Visualization and characterization of ribonucleoproteins in plants:
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| Main Author: | |
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2012
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| Subjects: | |
| Online Access: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-271116 Inhaltsverzeichnis |
| Physical Description: | IV, 131 Bl. Ill., graph. Darst. CD (12 cm) |
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MARC
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| 100 | 1 | |a Schönberger, Johannes Klaus |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Visualization and characterization of ribonucleoproteins in plants |c vorgelegt von Johannes Klaus Schönberger |
| 264 | 1 | |c 2012 | |
| 300 | |a IV, 131 Bl. |b Ill., graph. Darst. |e CD (12 cm) | ||
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| 502 | |a Regensburg, Univ., Diss., 2012 | ||
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| 650 | 0 | 7 | |a Schmalwand |g Arabidopsis |0 (DE-588)4179774-7 |2 gnd |9 rswk-swf |
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Record in the Search Index
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|---|---|
| adam_text | IMAGE 1
TABLE OF CONTENTS
TABLE OF CONTENTS
1. INTRODUCTION 1
1.1. POLARITY AND DIFFERENTIAL INHERITANCE 1
1.2. DEVELOPMENT O F THE EMBRYO 4
1.3. RNA LOCALIZATION AS A KEY FACTOR IN DEVELOPMENT 9
1.3.1. TRAPPING O F FREELY DIFFUSING RNA 11
1.3.2. LOCAL STABILIZATION/DEGRADATION 11
1.3.3. DIRECTED TRANSPORT O F RNA 11
1.4. VISUALIZING RNA IN PLANTS 13
1.5. AIMS O F THIS WORK 15
2. RESULTS 16
2.1. VISUALIZING RNA IN PLANTS 16
2.1.1. A VERSATILE GATEWAY* BASED VECTOR SERIES FOR RNA VISUALIZATION IN
PLANTS. 16
2.1.2. THE MS2 AND THE A .N22 SYSTEMS ARE BOTH SUITABLE FOR RNA
MONITORING IN
PLANTA 17
2.1.3. FURTHER CHARACTERIZATION O F THE VIABILITY O F THE AN22 AND THE
MS2 SYSTEM
AND THE INFLUENCE O F THE POSITION O F THE STEM LOOPS 22
2.1.4. RNA IS TRANSPORTED WITHIN MICROSCOPICALLY VISIBLE RNA TRANSPORT
GRANULES. 25
2.1.5. DUAL APPLICATION O F BOTH SYSTEMS 28
2.2. ELUCIDATING THE ROLE O F POLARLY DISTRIBUTED RNA IN THE ARABIDOPSIS
EGG CELL 30
2.2.1. SETTING UP THE VECTOR SYSTEM FOR RNA VISUALIZATION IN THE EGG
CELL 30
2.2.2. A LIST O F PUTATIVE POLAR RNAS WAS GENERATED FOR HIGH-THROUGHPUT
SCREENING 31
2.2.3. STABLY TRANSFORMED MS2CP- AND XN22-PLANTS SHOW DIFFERENT PATTERNS
OF
EXPRESSION AND LOCALIZATION 34
2.2.4. THE DISTRIBUTION O F THE XN22 CHANGES UPON EXPRESSION O F A
TARGET RNA
CARRYING BOXB STEM LOOPS 37
2.2.5. FURTHER CHARACTERIZATION O F THE CANDIDATE RNAS 38
2.3. CHARACTERIZATION O F AN ENDOGENOUS RNA BINDING PROTEIN 41
2.3.1. RBP1 AND ITS INTRIGUING SUBCELLULAR LOCALIZATION 41
2.3.2. STUDYING RBP1 AND ITS ROLE IN RNA TRANSPORT 45
2.3.3. HETEROLOGOUS EXPRESSION AND AFFINITY PURIFICATION O F RBP1 50
I
HTTP://D-NB.INFO/1029569061
IMAGE 2
TABLE OF CONTENTS
3. DISCUSSION 54
3.1. THE IN VIVO VISUALIZATION O F RNA IN PLANT CELLS 54
3.1.1. THE GENERATED VECTORS OFFER A BROAD SPECTRUM FOR IN VIVO
VISUALIZATION
O F RNA 54
3.1.2. MS2 AND XN22 ARE BOTH SUITABLE SYSTEMS FOR IN VIVO STUDIES O F
RNA
DISTRIBUTION IN PLANTS 55
3.1.3. THE POSITION O F THE STEM LOOPS INFLUENCES THE CAPABILITY O F THE
RNA TO BE
TRANSLATED 57
3.1.4. XN22 AND MS2CP BIND MRNA AND FORM MICROSCOPICALLY VISIBLE
TRANSPORT RNPS 58
3.1.5. AN22 AND MS2CP CAN BE SIMULTANEOUSLY USED TO MONITOR DIFFERENT
POOLS
O F RNPS IN PLANTA 60
3.2. THE DETECTION O F AN RNA GRADIENT WITHIN THE ARABIDOPSIS EGG CELL
62
3.2.1. A VERSATILE VECTOR SERIES FOR THE HIGH-THROUGHPUT STUDY O F RNA
VISUALIZATION IN THE ARABIDOPSIS EGG CELL 62
3.2.2. A CANDIDATE LIST O F PUTATIVE POLAR RNA CANDIDATES WAS GENERATED
BASED
ON SINGLE CELL MICROARRAY STUDIES FROM THE FEMALE GAMETOPHYTES OF
ARABIDOPSIS AND MAIZE 63
3.2.3. ^ 2 2 EXHIBITS A RELIABLE EXPRESSION PATTERN AND SUBCELLULAR
LOCALIZATION
UNDER EGG CELL SPECIFIC EXPRESSION 65
3.2.4. SUBCELLULAR LOCALIZATION STUDIES O F TRANSLATIONAL PRODUCTS O F
ALL CANDIDATE
RNAS PROVIDE SUPPORTING INFORMATION FOR SUBSEQUENT BIOLOGICAL STUDIES
O F POLARLY DISTRIBUTED TRANSCRIPTS 66
3.3. CHARACTERIZATION O F AN ENDOGENOUS RNA BINDING PROTEIN 67
3.3.1. RBP1 (AT4GL7520) FORMS CYTOPLASMIC FOCI RESEMBLING RNPS 67
3.3.2. RBP1 CONTAINING RNPS ARE TRANSPORTED ALONG THE CYTOSKELETON 68
3.3.3. RBP1 CAN BE PURIFIED FROM E. COLI 69
4. SUMMARY 71
5. ZUSAMMENFASSUNG 73
6. MATERIAL AND METHODS 75
I I
IMAGE 3
TABLE OF CONTENTS
6.1. CULTIVATION O F BACTERIA 75
6.2. MOLECULARBIOLOGICAL METHODS 76
6.2.1. POLYMERASE CHAIN REACTION (PCR) 76
6.2.2. ISOLATION O F HIGHLY PURE GENOMIC DNA FROM ARABIDOPSIS THALIANA
77
6.2.3. QUICK PREPARATION O F GENOMIC DNA FROM ARABIDOPSIS THALIANA 78
6.2.4. AGAROSE GELELECTROPHORESIS 79
6.2.5. RESTRICTION DIGESTS 79
6.2.6. DNA LIGATION 80
6.2.7. SUBCLONING WITH ZERO BLUNT TOPO PCR CLONING KIT 80
6.2.8. SUBCLONING WITH PENTR*/D-TOPO CLONING KIT 80
6.2.9. GENERATION O F CHEMICALLY COMPETENT E. COLI CELLS 80
6.2.10. TRANSFORMATION O F E. COLI 82
6.2.11. GENERATION O F COMPETENT AGROBACTERIA CELLS 82
6.2.12. TRANSFORMATION O F AGROBACTERIA : 82
6.2.13. MINIPREPARATION WITH INVITROGEN* PURELINK QUICK PLASMID
MINIPREP KIT 82
6.2.14. MIDIPREPARATION WITH INVITROGEN* PURELINK HIPURE PLASMID
MIDIPREP KIT 83
6.2.15. MRNA ISOLATION AND REVERSE TRANSCRIPTASE (RT)-PCR 83
6.2.16. QUANTITATIVE REAL-TIME PCR 85
6.2.17. GEL EXTRACTION O F DNA FRAGMENTS 83
6.2.18. SEQUENCING 84
6.3. BIOCHEMICAL METHODS 84
6.3.1. SDS-PAGE 84
6.3.2. COOMASSIE STAINING 85
6.3.3. WET BLOT 85
6.3.4. CRUDE PROTEIN EXTRACT FROM PLANTS 86
6.3.5. HETEROLOGOUS EXPRESSION O F RBP1 IN E. COLI 87
6.4. CELL BIOLOGICAL AND PLANT WORK 88
6.4.1. PLANT MATERIAL AND GROWTH CONDITIONS 88
6.4.2. DISSECTION O F OVULES 89
6.4.3. INFILTRATION O F N. BENTHAMIANA 89
III
IMAGE 4
TABLE OF CONTENTS
6.4.4. CONFOCAL MICROSCOPY 90
7. BIBLIOGRAPHY 91
8. APPENDIX 101
8.1. OLIGOS 101
8.2. PLASMID SEQUENCES 120
8.2.1. VECTORS USED FOR TRANSIENT ASSAY 121
8.2.2. VECTORS USED FOR STABLE TRANSFORMATION O F ARABDISDOPSIS THALIANA
123
8.2.3. VECTOR FOR HETEROLOGOUS EXPRESSION O F CBD-RBP1 -GFP 125
8.3. SUBCELLULAR LOCALIZATIONS 125
I V
|
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| spelling | Schönberger, Johannes Klaus Verfasser aut Visualization and characterization of ribonucleoproteins in plants vorgelegt von Johannes Klaus Schönberger 2012 IV, 131 Bl. Ill., graph. Darst. CD (12 cm) txt rdacontent n rdamedia nc rdacarrier Regensburg, Univ., Diss., 2012 Messenger-RNS (DE-588)4169499-5 gnd rswk-swf Zellteilung (DE-588)4190668-8 gnd rswk-swf Schmalwand Arabidopsis (DE-588)4179774-7 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Schmalwand Arabidopsis (DE-588)4179774-7 s Zellteilung (DE-588)4190668-8 s Messenger-RNS (DE-588)4169499-5 s b DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:355-epub-271116 http://epub.uni-regensburg.de/27111/ Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-271116 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025467801&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Schönberger, Johannes Klaus Visualization and characterization of ribonucleoproteins in plants Messenger-RNS (DE-588)4169499-5 gnd Zellteilung (DE-588)4190668-8 gnd Schmalwand Arabidopsis (DE-588)4179774-7 gnd |
| subject_GND | (DE-588)4169499-5 (DE-588)4190668-8 (DE-588)4179774-7 (DE-588)4113937-9 |
| title | Visualization and characterization of ribonucleoproteins in plants |
| title_auth | Visualization and characterization of ribonucleoproteins in plants |
| title_exact_search | Visualization and characterization of ribonucleoproteins in plants |
| title_full | Visualization and characterization of ribonucleoproteins in plants vorgelegt von Johannes Klaus Schönberger |
| title_fullStr | Visualization and characterization of ribonucleoproteins in plants vorgelegt von Johannes Klaus Schönberger |
| title_full_unstemmed | Visualization and characterization of ribonucleoproteins in plants vorgelegt von Johannes Klaus Schönberger |
| title_short | Visualization and characterization of ribonucleoproteins in plants |
| title_sort | visualization and characterization of ribonucleoproteins in plants |
| topic | Messenger-RNS (DE-588)4169499-5 gnd Zellteilung (DE-588)4190668-8 gnd Schmalwand Arabidopsis (DE-588)4179774-7 gnd |
| topic_facet | Messenger-RNS Zellteilung Schmalwand Arabidopsis Hochschulschrift |
| url | http://epub.uni-regensburg.de/27111/ https://nbn-resolving.org/urn:nbn:de:bvb:355-epub-271116 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025467801&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT schonbergerjohannesklaus visualizationandcharacterizationofribonucleoproteinsinplants |