Molecular analysis of PPR proteins in Chlamydomonas reinhardtii:
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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:19-150800 Inhaltsverzeichnis |
| Physical Description: | 122 Bl. Ill., graph. Darst. |
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MARC
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| 100 | 1 | |a Jalal, Abdullah |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Molecular analysis of PPR proteins in Chlamydomonas reinhardtii |c von Abdullah Jalal |
| 264 | 1 | |c 2012 | |
| 300 | |a 122 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a München, Univ., Diss., 2012 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
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| 912 | |a ebook | ||
| 999 | |a oai:aleph.bib-bvb.de:BVB01-025493556 | ||
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| _version_ | 1804149792850313216 |
|---|---|
| adam_text | IMAGE 1
TABLE OF CONTENTS
4
TABLE OF CONTENTS
ABSTRACT 2
ZUSAMMENFASSUNG 3
TABLE OF CONTENTS 4
ABBREVIATIONS 6
1 INTRODUCTION 8
1.1 ENDOSYMBIOSIS AND EVOLUTION O F CHLOROPLAST 8
1.2 PHOTOSYNTHESIS 9
1.3 CHLAMYDOMONAS REINHARDTII AS A MODEL ORGANISM 10
1.3.1 CHARACTERISTICS AND EXPRESSION O F THE CHLOROPLAST GENOME O F C.
REINHARDTII 11
1.4 ROLE O F NUCLEAR ENCODED FACTORS IN CHLOROPLAST GENE EXPRESSION 13
1.4.1 TPR PROTEINS 15
1.4.2 OPR PROTEINS 17
1.4.3 PPR PROTEINS 18
1.4.3.1 STRUCTURE AND CLASSES O F PPR PROTEINS 18
1.4.3.2 DISTRIBUTION AND EVOLUTION O F PPR PROTEINS 21
1.4.3.3 FUNCTIONS O F PPR PROTEINS IN RNA METABOLISM 22
1.5 AIMS O F THIS STUDY 29
2 MATERIALS AND METHODS 31
2.1 MATERIALS 31
2.1.1 ENZYMES 32
2.1.2 OLIGONUCLEOTIDES 32
2.1.3 DNA-VECTORS 32
2.1.4 REACTION SYSTEMS (KITS) 33
2.1.5 BACTERIAL STAINS 33
2.1.6 C. REINHARDTII STRAINS 33
2.2 METHODS 34
2.2.1 GROWTH O F BACTERIAL STRAINS 34
2.2.2 GROWTH O F C. REINHARDTII STRAINS 34
2.2.3 NUCLEIC ACIDS 34
2.2.3.1 ISOLATION O F NUCLEIC ACIDS 34
2.2.3.1.1 PLASMID ISOLATION FROM E. COLI 34
2.2.3.1.2 ISOLATION O F GENOMIC DNA FROM C. REINHARDTII 34
2.2.3.1.3 ISOLATION O F TOTAL CELLULAR RNA FROM C. REINHARDTII 35
2.2.3.2 DETERMINATION O F NUCLEIC ACID CONCENTRATIONS 35
2.2.3.3 NUCLEID ACID ELECTROPHORESES 35
2.2.3.3.1 AGAROSE GEL ELECTROPHORESIS O F DNA 35
2.2.3.3.2 AGAROSE GEL ELECTROPHORESIS O F RNA 35
2.2.3.4 CDNA SYNTHESIS AND RT-PCR 36
2.2.3.5 CLONING 36
2.2.3.5.1 TRANSFORMATION O F E. COLI 36
2.2.3.5.2 POLYMERASE CHAIN REACTION (PCR) 37
2.2.3.5.3 SEQUENCING 37
2.2.3.6 PROBE LABELLING AND TRANSCRIPT ACCUMULATION ANALYSES (NORTHERN
BLOT) 37
2.2.4 PROTEIN METHODS 39
2.2.4.1 DETERMINATION O F PROTEIN CONCENTRATIONS 39
2.2.4.2 SDS POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS PAGE) 39
2.2.4.3 IMMUNOBLOTTING 40
2.2.4.4 ISOLATION O F TOTAL PROTEIN EXTRACTS FROM C. REINHARDTII 41
2.2.4.5 ISOLATION O F TOTAL SOLUBLE PROTEIN EXTRACTS FROM C. REINHARDTII
41
2.2.4.6 CHLOROPLAST ISOLATION FROM C. REINHARDTII 41
2.2.4.7 CHLOROPLAST FRACTIONATION O F C. REINHARDTII 42
2.2.4.8 MITOCHONDRIA ISOLATION FROM C. REINHARDTII 42
2.2.4.9 SIZE EXCLUSION CHROMATOGRAPHY 43
2.2.4.10 EXPRESSION AND PURIFICATION O F RECOMBINANT PROTEINS 43
2.2.4.10.1 PLASMIDS FOR EXPRESSION O F RECOMBINANT PROTEINS 43
2.2.4.10.2 EXPRESSION AND PURIFICATION O F RECOMBINANT PROTEINS 44
HTTP://D-NB.INFO/1033964530
IMAGE 2
TABLE OF CONTENTS
5
2.2.4.11 ANTIBODY PRODUCTION AND PURIFICATION 45
2.2.5 NUCLEAR TRANSFORMATION O F C. REINHARDTII 45
2.2.6 GFP BASED SUBCELLULAR LOCALIZATION 45
2.2.6.1 GFP FUSION CONSTRUCTS 45
2.2.6.2 GFP FLUORESCENCE MICROSCOPY 46
2.2.7 GENERATION O F RNAI LINES FOR PPR7 46
2.2.8 CO-IMMUNOPRECIPITATION STUDIES 47
2.2.9 MICROARRAY DESIGN AND HYBRIDIZATION 48
2.2.10 UV CROSS-LINKING O F RNA AND RECOMBINANT PPR7 48
2.2.11 CHLOROPHYLL FLUORESCENCE MEASUREMENTS 49
2.2.12 CIRCULAR DICHROISM MEASUREMENTS 49
2.2.13 CRYSTALLIZATION O F HIS-PPR7 50
2.2.14 BIOINFORMATICS SOURCES 50
2.2.14.1 PREDICTION O F GENE MODELS 50
2.2.14.2 PREDICTION O F PROTEIN LOCALIZATION AND TRANSIT PEPTIDES 50
2.2.14.3 PROTEIN PROPERTIES AND REPEAT PREDICTIONS 51
2.2.14.4 ALPHA HELICAL STRUCTURE AND WHEEL MODEL PREDICTIONS 51
3 RESULTS 52
3.1 PPR PROTEINS IN C. REINHARDTII 52
3.2 SUBCELLULAR LOCALIZATION O F PPR PROTEINS IN C. REINHARDTII 54
3.3 STRUCTURE ANALYSIS O F THE PPR7 PROTEIN 57
3.3.1 PPR MOTIFS IN PPR7 AND THEIR HELICAL WHEEL MODELS 57
3.3.2 CIRCULAR DICHROISM MEASUREMENTS O F RECOMBINANT PPR7 PROTEIN 59
3.3.3 CRYSTAL STRUCTURE ANALYSIS O F RECOMBINANT PPR7 PROTEIN 60
3.4 ANALYSIS O F THE FUNCTION O F PPR7 IN C. REINHARDTII 61
3.4.1 ANALYSIS O F PPR7 RNA INTERFERENCE LINES 61
3.4.2 CHARACTERIZATION O F RNA BINDING PROPERTY O F PPR7 64
3.4.2.1 PPR7 IS A COMPONENT O F A HIGH MOLECULAR WEIGHT RNASE-SENSITIVE
COMPLEX 65 3.4.2.2 THE RECOMBINANT PPR7 PROTEIN REVEALS INTRINSIC RNA
BINDING ACTIVITY 65
3.4.2.3 IDENTIFICATION O F TARGET RNAS O F PPR7 BY RIP-CHIP ANALYSIS 66
3.4.2.4 SEMI QUANTITATIVE RT-PCR O F PPR7 CO-IMMUNOPRECIPITATED RNAS 68
3.4.2.5 THE ROLE O F PPR7 AT THE IDENTIFIED PUTATIVE TARGET RNAS 70
3.4.3 PHOTOSYNTHETIC STRESS RESPONSE O F PPR7 80
4 DISCUSSION 81
4.1 PPR PROTEINS IN C. REINHARDTII 81
4.2 PPR7 IS PART O F AN RNASE SENSITIVE COMPLEX 82
4.3 PPR7 IS ASSOCIATED WITH MULTIPLE CHLOROPLAST RNAS 84
4.3.1 ROLE O F PPR7 AS A STABILITY FACTOR 85
4.3.2 ROLE O F PPR7 AS A PROCESSING FACTOR 87
4.4 PPR7 DEFICIENCY CAUSES A LIGHT SENSITIVE PHENOTYPE 91
5 REFERENCES 93
6 ANNEX 109
ANNEX A: 109
ANNEX B: 112
CURRICULUM VITAE 120
PUBLICATIONS AND CONFERENCE ABSTRACTS 121
ACKNOWLEDGMENT 122
|
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| spelling | Jalal, Abdullah Verfasser aut Molecular analysis of PPR proteins in Chlamydomonas reinhardtii von Abdullah Jalal 2012 122 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Univ., Diss., 2012 (DE-588)4113937-9 Hochschulschrift gnd-content Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:19-150800 http://edoc.ub.uni-muenchen.de/15080/ Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:19-150800 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025493556&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Jalal, Abdullah Molecular analysis of PPR proteins in Chlamydomonas reinhardtii |
| subject_GND | (DE-588)4113937-9 |
| title | Molecular analysis of PPR proteins in Chlamydomonas reinhardtii |
| title_auth | Molecular analysis of PPR proteins in Chlamydomonas reinhardtii |
| title_exact_search | Molecular analysis of PPR proteins in Chlamydomonas reinhardtii |
| title_full | Molecular analysis of PPR proteins in Chlamydomonas reinhardtii von Abdullah Jalal |
| title_fullStr | Molecular analysis of PPR proteins in Chlamydomonas reinhardtii von Abdullah Jalal |
| title_full_unstemmed | Molecular analysis of PPR proteins in Chlamydomonas reinhardtii von Abdullah Jalal |
| title_short | Molecular analysis of PPR proteins in Chlamydomonas reinhardtii |
| title_sort | molecular analysis of ppr proteins in chlamydomonas reinhardtii |
| topic_facet | Hochschulschrift |
| url | http://edoc.ub.uni-muenchen.de/15080/ https://nbn-resolving.org/urn:nbn:de:bvb:19-150800 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025493556&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT jalalabdullah molecularanalysisofpprproteinsinchlamydomonasreinhardtii |