Verfügbarkeit: RNA-binding proteins in the early Drosophila embryo

RNA-binding proteins in the early Drosophila embryo:

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Bibliographische Detailangaben
1. Verfasser:	Sysoev, Vasily (VerfasserIn)
Format:	Abschlussarbeit Buch
Sprache:	English
Veröffentlicht:	Heidelberg 2016
Schlagworte:	Drosophila RNS-Bindungsproteine Embryonalentwicklung Hochschulschrift
Online-Zugang:	Inhaltsverzeichnis Inhaltsverzeichnis
Beschreibung:	150 Seiten Illustrationen, Diagramme 30 cm

Internformat

MARC


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Datensatz im Suchindex

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adam_text	RNA-BINDING PROTEINS IN THE EARLY DROSOPHILA EMBRYO EXAMINERS: DR. ALEXANDER AULEHLA DR. MICHAEL BOUTROS TABLE OF CONTENTS ABSTRACT.....................................................................................................................5 ZUSAMMENFASSUNG.................................................................................................7 ACKNOWLEDGEMENTS...............................................................................................9 TABLE OF CONTENTS................................................................................................11 LIST OF FIGURES.......................................................................................................15 1 INTRODUCTION..................................................................................................17 1.1 THE EARLY DROSOPHILA EMBRYO IS A SELF-ORGANIZING SYSTEM IN WHICH RNA-PROTEIN INTERACTIONS PLAY AN IMPORTANT ROLE .................................................... 19 1.2 RBPS IN MRNA LOCALIZATION AND TRANSLATIONAL CONTROL......................20 1.3 RBPS DRIVE THE MATERNAL-TO-ZYGOTIC TRANSITION..................................23 1.4 DIVERSITY OF RNA-BINDING PROTEINS.......................................................26 2 GOALS OF THE PROJECT.....................................................................................33 3 RNA INTERACTOME OF A DROSOPHILA EMBRYONIC CELL LINE.....................37 3.1 BACKGROUND AND AIMS.............................................................................39 3.2 CONTRIBUTIONS............................................................................................40 3.3 OPTIMIZATION OF RNA INTERACTOME CAPTURE FOR THE DROSOPHILA EMBRYO-DERIVED 82 CELL LINE..........................................................................................40 3.4 DETERMINATION OF OPTIMAL CROSS-LINKING CONDITIONS IN S2R+ CELLS. 43 3.5 THE INTERACTOME CAPTURE METHOD ALLOWS SELECTIVE PRECIPITATION OF RNA RATHER THAN DNA.....................................................................................................47 3.6 THE CO-PRECIPITATED RRNA IS NOT POLYADENYLATED..............................48 3.7 PRELIMINARY MASS-SPECTROMETRY ANALYSIS OF SAMPLES CAPTURED FROM S2R+ CELLS 50 3.8 TWO ROUNDS OF OLIGO(DT) PULL-DOWN IMPROVES SAMPLE PURITY ....... 52 3.9 RNA INTERACTOME OF THE DROSOPHILA S2R+ CELL LINE ............................ 55 3.10 CONCLUSIONS.............................................................................................57 3.11 MATERIALS AND METHODS ........................................................................ 58 CELL CULTURE........................................................................................................58 UV IRRADIATION OF S2 AND S2R+ CELLS................................................................58 OLIGO(DT) PULL-DOWN........................................................................................58 RNA AND PROTEIN ANALYSIS................................................................................59 RT-QPCR............................................................................................................60 SEQUENCING OF 185 3 END.................................................................................60 MASS-SPECTROMETIY..........................................................................................61 3.12 SUPPLEMENTARY DATASET: RNA INTERACTOME OF S2R+ CELLS ............... 61 4 RNA INTERACTOME OF THE EARLY DROSOPHILA EMBRYO...........................63 4.1 BACKGROUND AND AIMS.............................................................................65 4.2 CONTRIBUTIONS........................................................................................... 65 4.3 DETERMINATION OF OPTIMAL CROSS-LINKING CONDITIONS IN DROSOPHILA EMBRYOS 65 4.4 APPROACHES USED TO REDUCE RRNA CONTAMINATION DURING RNA INTERACTOME CAPTURE IN EARLY DROSOPHILA EMBRYOS.................................................67 4.5 LYSIS AT ELEVATED TEMPERATURE AND UNDER REDUCTIVE CONDITIONS IMPROVES QUALITY OF CAPTURED SAMPLES......................................................................70 4.6 SAMPLES USED FOR DETERMINATION OF HIGH CONFIDENCE EMBRYO RNA BINDING PROTEINS............................................................................................................ 74 4.7 MASS-SPECTROMETRY AND DETERMINATION OF THE EMBRYO RNA INTERACTOME 76 4.8 OVERVIEW OF THE DROSOPHILA EMBRYO RNA INTERACTOME .................... 79 4.9 FEATURES OF NOVEL AND PREVIOUSLY KNOWN DROSOPHILA RBPS............82 4.10 COMPARISON OF FIVE EUKARYOTIC RNA INTERACTOMES ......................... 87 4.11 NOVEL DROSOPHILA RNA-BINDING PROTEINS ARE LINKED TO DEVELOPMENT 88 4.12 VALIDATION OF RNA-BINDING CAPACITY OF INTERACTOME PROTEINS BY PNK ASSAY 91 4.13 CONCLUSIONS AND OUTLOOK ..................................................................... 97 4.14 MATERIALS AND METHODS........................................................................98 EMBRYO COLLECTION AND UV IRRADIATION............................................................98 OLIGO(DT) PULL-DOWN........................................................................................99 PRECIPITATION OF RIBOSOMES PRIOR TO OLIGO(DT) PULL-DOWN.............................99 RNA AND PROTEIN ANALYSIS................................................................................99 RT-QPCR..........................................................................................................100 STAGING OF EMBRYOS.........................................................................................100 VALIDATION OF RNA-BINDING ABILITY BY PNK ASSAY ......................................... 100 SAMPLE PREPARATION FOR MS...........................................................................101 LC-MS/MS.......................................................................................................102 PEPTIDE IDENTIFICATION AND QUANTIFICATION....................................................102 5 RNA-BOUND PROTEOME DYNAMICITY DURING THE MATERNAL-TO-ZYGOTIC TRANSITION 105 5.1 BACKGROUND AND GOALS..........................................................................107 5.2 CONTRIBUTIONS.........................................................................................107 5.3 EXPERIMENTAL DESIGN AND SAMPLES USED IN THE ANALYSIS.................107 5.4 PROTEIN ABUNDANCE CHANGES IN THE TOTAL EMBRYO AND IN THE RNA BOUND FRACTIONS.............................................................................................................108 5.5 COMPARISON OF TOTAL PROTEOMES AND RNA INTERACTOMES IN PRE- AND POST-MZT EMBRYOS REVEALS GLOBAL CHANGES.............................................................109 5.6 CLASSIFICATION OF RBPS BASED ON THEIR DYNAMIC BEHAVIOR DURING THE MZT 112 5.7 POTENTIAL ROLES OF ALTERNATIVE SPLICING IN REGULATION OF RNA-BINDING ACTIVITY 114 5.8 EVIDENCE FOR ADDITIONAL MECHANISMS AFFECTING OBSERVED RNA BINDING ACTIVITY OF SOME PROTEINS ............................................................................. 120 5.9 CONCLUSIONS AND OUTLOOK......................................................................122 5.10 MATERIALS AND METHODS......................................................................123 6 CONCLUSIONS AND OUTLOOK ............................................................. 125 6.1 BACKGROUND............................................................................................127 6.2 OPTIMIZATION OF THE RNA INTERACTOME CAPTURE ................................ 127 6.3 RNA INTERACTOME OF THE EARLY DROSOPHILA EMBRYO .......................... 128 6.4 NEW LINKS BETWEEN DEVELOPMENT AND RNA METABOLISM ................ 129 6.5 DYNAMICITY OF THE RNA BOUND PROTEOME DURING THE MZT ............. 129 6.6 ROLES OF DISORDERED REGIONS IN RBPS..................................................130 6.7 FUTURE DIRECTIONS...................................................................................131 APPENDIX............................................................................................133 TABLE AL. ANTIBODIES USED FOR IMMUNOBLOTTING.........................................133 TABLE A2. OLIGONUCLEOTIDES USED FOR QPCR..................................................134 LIST OF ABBREVIATIONS.........................................................................135 REFERENCES..........................................................................................137
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author	Sysoev, Vasily
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building	Verbundindex
bvnumber	BV044316956
ctrlnum	(OCoLC)969453147 (DE-599)DNB1119086531
discipline	Biologie
format	Thesis Book
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spelling	Sysoev, Vasily Verfasser aut RNA-binding proteins in the early Drosophila embryo presented by Vasily Sysoev Heidelberg 2016 150 Seiten Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Ruperto-Carola University of Heidelberg 2016 Drosophila (DE-588)4150708-3 gnd rswk-swf RNS-Bindungsproteine (DE-588)4178253-7 gnd rswk-swf Embryonalentwicklung (DE-588)4070792-1 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Drosophila (DE-588)4150708-3 s Embryonalentwicklung (DE-588)4070792-1 s RNS-Bindungsproteine (DE-588)4178253-7 s DE-604 B:DE-101 application/pdf http://d-nb.info/1119086531/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029720506&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis
spellingShingle	Sysoev, Vasily RNA-binding proteins in the early Drosophila embryo Drosophila (DE-588)4150708-3 gnd RNS-Bindungsproteine (DE-588)4178253-7 gnd Embryonalentwicklung (DE-588)4070792-1 gnd
subject_GND	(DE-588)4150708-3 (DE-588)4178253-7 (DE-588)4070792-1 (DE-588)4113937-9
title	RNA-binding proteins in the early Drosophila embryo
title_auth	RNA-binding proteins in the early Drosophila embryo
title_exact_search	RNA-binding proteins in the early Drosophila embryo
title_full	RNA-binding proteins in the early Drosophila embryo presented by Vasily Sysoev
title_fullStr	RNA-binding proteins in the early Drosophila embryo presented by Vasily Sysoev
title_full_unstemmed	RNA-binding proteins in the early Drosophila embryo presented by Vasily Sysoev
title_short	RNA-binding proteins in the early Drosophila embryo
title_sort	rna binding proteins in the early drosophila embryo
topic	Drosophila (DE-588)4150708-3 gnd RNS-Bindungsproteine (DE-588)4178253-7 gnd Embryonalentwicklung (DE-588)4070792-1 gnd
topic_facet	Drosophila RNS-Bindungsproteine Embryonalentwicklung Hochschulschrift
url	http://d-nb.info/1119086531/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029720506&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
work_keys_str_mv	AT sysoevvasily rnabindingproteinsintheearlydrosophilaembryo

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