBUNITS 21
1.2.4. THE G PROTEIN CYCLE 23
1.2.5. THE G PROTEIN COUPLED RECEPTORS 23
1.2.6. EFFECTORS 25
1.2.7. THE GAS GENE 25
1.2.8. TRIMENC G-PROTEINS AND MEMBRANE TRAFFIC 26
1.3. XLAS, A NOVEL TYPE OF G-PROTEIN 27
2. RESULTS
30
2.1. CHARACTERIZATION OF THE RAT AND HUMAN XLAS/GAS GENE 30
2.1.1. ISOLATION OF THE XLAS GENE FROM A RAT GENOMIC LIBRARY 30
2.1.1.1 THE XLAS GENE IS A SINGLE COPY GENE IN THE RAT GENOME 30
2.1.1.2 ISOLATION AND CHARACTERIZATION OF RAT XLAS GENOMIC CLONES 31
2.1.1.3 THE XL-DOMAIN IS ENCODED BY THE XL-EXON 34
2.1.1.4 DETERMINATION OF THE 5' BOUNDARY OF THE XLAS MRNA BY
PRIMER EXTENSION ANALYSIS 34
2.1.1.5 ANALYSIS OF THE PROMOTER REGION OF THE XLAS GENE 35
2.1.1.6 THE SECOND OPEN READING FRAME OF THE XL-EXON 38
2.1.2. CHROMOSOME LOCALIZATION OF THE XLAS GENE 38
2.1.3. DISCOVERY OF NEW XL-DOMAIN CONTAINING PROTEINS 41
2.1.3.1 DISCOVERY OF NEW XL-EXON CONTAINING EDNA CLONES 42
2.1.3.2 NEUROENDOCRINE SPECIFIC EXPRESSION OL THE NEW XL-DOMAIN
CONTAINING PROTEINS 44
I
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/956905307
_CONTENTS
2.1.4. CLONING OF THE XL2 CDNAS 45
2.1.4.1 RAT XL2 CDNA CLONING AND DNA SEQUENCING 45
2.1.4.2 CLONING MOUSE XL2 CDNAS 47
2.1.4.3 THE XL2 PROTEIN SEQUENCE IS CONSERVED BETWEEN DIFLERENT
SPECIES 50
2.1.5. THE ORGANIZATION OF THE XLAS/GIXS GENE AND ITS MRNA AND PROTEIN
PRODUCTS 50
2.1.5.1 ISOLATION OF THE XLUS GENE FROM A HUMAN PAC LIBRARY 50
2.1.5.2 THE XL2 PROTEIN IS ENCODED BY THE XL2-EXON 52
2.1.5.3 GENOMIC MAPPING OF THE HUMAN XLAS GENE 53
2.1.5.4 THE ORGANIZATION OF THE XLOCS/GAS GENE AND ITS MRNA AND
PROTEIN PRODUCTS 53
2.2. MOLECULAR CHARACTERIZATION OF THE XL2 PROTEIN, A LUMINAL GOLGI
RESIDENT PROTEIN
IN NEUROENDOCRINE CELLS 56
2.2.1. NEUROENDOCRINE SPECIFIC EXPRESSION OF THE XL2 MRNAS IN RAT
TISSUES 56
2.2.2. THE XL2AS MRNA CAN GENERATE BOTH THE XL2 PROTEIN AND THE NTAS
PROTEINS IN VITRO 56
2.2.3. XL2 IS A NEUROENDOCRINE SPECIFIC PROTEIN 59
2.2.3.1 PREPARATION AND PURIFICATION OF AN ANTIBODY AGAINST AN XL2
GST FUSION PROTEIN 59
2.2.3.2 PREPARATION OF ANTIBODIES AGAINST SNYTHETIC XL2 PEPTIDES 59
2.2.3.3 THE XL2 PROTEIN IS EXPRESSED IN NEUROENDOCRINE CELLS 60
2.2.4. XL2 IS A MEMBRANE-BOUND PROTEIN 61
2.2.5. XL2 IS A GOLGI PROTEIN 62
2.2.5.1 XL2 COLOCALIZES WITH GOLGI MARKERS 62
2.2.5.2 XI,2 IS REDISTRIBUTED TO THE ER BY BREFELDIN A TREATMENT 66
2.2.53 XL2 IS A GOLGI PROTEIN AS SHOWN BY ELECTRON MICROSCOPY 67
2.2.5.4 XL2 IS A GOLGI PROTEIN SHOWN BY SUBCELLULAR FRACTIONATION 68
2.2.5.5 SOME OF THE XL2 PROTEIN IS RELEASED FROM THE GOLGI INTO THE
GRANULES IN BOVINE ADRENAL MEDULLA CHROMAFFIN CELLS 70
2.2.6. XL2 IS A GOLGI LUMINAL PROTEIN 23
2.2.6.1 XL2 IS PROTECTED FROM PROTEINASE K DIGESTION BY THE MEMBRANE 73
2.2.6.2 XL2 IS AN N-GLYCOSYLATED PROTEIN 73
2.2.6.3 A PORTION OF THE XL2 PROTEIN IS SULFATED 75
2.2.7. XL2 IS A GOLGI RESIDENT PROTEIN 77
2.2.8. THE XL2 PROTEIN CONTAINS A GOLGI RETENTION SIGNAL 79
2.2.8.1 EXOGENOUSLY EXPRESSED XL2 IN VARIOUS CELL TYPES IS LOCALIZED
TO THE GOLGI 79
II
CONTENTS
2.2.8.2 THE N-TERMINAL PART OF XL2 IS NOT SULTICIENI TORELTICIEM
GOLGI LOCALIZATION OF THE PROTEIN 82
2.2.8.3 THE N-TERMINUS OF XL2 DOES NOT CONTAIN THE GOLGI LOCALIZATION
SIGNAL OF THE PROTEIN 82
2.2.9. XL2 IS A TYPE II MEMBRANE PROTEIN IN THE GOLGI 84
2.2.9.1 XL2 CONTAINS A MEMBRANE ANCHOR 85
2.2.9.2 THE GOLGI LOCALIZATION OF XL2 IS INDEPENDENT OF CHOLESTEROL 85
2.2.9.3 THE N-TERMINUS OF XL2 CONTAINS A MEMBRANE ANCHOR 86
2.2.9.4 XL2 IS A TYPE II MEMBRANE PROTEIN 88
2.2.10. TRANSLOCATION OF XL2 INTO THE LUMEN OF THE SECRETORY PATHWAY
OCCURS
AT THE LEVEL OF THE ER 89
2.3. A FUNCTIONAL STUDY OF THE ROLE OF CHOLESTEROL IN THE SECRETORY
PATHWAY
USING XL2 AS A GOLGI MARKER ^1
2.3.1. CHOLESTEROL DEPLETION FROM ATT20 CELLS INHIBITS VESICLE
BIOGENESIS
AT THE LEVEL OF THE TGN 91
2.3.2. DECREASING CELLULAR CHOLESTEROL LEVEL DOES NOT AFFECT ER-CGN
TRANSPORT 97
2.3.3. CHOLESTEROL DEPLETION/REPLENISHMENT DOES NOT AFFECT THE
CYTOSKELETON 98
2.3.4. CHOLESTEROL SUPPLEMENTATION PROMOTES VESICLE BIOGENESIS 98
3. DISCUSSION
103
3.1. THE XLOTS GENE - INCREASING THE COMPLEXITY OF THE GAS GENE 103
3.1.1. THE XLAS GENE CONTAINS TWO ADDITIONAL EXONS BESIDES THOSE
ENCODING GAS
3.1.2. THE XLAS GENE CONTAINS THREE ALTERNATIVE TRANSCRIPTION INITIATION
SITES 103
3.1.3. THE XLAS GENE CONTAINS TWO ALTERNATIVE TRANSCRIPTIONAL
TERMINATION SITES 104
3.1.4. ALTERNATIVE SPLICING OF THE PRE-MRNAS TRANSCRIBED FROM THE XLAS
GENE 105
3.1.5. THE SECOND OPEN READING FRAMES OF THE XL-EXON AND THE XL2-EXON
105
3.1.6. MULTIPLE MRNA AND PROTEIN PRODUCTS OF THE XLAS GENE 108
3.1.7. MECHANISM OF GENERATION OF THE MULTIPLE MRNA PRODUCTS FROM
THE XLAS GENE: THE SCAN-SELECTION-SPLICING MODEL 109
3.1.8. DIFFERENT SUBCELLULAR LOCALIZATION OF THE XLAS GENE PRODUCTS 110
3.1.9. IMPRINTING OF THE XLAS GENE 110
3.1.10. POSSIBLE FUNCTIONS OF XLAS 111
3.2. XL2, A LUMINAL GOLGI RESIDENT PROTEIN IN NEUROENDOCRINE CELLS 112
3.2.1 XL2 IS A CONSERVED PROTEIN IN DIFFERENT SPECIES 112
3.2.2 XL2 IS A NEUROENDOCRINE-SPECIFIC PROTEIN 113
3.2.3. SUBCELLULAR LOCALIZATION OF THE XL2 PROTEIN IS MAINLY IN THE
EARLY GOLGI 114
III
CONTENTS
3.2.4. XL2 IS LOCATED IN THE LUMEN OF THE GOLGI 116
3.2.5. XL2. A GOLGI RESIDENT PROTEIN OR A GRANIN PRECURSOR? 116
3.2.6. XL2 CONTAINS A GOLGI LOCALIZATION SIGNAL AND A MEMBRANE ANCHOR
118
3.2.7. TRANSLOCATION OF XL2 INTO THE LUMEN OF THE SECRETORY PATHWAY
OCCURS
AT THE LEVEL OF THE HR , 1-6
3.2.8. FUNCTIONS OF THE XL2 PROTEIN - A SPECULATION 122
3.3. CHOLESTEROL IS AN ESSENTIAL COMPONENT FOR SECRETORY VESICLE
BIOGENESIS
FROM THE TGN * -6
3.4. OUTLOOK 128
4. MATERIALS AND METHODS
130
4.1. MATERIALS 130
4.1.1. CHEMICALS 130
4.1.2. PLASTIC WARE, FILMS AND FILTERS 131
4.1.3. TECHNICAL EQUIPMENT 132
4.1.4. VECTORS AND BACTERIAL STRAINS 133
4.1.4.1 VECTORS 133
4.1.4.2 BACTERIAL STRAINS 134
4.1.5. OLIGONUCLEOTIDES 135
4.1.6. DNA LIBRARIES 137
4.1.7. CELLS IN CULTURE 137
4.1.8. ANTIBODIES 138
4.2. BUFFERS AND STOCK SOLUTIONS 140
4.2.1. BUFFERS AND SOLUTIONS FOR BACTERIAL CULTURE 140
4.2.2. BUFFERS AND SOLUTIONS FOR DNA PREPARATION AND ANALYSIS 140
4.2.3. BUFFERS AND SOLUTIONS FOR RNA-PREPARATION AND NORTHERN BLOT
ANALYSIS 141
4.2.4. SOLUTIONS AND BUFFERS FOR BIOCHEMISTRY 142
4.2.5. SOLUTIONS FOR CELL BIOLOGY 142
4.3. MOLECULAR BIOLOGY 143
4.3.1. DNA PREPARATION 143
4.3.1.1 PLASMID MINI PREPARATION 143
4.3.1.2 PLASMID MIDI PREPARATION AND PURIFICATION 143
4.3.1.3 GENOMIC DNA PREPARATION FROM EUKARYOTIC CELLS 144
4.3.1.4 LAMBDA PHAGE AMPLIFICATION 145
4.3.1.5 LAMBDA PHAGE DNA PREPARATION 145
_CONTENTS
4.3.1.6 PAC DNA PREPARATION 146
4.3.2. RNA PREPARATION 146
4.3.2.1 TOTAL RNA PREPARATION 146
4.3.2.2 MRNA PREPARATION 146
4.3.3. POLYMERASE CHAIN REACTION (PCR) 147
4.3.4. PRECIPITATION AND PHENOL EXTRACTION OF NUCLEIC ACIDS 147
4.3.3. RESTRICTION ENZYME DIGESTION 148
4.3.6. AGAROSE GEL ELECTROPHORESIS OF DNA 149
4.3.7. RECOVERING OF DNA FRAGMENTS FROM AGAROSE GELS 149
4.3.8. LIGATION OF DNA FRAGMENTS 149
4.3.9. PREPARATION OF COMPETENT BACTERIA FOR TRANSFORMATION 149
4.3.10. TRANSFORMATION OF BACTERIA 150
4.3.11. COLONY TRANSFER ONTO MEMBRANE FILTERS 150
4.3.12. [A32P]-LABELING OF DNA PROBES 151
4.3.13. SOUTHERN BLOT ANALYSIS 151
4.3.14. RNA GEL ELECTROPHORESIS AND NORTHERN BLOTS 151
4.3.15. HYBRIDIZATION OF DNA OR RNA BINDING FILTERS 152
4.3.16. DNA SEQUENCING 153
4.3.17. PRIMER EXTENSION 153
4.3.18. RNASC PROTECTION 154
4.3.19. IN VITRO TRANSCRIPTION 155
4.3.20. IN VITRO TRANSLATION 155
4.3.21. DNA NICK TRANSLATION 155
4.3.22. FLUORESCENCE IN SITU HYBRIDIZATION (FISH) OF CHROMOSOMES 156
4.3.23. BIOCOMPUTING
156
4.4. BIOCHEMISTRY J-57
4.4.1. DETERMINATION OF PROTEIN CONCENTRATION 157
4.4.2. PROTEIN PRECIPITATION 157
4.4.3. SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) 158
4.4.4. WESTERN BLOTTING 158
4.4.5. STRIPPING OF NITROCELLULOSE MEMBRANES 159
4.4.6. ENDOGLYCOSIDASE DIGESTION 159
4.4.7. IMMUNOPRECIPITATION 159
4.4.8. GST-FUSION PROTEIN PREPARATION 160
4.4.9. IMMUNIZATION OF RABBITS WITH FUSION PROTEINS 160
4.4.10. AFFINITY PURIFICATION OF POLYCLONAL ANTIBODIES 161
4.4.11. IMMUNIZATION OF RABBITS WITH SYNTHETIC PEPTIDES 161
4.4.12. ELISA 162
V
CONTENTS
4.5. CELL BIOLOGY 162
4.5.1. CELL CULTURE 162
4.5.2. CELL TRANSFECTION 162
4.5.3. SELECTION OF STABLY TRANSFECTED CELLS 163
4.5.4. POST-NUCLEAR SUPERNATANT (PNS) PREPARATION , 164
4.5.4.1 PNS PREPARATION FROM CULTURED CELLS 164
4.5.4.2 TISSUE HOMOGENIZATION 164
4.5.4.3. MEMBRANE PREPARATION 164
4.5.5. MEMBRANE CARBONATE TREATMENT 165
4.5.6. TRITON X-114 PHASE PARTITION 165
4.5.7. HEAT STABILITY TEST OF PROTEINS 166
4.5.8. PROTEINASE K PROTECTION ANALYSIS 166
4.5.9. PULSE-CHASE LABELING OF ATT20 CELLS AND SUBSEQUENT SUBCELLULAR
FRACTIONATION BY SUCROSE GRADIENTS 166
4.5.10. FRACTIONATION OF SUBCELLULAR COMPARTMENTS OF BOVINE ADRENAL
MEDULLA 167
4.5.11. CHARACTERIZATION OF THE XL2 PROTEIN IN BOVINE ADRENAL MEDULLA
SUBCELLULAR FRACTIONS 167
4.5.12. IMMUNOFLUORESCENCE 168
4.5.12.1 IMMUNOFLUORESCENCE OF CULTURED CELLS 168
4.5.12.2 IMMUNOFLUORESCENCE OF TISSUES 169
4.5.13. BREFELDIN A TREATMENT OF ATT20 CELLS 169
4.5.14. CYCLOHEXIMIDE TREATMENT OF ATT20 CELLS 169
4.5.15. TEMPERATURE TREATMENT OF ATT20 CELLS 170
4.5.16. DETERMINATION OF THE XL2 GOLGI LOCALIZATION SIGNAL 170
4.5.17. ELECTRON MICROSCOPY 170
4.5.18. MANIPULATION OF THE CELLULAR CHOLESTEROL LEVEL 171
5. REFERENCES
172
6. ABBREVIATIONS 193
7. APPENDIX INFORMATION OF XLAS CDNA AND GENOMIC CLONES
195
8.
SUMMARY
196
V |
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| indexdate | 2024-10-09T18:14:31Z |
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| language | German |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-008668917 |
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| spelling | Wang, Yanzhuang Verfasser aut XL2 - a novel neuroendocrine-specific luminal Golgi-resident protein derived from the XL alpha s/G alpha s gene by Yanzhuang Wang 1999 VI, 197 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Heidelberg, Univ., Diss., 1999 GTP-bindende Proteine (DE-588)4248354-2 gnd rswk-swf Golgi-Apparat (DE-588)4192795-3 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Golgi-Apparat (DE-588)4192795-3 s GTP-bindende Proteine (DE-588)4248354-2 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008668917&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Wang, Yanzhuang XL2 - a novel neuroendocrine-specific luminal Golgi-resident protein derived from the XL alpha s/G alpha s gene GTP-bindende Proteine (DE-588)4248354-2 gnd Golgi-Apparat (DE-588)4192795-3 gnd |
| subject_GND | (DE-588)4248354-2 (DE-588)4192795-3 (DE-588)4113937-9 |
| title | XL2 - a novel neuroendocrine-specific luminal Golgi-resident protein derived from the XL alpha s/G alpha s gene |
| title_auth | XL2 - a novel neuroendocrine-specific luminal Golgi-resident protein derived from the XL alpha s/G alpha s gene |
| title_exact_search | XL2 - a novel neuroendocrine-specific luminal Golgi-resident protein derived from the XL alpha s/G alpha s gene |
| title_full | XL2 - a novel neuroendocrine-specific luminal Golgi-resident protein derived from the XL alpha s/G alpha s gene by Yanzhuang Wang |
| title_fullStr | XL2 - a novel neuroendocrine-specific luminal Golgi-resident protein derived from the XL alpha s/G alpha s gene by Yanzhuang Wang |
| title_full_unstemmed | XL2 - a novel neuroendocrine-specific luminal Golgi-resident protein derived from the XL alpha s/G alpha s gene by Yanzhuang Wang |
| title_short | XL2 - a novel neuroendocrine-specific luminal Golgi-resident protein derived from the XL alpha s/G alpha s gene |
| title_sort | xl2 a novel neuroendocrine specific luminal golgi resident protein derived from the xl alpha s g alpha s gene |
| topic | GTP-bindende Proteine (DE-588)4248354-2 gnd Golgi-Apparat (DE-588)4192795-3 gnd |
| topic_facet | GTP-bindende Proteine Golgi-Apparat Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008668917&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT wangyanzhuang xl2anovelneuroendocrinespecificluminalgolgiresidentproteinderivedfromthexlalphasgalphasgene |