Biochemical characterization of GAS2L3, a target gene of the DREAM complex: = Biochemische Charakterisierung von GAS2L3, ein Zielgen des DREAM Komplex
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2014
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| Beschreibung: | V, 125 Blätter Illustrationen, Diagramme |
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| 245 | 1 | 0 | |a Biochemical characterization of GAS2L3, a target gene of the DREAM complex |b = Biochemische Charakterisierung von GAS2L3, ein Zielgen des DREAM Komplex |c vorgelegt von Marc Fackler aus Memmingen |
| 246 | 1 | 1 | |a Biochemische Charakterisierung von GAS2L3, ein Zielgen des DREAM Komplex |
| 264 | 1 | |a Würzburg |c 2014 | |
| 300 | |a V, 125 Blätter |b Illustrationen, Diagramme | ||
| 336 | |b txt |2 rdacontent | ||
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| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Julius-Maximilians-Universität Würzburg |d 2014 | ||
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Datensatz im Suchindex
| _version_ | 1804176717043990528 |
|---|---|
| adam_text | BIOCHEMICAL CHARACTERIZATION
OF GAS2L3,
A TARGET GENE OF THE
DREAM COMPLEX
DISSERTATION ZUR ERLANGUNG DES
NATURWISSENSCHAFTLICHEN DOKTORGRADES
DER BAYERISCHEN JULIUS-MAXIMILIANS-UNIVERSITAET WUERZBURG
VORGELEGT VON
MARC FACKLER
AUS
MEMMINGEN
WUERZBURG, 2014
TABLE OF CONTENTS
1.
INTRODUCTION................................................................................................................................................
1
1.1. MAMMALIAN CELL CYCLE AND ITS
REGULATION....................................................................................
1
1.2. DREAM COM
PLEX.............................................................................................................................
4
1.3. GROWTH ARREST SPECIFIC 2 PROTEIN
FAMILY......................................................................................
6
1.3.1. G A S 2
...............................................................................................................................................8
1.3.2. G A S 2L
1..........................................................................................................................................8
1.3.3. G A S
2L2..........................................................................................................................................9
1.3.4. G A S
2L3.........................................................................
9
1.4. CHROMOSOMAL PASSENGER COMPLEX (C P C
)...............................................................................9
1.4.1. BOREALIN
.......
.
................................................................................................................................
10
1.4.2.
SURVIVIN.........................................................................................................................................10
1.4.3. AURORA
B........................................................................................................................................11
1.4.4. CPC LOCALIZATION DURING INTERPHASE, MITOSIS AND CYTOKINESIS
........................................
13
1.4.5. FUNCTIONS OF THE CPC DURING MITOSIS AND
CYTOKINESIS.....................................................14
1.4.5.1. REGULATION OF KINETOCHORE-MICROTUBULE ATTACHMENTS
.....................................................
14
1.4.5.2. REGULATION OF
ABSCISSION......................................................................................................
15
1.5. AIM OF THE
PROJECT.........................................................................................................................15
2. MATERIALS AND
METHODS.........................................................................................................................16
2.1. M
ATERIALS..........................................................................................................................................16
2.1.1. CHEMICAL STOCKS &
REAGENTS....................................................................................................
16
2.1.2. ENZYMES
...........................................................
17
2.1.3.
ANTIBIOTICS....................................................................................................................................
18
2.1.4. BUFFERS &
SOLUTIONS....................................................................................................................
18
2.1.4.1. GENERAL
BUFFERS........................................................................................................................18
2.1.4.2. BUFFERS FOR WHOLE CELL
LYSATES...............................................................................................
20
2.1.4.3. BUFFERS FOR TANDEM AFFINITY PURIFICATION (T A P
)...................................................................21
2.1.4.4. BUFFERS FOR
IMMUNOBLOTTING...................................................................................................21
2.1.4.5. BUFFERS FOR
IMMUNOFLUORESCENCE........................................................................................
23
2.1.4.6. BUFFERS FOR RECOMBINANT PROTEIN PURIFICATION
...................
23
2.1.4.7. BUFFERS FOR KINASE
ASSAYS.....................................................................................................24
2.1.4.8. BUFFERS FOR GENOMIC DNA EXTRACTION
..................................................................................
24
2.1.4.9. BUFFERS FOR ANTIBODY
PURIFICATION.........................................................................................
25
2.1.4.10. STAINING
SOLUTIONS................................................................................................................
25
2.1.5.
PLASMIDS....................................................................................................................................26
2.1.5.1. PLASMIDS FOR BACTERIAL
OVEREXPRESSION............................................................................26
2.1.5.2. PLASMIDS FOR TRANSIENT OVEREXPRESSION IN MAMMALIAN CELLS
.........................................
27
2.1.5.3. PLASMIDS FOR ESTABLISHMENT OF STABLE CELL LINES
...............................................................
27
2.1.5.4. PLASMIDS FOR RETROVIRAL INFECTION OF M E F S
..........................................................................27
2.1.6.
ANTIBODIES....................................................................................................................................
28
2.1.6.1. PRIMARY
ANTIBODIES..................................................................................................................28
2.1.6.2. SECONDARY
ANTIBODIES............................................................................................................
29
2.1.7. PRIMER
SEQUENCES......................................................................................................................
29
2.1.7.1. PRIMERS FOR
CLONING.................................................................................................................29
2.1.7.2. PRIMERS FOR Q P C R
...................................................................................................................31
2.1.7.3. PRIMERS FOR
GENOTYPING.......................................................................................................
32
2.1.7.4. PRIMERS FOR LONG RANGE P C R
.................................................................................................33
2.1.8. SIRNA
SEQUENCES......................................................................................................................
33
2.1.9. TRANSFECTION
REAGENTS...............................................................................................................
34
2.1.10.
MARKERS......................................................................................................................................
34
2.1.11.
KITS..............................................................................................................................................
34
2.1.12.
BEADS..........................................................................................................................................34
2.1.13. ESCHERICHIA COII
STRAINS...........................................................................................................35
2.1.14. REAGENTS FOR STANDARD CELL
CULTURE......................................................................................
35
2.1.15. REAGENTS FOR SILAC CELL
CULTURE...........................................................................................35
2.1.16. CELL CULTURE MEDIUM & CELL
LINES...........................................................................................36
2.1.17. MOUSE
STRAINS...........................................................................................................................38
2.2.
METHODS.........................................................................................................................................39
2.2.1. CELL BIOLOGICAL M
ETHODS............................................................................................................39
2.2.1.1. PASSAGING OF
CELLS..................................................................................................................39
2.2.1.2. FREEZING AND THAWING OF
CELLS..............................................................................................
39
2.2.1.3. COUNTING OF
CELLS.....................................................................................................................
39
2.2.1.4. TRANSIENT
TRANSFECTION............................................................................................................39
2.2.1.5. GENERATION OF STABLE CELL
LINES.............................................................................................40
2.2.1.6. RECONSTITUTION
ASSAYS...........................................................................................................40
2.2.1.7. GENERATION OF MOUSE EMBRYONIC FIBROBLASTS (M E F S )
..................................................
41
2.2.1.8. PRODUCTION OF RETROVIRUS IN PLAT-E
CELLS..............................................................................41
2.2.1.9. INFECTION AND IMMORTALIZATION OF M E
FS..............................................................................41
2.2.1.10. INDUCTION OF CRE RECOMBINASE BY TAM OXIFEN
.................................................................
42
2.2.1.11. SYNCHRONIZATION OF C
ELLS.....................................................................................................42
2.2.1.12. PHARMACOLOGICAL INHIBITION OF THE C P C
..........................................................................
42
2.2.1.13. DETERMINATION OF CPC
ACTIVITY...........................................................................................42
2.2.1.14.
IMMUNOFLUORESCENCE....................................................................................
2.2.2. MOLECULAR BIOLOGICAL
METHODS.............................................................................
2.2.2.1. RNA
EXTRACTION...................................................................................................
2.2.2 2. REVERSE TRANSCRIPTION
.......................................................................................
2 2.2.3. QUANTITATIVE P C R
................................................................................................
2 .2 .2 A SMALL SCALE PREPARATION OF PLASMID DNA FROM
BACTERIA............................
2.2.2.5. LARGE SCALE PREPARATION OF PLASMID DNA FROM
BACTERIA............................
2.2.2 6. EXTRACTION OF GENOMIC DNA FROM TISSUE/CELLS AND GENOTYPING VIA
PCR
2 .2 .2 7 . EXTRACTION OF DNA FRAGMENTS FROM AGAROSE G E LS
.......................................
2.2.2.8. PURIFICATION OF PCR FRAGMENTS VIA COLUMN PURIFICATION
.............................
2.2.2.9. DETERMINATION OF NUCLEIC ACID CONCENTRATION
...............................................
2.2.2.10. AGAROSE GEL
ELECTROPHORESIS..........................................................................
2.2.2.11. RESTRICTION OF DNA WITH RESTRICTION ENDONUCLEASES
.................................
2.2.2.12. STANDARD CLONING PROCEDURE
2.2.2.13. PROOFREADING PCR USING PFU POLYMERASE
......................................
2.2.2.14. LONG RANGE P C R
...................................................................................
2.2.3. BIOCHEMICAL METHODS
...............................................................................
2.2.3.1. WHOLE CELL LYSATES
...................................................................................
2.2.3 2. DETERMINATION OF PROTEIN
CONCENTRATION..............................................
2.2.3.3.
IMMUNOPRECIPITATION...............................................................................
2.2.3.4. STABLE ISOTOPE LABELLING BY AMINO ACIDS IN CELL CULTURE (S IL A
C )...
2 2.3.5. TANDEM AFFINITY PURIFICATION OF HA-/STREP-/STREP-TAGGED
PROTEINS
2.2.3 6. SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS
........
2.2.3.7. NUPAGE BIS-TRIS GEL ELECTROPHORESIS
...................................................
2.2.3 6. COOMASSIE BLUE STAINING OF POLYACRYLAMIDE GELS
............................
2.2.3 9.
IMMUNOBLOTTING........................................................................................
2.2.3.10. TRANSFORMATION OF CHEMICALLY COMPETENT BACTERIA
........................
2.2.3.11. PREPARATION OF GLYCEROL STOCKS FROM BACTERIA
.................................
2.2.3.12. OVEREXPRESSION OF RECOMBINANT PROTEINS IN BACTERIA
...................
2.2.3.13. PURIFICATION OF RECOMBINANT GST FUSION PROTEINS FROM
BACTERIA..
2.2.3.14. ACTIN CO-SEDIMENTATION ASSAY
...........................................................
2.2.3.15. MICROTUBULE CO-SEDIMENTATION A S S A Y
...............................................
2.2.3.16. MICROTUBULE-F-ACTIN CROSSLINKING ASSAY
...........................................
2.2.3.17. MICROTUBULE BUNDLING
ASSAY................................................................
2.2.3.18. GST PULLDOWN A S S A Y
...........................................................................
2.2.3.19. HIS-TAG PULLDOWN
ASSAY......................................................................
42
43
43
43
44
45
45
45
46
46
46
46
47
47
47
48
48
48
49
49
49
49
50
51
51
51
51
52
52
52
53
53
53
54
54
54
2.2.3.20. KINASE
ASSAY.........................................................................................................................54
2.2.3.21. ANTIGEN AFFINITY PURIFICATION OF POLYCLONAL GAS2L3 ANTIBODY
.....................................
55
3.
RESULTS.....................................................................................................................................................
56
3.1. GAS2L3 IS REGULATED ON PROTEIN LEVEL DURING CELL
CYCLE.....................................................56
3.2. GAS2L3 IS IMPORTANT FOR PROPER
CYTOKINESIS........................................................................
56
3.3. GAS2L3 IS A CYTOSKELETON ASSOCIATED PROTEIN
.......................................................................
57
3.4. GAS2L3 INTERACTS WITH THE CHROMOSOMAL PASSENGER
COMPLEX..........................................63
3.5. GAS2L3 IS PHOSPHORYLATED BY CDK1 BUT NOT BY AURORA B
................................................
69
3.6. DEPLETION OF GAS2L3 INFLUENCES STABILITY AND ACTIVITY OF THE C P C
....,
..............................
70
3.7. LOCALIZATION OF GAS2L3 TO THE CONSTRICTION ZONE DEPENDS ON THE GAR
DOMAIN
..........
73
3.8. THE GAR DOMAIN IS NECESSARY FOR PROPER FUNCTION OF GAS2L3
........................................
74
3.9. IDENTIFICATION OF NEW INTERACTING PROTEINS OF G
AS2L3...........................................................75
3.10. ESTABLISHMENT OF AN IN VIVO SYSTEM TO STUDY GAS2L3 FUNCTION
........................................
79
4.
DISCUSSION..............................................................................................................................................
87
4.1. GAS2L3 IS REGULATED DURING CELL CYCLE
..................................................................................
87
4.2. GAS2L3 IS IMPORTANT FOR PROPER
CYTOKINESIS........................................................................
87
4.3. GAS2L3 IS A CYTOSKELETON ASSOCIATED
PROTEIN.......................................................................
87
4.4. GAS2L3 INTERACTS WITH THE CHROMOSOMAL PASSENGER
COMPLEX...........................................89
4.5. GAS2I3 IS PHOSPHORYLATED BY CDK1 BUT NOT BY AURORA B
..................................................90
4.6. DEPLETION OF GAS2L3 INFLUENCES STABILITY AND ACTIVITY OF THE C P C
..........................
.........
90
4.7. LOCALIZATION OF GAS2L3 TO THE CONSTRICTION ZONE DEPENDS ON THE GAR
DOMAIN
..........
91
4.8. THE GAR DOMAIN IS NECESSARY FOR PROPER FUNCTION OF GAS2L3
.........................................
92
4.9. IDENTIFICATION OF NEW INTERACTING PROTEINS OF G
AS2L3............................................................93
4.10. ESTABLISHMENT OF AN IN VIVO SYSTEM TO STUDY GAS2L3 FUNCTION
.........................................
94
4.11.
CONCLUSION....................................................................................................................................
96
5. SUM M
ARY................................................................................................................................................
98
6.
ZUSAMMENFASSUNG.........................................................................................................................
99
7.
REFERENCES...........................................................................................................................................100
8.
APPENDIX................................................................................................................................................119
8.1. LIST OF
FIGURES...............................................................................................................................
119
8.2.
ABBREVIATIONS...............................................................................................................................
121
8.3. CURRICULUM
VITAE...........................................................................................................................123
8.4.
PUBLICATIONS..................................................................................................................................124
8.5. EIDESSTATTLICHE
ERKLAERUNG...........................................................................................................125
|
| any_adam_object | 1 |
| author | Fackler, Marc 1983- |
| author_GND | (DE-588)1117094146 |
| author_facet | Fackler, Marc 1983- |
| author_role | aut |
| author_sort | Fackler, Marc 1983- |
| author_variant | m f mf |
| building | Verbundindex |
| bvnumber | BV043846821 |
| collection | ebook |
| ctrlnum | (OCoLC)962085094 (DE-599)BVBBV043846821 |
| dewey-full | 570 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 570 - Biology |
| dewey-raw | 570 |
| dewey-search | 570 |
| dewey-sort | 3570 |
| dewey-tens | 570 - Biology |
| discipline | Biologie |
| format | Thesis Book |
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| genre_facet | Hochschulschrift |
| id | DE-604.BV043846821 |
| illustrated | Illustrated |
| indexdate | 2024-07-10T07:36:37Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-029257247 |
| oclc_num | 962085094 |
| open_access_boolean | 1 |
| owner | DE-384 DE-473 DE-BY-UBG DE-703 DE-1051 DE-824 DE-29 DE-12 DE-91 DE-BY-TUM DE-19 DE-BY-UBM DE-1049 DE-92 DE-739 DE-898 DE-BY-UBR DE-355 DE-BY-UBR DE-706 DE-20 DE-1102 DE-860 DE-2174 |
| owner_facet | DE-384 DE-473 DE-BY-UBG DE-703 DE-1051 DE-824 DE-29 DE-12 DE-91 DE-BY-TUM DE-19 DE-BY-UBM DE-1049 DE-92 DE-739 DE-898 DE-BY-UBR DE-355 DE-BY-UBR DE-706 DE-20 DE-1102 DE-860 DE-2174 |
| physical | V, 125 Blätter Illustrationen, Diagramme |
| psigel | ebook |
| publishDate | 2014 |
| publishDateSearch | 2014 |
| publishDateSort | 2014 |
| record_format | marc |
| spelling | Fackler, Marc 1983- Verfasser (DE-588)1117094146 aut Biochemical characterization of GAS2L3, a target gene of the DREAM complex = Biochemische Charakterisierung von GAS2L3, ein Zielgen des DREAM Komplex vorgelegt von Marc Fackler aus Memmingen Biochemische Charakterisierung von GAS2L3, ein Zielgen des DREAM Komplex Würzburg 2014 V, 125 Blätter Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier Dissertation Julius-Maximilians-Universität Würzburg 2014 Zusammenfassung in deutscher und englischer Sprache Zellzyklus (DE-588)4129960-7 gnd rswk-swf Molekulargenetik (DE-588)4039987-4 gnd rswk-swf Zellteilung (DE-588)4190668-8 gnd rswk-swf Regulation (DE-588)4049075-0 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Zellzyklus (DE-588)4129960-7 s Zellteilung (DE-588)4190668-8 s Regulation (DE-588)4049075-0 s Molekulargenetik (DE-588)4039987-4 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-103394 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-103394 Resolving-System kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029257247&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Fackler, Marc 1983- Biochemical characterization of GAS2L3, a target gene of the DREAM complex = Biochemische Charakterisierung von GAS2L3, ein Zielgen des DREAM Komplex Zellzyklus (DE-588)4129960-7 gnd Molekulargenetik (DE-588)4039987-4 gnd Zellteilung (DE-588)4190668-8 gnd Regulation (DE-588)4049075-0 gnd |
| subject_GND | (DE-588)4129960-7 (DE-588)4039987-4 (DE-588)4190668-8 (DE-588)4049075-0 (DE-588)4113937-9 |
| title | Biochemical characterization of GAS2L3, a target gene of the DREAM complex = Biochemische Charakterisierung von GAS2L3, ein Zielgen des DREAM Komplex |
| title_alt | Biochemische Charakterisierung von GAS2L3, ein Zielgen des DREAM Komplex |
| title_auth | Biochemical characterization of GAS2L3, a target gene of the DREAM complex = Biochemische Charakterisierung von GAS2L3, ein Zielgen des DREAM Komplex |
| title_exact_search | Biochemical characterization of GAS2L3, a target gene of the DREAM complex = Biochemische Charakterisierung von GAS2L3, ein Zielgen des DREAM Komplex |
| title_full | Biochemical characterization of GAS2L3, a target gene of the DREAM complex = Biochemische Charakterisierung von GAS2L3, ein Zielgen des DREAM Komplex vorgelegt von Marc Fackler aus Memmingen |
| title_fullStr | Biochemical characterization of GAS2L3, a target gene of the DREAM complex = Biochemische Charakterisierung von GAS2L3, ein Zielgen des DREAM Komplex vorgelegt von Marc Fackler aus Memmingen |
| title_full_unstemmed | Biochemical characterization of GAS2L3, a target gene of the DREAM complex = Biochemische Charakterisierung von GAS2L3, ein Zielgen des DREAM Komplex vorgelegt von Marc Fackler aus Memmingen |
| title_short | Biochemical characterization of GAS2L3, a target gene of the DREAM complex |
| title_sort | biochemical characterization of gas2l3 a target gene of the dream complex biochemische charakterisierung von gas2l3 ein zielgen des dream komplex |
| title_sub | = Biochemische Charakterisierung von GAS2L3, ein Zielgen des DREAM Komplex |
| topic | Zellzyklus (DE-588)4129960-7 gnd Molekulargenetik (DE-588)4039987-4 gnd Zellteilung (DE-588)4190668-8 gnd Regulation (DE-588)4049075-0 gnd |
| topic_facet | Zellzyklus Molekulargenetik Zellteilung Regulation Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-103394 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029257247&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT facklermarc biochemicalcharacterizationofgas2l3atargetgeneofthedreamcomplexbiochemischecharakterisierungvongas2l3einzielgendesdreamkomplex AT facklermarc biochemischecharakterisierungvongas2l3einzielgendesdreamkomplex |