Characterization of telomere protein complexes in Trypanosoma brucei: = Charakterisierung von telomerischen Proteinkomplexen in Trypanosoma brucei
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Würzburg
2017
|
| Schlagworte: | |
| Online-Zugang: | kostenfrei http://d-nb.info/1137835990/34 Inhaltsverzeichnis |
| Beschreibung: | 114 Seiten Illustrationen |
| Format: | Langzeitarchivierung gewährleistet, LZA |
Internformat
MARC
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| 100 | 1 | |a Reis, Helena |e Verfasser |0 (DE-588)1138642037 |4 aut | |
| 245 | 1 | 0 | |a Characterization of telomere protein complexes in Trypanosoma brucei |b = Charakterisierung von telomerischen Proteinkomplexen in Trypanosoma brucei |c vorgelegt von Helena Reis aus Pawlodar (Kasachstan) |
| 246 | 1 | 1 | |a Charakterisierung von telomerischen Proteinkomplexen in Trypanosoma brucei |
| 264 | 1 | |a Würzburg |c 2017 | |
| 300 | |a 114 Seiten |b Illustrationen | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Julius-Maximilians-Universität Würzburg |d 2017 | ||
| 538 | |a Langzeitarchivierung gewährleistet, LZA | ||
| 650 | 0 | 7 | |a Telomer |g Molekulargenetik |0 (DE-588)4310952-4 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Trypanosoma brucei |0 (DE-588)4186350-1 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Genexpression |0 (DE-588)4020136-3 |2 gnd |9 rswk-swf |
| 653 | |a telomere-binding protein | ||
| 653 | |a chromatin remodeling | ||
| 653 | |a developmental differentiation | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 689 | 0 | 0 | |a Trypanosoma brucei |0 (DE-588)4186350-1 |D s |
| 689 | 0 | 1 | |a Telomer |g Molekulargenetik |0 (DE-588)4310952-4 |D s |
| 689 | 0 | 2 | |a Genexpression |0 (DE-588)4020136-3 |D s |
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| 912 | |a ebook | ||
| 999 | |a oai:aleph.bib-bvb.de:BVB01-029857563 | ||
Datensatz im Suchindex
| _version_ | 1804177777451073536 |
|---|---|
| adam_text | TABLE OF CONTENTS
ACKNOWLEDGMENTS.................................................................................................................................
4
ZUSAMMENFASSUNG.................................................................................................................................
5
SUMMARY......................................... 7
ABBREVIATIONS...........................................................................................................................................9
FIGURES AND TABLES
INDEX...................................................................................................................
14
1
INTRODUCTION....................................................................................................................................17
1.1 TELOMERE
BIOLOGY.......................................................................................................................
17
1.1.1 MAJOR MILESTONES IN TELOMERE
RESEARCH............................................................................
17
1.1.2 TELOMERE ORGANIZATION AND
FUNCTION................................................................................
18
1.1.3 YEAST AS A MODEL ORGANISM FOR TELOMERE
STUDIES...........................................................20
1.1.4 PATHOGENS AND TELOMERE-DEPENDENT REGULATION OF CONTINGENCY GENES
.......................
22
1.2 TRYPANOSOMA
BRUCEI..................................................................................................................24
1.2.1 AFRICAN
TRYPANOSOMIASIS...................................................................................................24
1.2.2 LIFE CYCLE OF
T. BRUCEI
.......................................................................................................
25
1.2.3 GENOME ORGANIZATION AND GENE EXPRESSION REGULATION IN
T. BRUCEI
............................
27
1.2.4 ANTIGENIC VARIATION IN BLOODSTREAM FORM TRYPANOSOMES
.................................................
29
1.2.5 DEVELOPMENTAL^ REGULATED VSG SILENCING MECHANISMS
...............................................
38
1.3 AIM OF THIS
STUDY........................................................................................................................40
2 MATERIALS AND
METHODS..............................................................................................................
41
2.1 BACTERIAL CULTURE AND DNA
PLASMIDS.......................................................................................
41
2.1.1 BACTERIAL STRAINS
.............................
41
2.1.2 GROWTH OF
E. COLI.
...............................................................................................................
41
2.1.3 TRANSFORMATION OF
E. COLI
............................................. 41
2.1.4 DNA EXTRACTION FROM E.
COLI.
..............................................................................................
41
2.1.5 PLASMIDS USED AND GENERATED IN THIS
STUDY...................................................................
42
2.2 TRYPANOSOME CULTURE AND
ANALYSES........................................................................................
44
2.2.1
TRYPANOSOMA BRUCEI BRUCEI
STRAINS.................................................................................44
2.2.2 TRYPANOSOME
GROWTH........................................................................................
45
2.2.3 STABILATE PREPARATION AND
RETRIEVAL..................................................................................46
2.2.4 STABLE TRANSFORMATION OF TRYPANOSOMES
..........................................................................
46
2.2.5 TRANSIENT TRANSFORMATION OF BSF
TRYPANOSOMES..............................................................46
2.2.6 DIFFERENTIATION OF MONOMORPHIC BLOODSTREAM FORM TO PROCYCLIC FORM
TRYPANOSOMES...................................................................................................................
47
2.2.7 ISOLATION OF GENOMIC DNA OF
T. BRUCEI
.............................................................................47
2.2.8 ISOLATION OF RNA FROM
T. BRUCEI
AND CDNA SYNTHESIS
....................................................
48
2.2.9 IMMUNOFLUORESCENCE
ANALYSIS..........................................................................................48
2.2.10 DUAL-LUCIFERASE
ASSAY......................................................................................................
49
2.3 DNA M
ETHODS.............................................................................................................................49
2.3.1 POLYMERASE CHAIN REACTION
(PCR)..................................................................................
49
2.3.2 SITE-DIRECTED
MUTAGENESIS................................................................................................51
2.3.3 PCR
PURIFICATION.................................................................................................................51
2.3.4 RESTRICTION
DIGEST...............................................................................................................51
2.3.5 ISOPROPANOL PRECIPITATION OF D N A
....................................................................................
51
2.3.6 AGAROSE GEL ELECTROPHORESIS AND GEL
EXTRACTION.............................................................51
2.3.7 MEASUREMENT OF DNA
CONCENTRATION................................................................................52
2.3.8
LIGATION...............................................................................................................................52
2.3.9 DNA
SEQUENCING................................................................................................................52
2.3.10 TELOMERE AND VSG SOUTHERN B LO
T....................................................................................
52
2.4 PROTEIN
METHODS.........................................................................................................................
53
2.4.1 TELOMERE
PULL-DOWN..........................................................................................................
53
2.4.2
CO-LMMUNOPRECIPITATION...................................................................................................
54
2.4.3 MASS SPECTROMETRY AND DATA
ANALYSIS.............................................................................54
2.4.4 PREPARATION OF CELL LYSATES FOR SDS PAGE
............................
55
2.4.5 SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS
(SDS-PAGE)
.................
55
2.4.6 COOMASSIE
STAINING............................................................................................................55
2.4.7 WESTERN
BLOT.......................................................................................................................
55
2.4.8 ELECTROMOBILITY SHIFT ASSAY
(EMSA)..................................................................................56
2.4.9 RECOMBINANT PROTEIN
EXPRESSION.....................................................................................
56
2.4.10 ANTIBODY
PRODUCTION.........................................................................................................
57
3
RESULTS...............................................................................................................................................
58
3.1 IDENTIFICATION OF NOVEL TELOMERE-BINDING PROTEINS IN
TRYPANOSOMA BRUCEI.
...........................
58
3.2 BIOINFORMATIC ANALYSIS OF IDENTIFIED POTENTIAL TELOMERE-BINDING
PROTEINS
..............................
61
3.3 TOOLS FOR TB927.11.9870 (TELBPI)
CHARACTERIZATION...............................................................65
3.3.1 PRODUCTION OF TELBPI AND TBTRF MONOCLONAL
ANTIBODIES.............................................65
3.3.2 GENERATION OF TELBPI RNAI CELL LINES IN BOTH LIFE CYCLE STAGES OF
THE PARASITE
............
66
3.3.3 GENERATION OF TELBPI BSF AND PCF KNOCKOUT MUTANTS
.................................................
66
3.3.4 OVEREXPRESSION OF TELBPI REVEALS TELBPI PROCESSING INTO SMALLER
ISOFORMS
.............
67
3.3.5 ANALYSIS OF TELBPI
ISOFORMS..............................................................................................68
3.4 TELBPI IS A COMPONENT OF THE
T. BRUCEI
TELOMERE PROTEIN COMPLEX
......................................
74
3.5 RECOMBINANT TELBPI DOES NOT INTERACT DIRECTLY WITH TELOMERIC DNA
....................................
78
3.6 TELBPI SEEMS TO BE DISPENSABLE FOR TELOMERE MAINTENANCE IN BSF CE
LLS...........................79
3.7 EXPRESSION OF TELBPI IS STAGE-SPECIFICALLY
REGULATED.............................................................81
3.8 TELOMERE PROTEIN COMPLEX COMPOSITION IS STAGE-SPECIFICALLY REGULATED
..............................
81
3.9 TELBPI REGULATES BES SILENCING KINETICS DURING DEVELOPMENTAL
DIFFERENTIATION OF BSF
TO PCF CELLS
..........................
83
4 DISCUSSION
88
4.1 BIOCHEMICAL APPROACH TO IDENTIFY NOVEL TELOMERE-BINDING PROTEINS IN
7.
BRUCEI
....................
88
4.2 DISTINCT TELOMERE-BINDING PROTEIN COMPLEXES ARE PRESENT IN 7.
BRUCEI
.................................
89
4.3 STAGE-SPECIFIC REGULATION OF TELOMERE PROTEIN COMPLEXES IN
TRYPANOSOMES
........................
90
4.4 TELBPI IS A NOVEL TELOMERE-BINDING PROTEIN AND MIGHT FUNCTION
INDEPENDENTLY FROM
TBTRF, TBTIF2 AND
TBRAPI.................................................................................
91
4.5 TELBPI IS PROCESSED INTO SMALLER
ISOFORMS..............................................................................
92
4.6 TELBPI FINE TUNES DEVELOPMENTAL SILENCING OF THE ACTIVE BBS
..............................................
93
4.7 CONCLUSION AND FUTURE
PERSPECTIVE..........................................................................................94
5
REFERENCES........................................................................................................................................96
6 APPENDIX
..........
.................................................................................................................................
110
6.1 SUPPLEMENTARY
FIGURES............................................................................................................110
6.2 SUPPLEMENTARY
TABLES..............................................................................................................113
|
| any_adam_object | 1 |
| author | Reis, Helena |
| author_GND | (DE-588)1138642037 |
| author_facet | Reis, Helena |
| author_role | aut |
| author_sort | Reis, Helena |
| author_variant | h r hr |
| building | Verbundindex |
| bvnumber | BV044456777 |
| collection | ebook |
| ctrlnum | (OCoLC)1159311207 (DE-599)DNB1137835990 |
| dewey-full | 590 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 590 - Animals |
| dewey-raw | 590 |
| dewey-search | 590 |
| dewey-sort | 3590 |
| dewey-tens | 590 - Animals |
| discipline | Biologie |
| format | Thesis Book |
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| spelling | Reis, Helena Verfasser (DE-588)1138642037 aut Characterization of telomere protein complexes in Trypanosoma brucei = Charakterisierung von telomerischen Proteinkomplexen in Trypanosoma brucei vorgelegt von Helena Reis aus Pawlodar (Kasachstan) Charakterisierung von telomerischen Proteinkomplexen in Trypanosoma brucei Würzburg 2017 114 Seiten Illustrationen txt rdacontent n rdamedia nc rdacarrier Dissertation Julius-Maximilians-Universität Würzburg 2017 Langzeitarchivierung gewährleistet, LZA Telomer Molekulargenetik (DE-588)4310952-4 gnd rswk-swf Trypanosoma brucei (DE-588)4186350-1 gnd rswk-swf Genexpression (DE-588)4020136-3 gnd rswk-swf telomere-binding protein chromatin remodeling developmental differentiation (DE-588)4113937-9 Hochschulschrift gnd-content Trypanosoma brucei (DE-588)4186350-1 s Telomer Molekulargenetik (DE-588)4310952-4 s Genexpression (DE-588)4020136-3 s DE-604 Erscheint auch als Online-Ausgabe, PDF urn:nbn:de:bvb:20-opus-151323 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-151323 Resolving-System kostenfrei Volltext http://d-nb.info/1137835990/34 Langzeitarchivierung Nationalbibliothek DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029857563&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Reis, Helena Characterization of telomere protein complexes in Trypanosoma brucei = Charakterisierung von telomerischen Proteinkomplexen in Trypanosoma brucei Telomer Molekulargenetik (DE-588)4310952-4 gnd Trypanosoma brucei (DE-588)4186350-1 gnd Genexpression (DE-588)4020136-3 gnd |
| subject_GND | (DE-588)4310952-4 (DE-588)4186350-1 (DE-588)4020136-3 (DE-588)4113937-9 |
| title | Characterization of telomere protein complexes in Trypanosoma brucei = Charakterisierung von telomerischen Proteinkomplexen in Trypanosoma brucei |
| title_alt | Charakterisierung von telomerischen Proteinkomplexen in Trypanosoma brucei |
| title_auth | Characterization of telomere protein complexes in Trypanosoma brucei = Charakterisierung von telomerischen Proteinkomplexen in Trypanosoma brucei |
| title_exact_search | Characterization of telomere protein complexes in Trypanosoma brucei = Charakterisierung von telomerischen Proteinkomplexen in Trypanosoma brucei |
| title_full | Characterization of telomere protein complexes in Trypanosoma brucei = Charakterisierung von telomerischen Proteinkomplexen in Trypanosoma brucei vorgelegt von Helena Reis aus Pawlodar (Kasachstan) |
| title_fullStr | Characterization of telomere protein complexes in Trypanosoma brucei = Charakterisierung von telomerischen Proteinkomplexen in Trypanosoma brucei vorgelegt von Helena Reis aus Pawlodar (Kasachstan) |
| title_full_unstemmed | Characterization of telomere protein complexes in Trypanosoma brucei = Charakterisierung von telomerischen Proteinkomplexen in Trypanosoma brucei vorgelegt von Helena Reis aus Pawlodar (Kasachstan) |
| title_short | Characterization of telomere protein complexes in Trypanosoma brucei |
| title_sort | characterization of telomere protein complexes in trypanosoma brucei charakterisierung von telomerischen proteinkomplexen in trypanosoma brucei |
| title_sub | = Charakterisierung von telomerischen Proteinkomplexen in Trypanosoma brucei |
| topic | Telomer Molekulargenetik (DE-588)4310952-4 gnd Trypanosoma brucei (DE-588)4186350-1 gnd Genexpression (DE-588)4020136-3 gnd |
| topic_facet | Telomer Molekulargenetik Trypanosoma brucei Genexpression Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-151323 http://d-nb.info/1137835990/34 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029857563&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT reishelena characterizationoftelomereproteincomplexesintrypanosomabruceicharakterisierungvontelomerischenproteinkomplexenintrypanosomabrucei AT reishelena charakterisierungvontelomerischenproteinkomplexenintrypanosomabrucei |