Diurnal regulation of chloroplast gene expression in the green alga Chlamydomonas reinhardtii:
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| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
Potsdam
Univ.-Bibl.
2013
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | 137 Bl. Ill., graph. Darst. |
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MARC
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| 245 | 1 | 0 | |a Diurnal regulation of chloroplast gene expression in the green alga Chlamydomonas reinhardtii |c von Adam Dolbel Idoine |
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|---|---|
| adam_text | CONTENTS
1 SUMMARY 7
2 DEUTSCHE ZUSAMMENFASSUNG 8
3 INTRODUCTION 9
3.1 CHLAMYDOMONAS REINHARDTII AS A MODEL ORGANISM 9
3.2 THE PLASTOME 10
3.3 PLASTID GENE EXPRESSION 12
3.3.1 TRANSCRIPTION 12
3.3.2 TRANSCRIPT MATURATION 13
3.3.3 TRANSCRIPT STABILITY/DEGRADATION 15
3.3.4 TRANSLATION 16
3.3.5 CONTROL BY EPISTASY OF SYNTHESIS 18
3.3.6 PROTEIN TURNOVER 18
3.4 DIURNAL AND CIRCADIAN FLUCTUATIONS 18
3.4.1 PHYSIOLOGICAL PROCESSES 19
3.4.2 PLASTID PROCESSES 19
3.4.3 CIRCADIAN CLOCK IN C. REINHARDTII 22
3.5 METHODS USED IN GLOBAL PLASTID GENE EXPRESSION STUDIES 23
3.5.1 MACRO- AND MICROARRAYS 26
3.5.2 QUANTITATIVE REAL TIME POLYMERASE CHAIN REACTION 27
3.5.3 DEEP SEQUENCING AND OTHER MODERN METHODS 27
4 PERSPECTIVE 28
5 AIMS 28
6 METHODS 29
6.1 EQUIPMENT 29
6.1.1 GENERAL LAB EQUIPMENT 29
6.1.2 CENTRIFUGES 29
2
HTTP://D-NB.INFO/1046086057
6.1.3 NUCLEIC ACID GEL EQUIPMENT 30
6.1.4 MICROARRAY EQUIPMENT 30
6.1.5 QRT-PCR EQUIPMENT 30
6.1.6 CELL GROWTH 30
6.1.7 FERMENTER COMPONENTS 31
6.2 MOLECULAR BIOLOGY SUPPLIES 32
6.3 SOLUTIONS 33
6.3.1 GROWTH MEDIA 33
6.3.2 OTHER SOLUTIONS 34
6.4 GROWTH CONDITIONS 35
6.4.1 BIOREACTOR 36
6.4.2 STRAINS 36
6.5 CELL HARVEST 36
6.5.1 STANDARD 36
6.5.2 QUENCHING 36
6.5.3 BIOREACTOR SAMPLING 38
6.6 CELL COUNTING 39
6.7 RNA PROCEDURES 39
6.7.1 RNA EXTRACTION 39
6.7.2 DNASE TREATMENT 40
6.7.3 RNA QUANTIFICATION 40
6.7.4 RNA CONCENTRATION 40
6.7.5 DENATURING AGAROSE GEL ANALYSIS 40
6.7.6 NORTHERN BLOTTING 41
6.8 DNA PROCEDURES 41
6.8.1 POLYMERASE CHAIN REACTION 41
6.8.2 DNA AGAROSE GEL ANALYSIS 41
6.9 MICROARRAY HYBRIDISATION 42
6.9.1 OLIGONUCLEOTIDE DESIGN 42
6.9.2 MICROARRAY SPOTTING 42
3
6.9.3 REVERSE TRANSCRIPTION 43
6.9.4 CDNA LABELLING 43
6.9.5 HYBRIDISATION PROBE PREPARATION 43
6.9.6 MICROARRAY SLIDE PREPARATION 44
6.9.7 HYBRIDISATION 44
6.9.8 SCANNING 44
6.9.9 QUANTIFICATION AND NORMALISATION 44
6.9.10 DATA ANALYSIS 45
6.10 QUANTITATIVE REAL TIME REVERSE TRANSCRIPTION PCR 45
6.10.1 QRT-PCR PRIMER DESIGN 45
6.10.2 REVERSE TRANSCRIPTION 45
6.10.3 REAL TIME PCR REACTION 46
6.10.4 DATA ANALYSIS 46
6.11 METABOLITE ANALYSIS 47
6.11.1 METABOLITE EXTRACTION 47
6.11.2 METABOLITE MEASUREMENT 47
6.11.3 CHLOROPHYLL CONTENT DETERMINATION 47
6.12 DIURNAL DATA META-ANALYSIS 48
6.13 TRANSCRIPT CLUSTERING 48
6.14 COCLUSTERING ANALYSIS 49
6.15 RHYTHMICITY ANALYSIS 49
6.16 SOFTWARE 49
7 RESULTS 51
7.1 MICROARRAY DESIGN 51
7.2 MICROARRAY EXPERIMENTS 51
7.2.1 GOFORSYS LIGHT SHIFT EXPERIMENT 51
7.2.2 DIURNAL GENE REGULATION IN BATCH BIOREACTOR CULTURES 53
7.2.3 TRANSCRIPT REGULATION BY LIGHT IN BATCH FLASK CULTURES 55
7.3 QRT-PCR PLATFORM 59
7.3.1 ESTABLISHMENT 59
4
7.3.2 VALIDATION 60
7.4 BIOREACTOR CELL GROWTH 61
7.5 QRT-PCR EXPERIMENTS 65
7.5.1 DIURNAL FLUCTUATIONS IN TRANSCRIPT LEVELS 65
7.5.2 LIGHT-DEPENDENT FLUCTUATIONS IN TRANSCRIPT LEVELS 74
7.5.3 CIRCADIAN FLUCTUATIONS IN TRANSCRIPT LEVELS 76
7.6 METABOLITE ANALYSIS 82
7.7 AVERAGE EXPRESSION LEVELS 85
8 DISCUSSION 88
8.1 EXPERIMENTAL MOTIVATION 88
8.2 THE METHODS AND GROWTH CONDITIONS ARE HIGH QUALITY 89
8.2.1 MICROARRAY 89
8.2.2 QUANTITATIVE REAL TIME PCR 90
8.2.3 EVIDENCE THAT THE PLASTID TRANSCRIPTOMICS PLATFORMS PERFORM WELL .
. 90
8.2.4 GROWTH CONDITIONS 91
8.3 FUNCTIONALLY-RELATED TRANSCRIPTS ARE CO-REGULATED IN DIURNAL
CONDITIONS ... 93
8.4 TRANSCRIPTS PEAK WHEN THEY ARE NEEDED IN DIURNAL CONDITIONS 95
8.5 TRANSCRIPT PROFILES DEPEND ON THE STRAIN AND ORGANELLE FROM WHICH
THEY ARE
EXPRESSED 97
8.6 TRANSCRIPT LEVELS ARE NOT STRONGLY REGULATED BY LIGHT 99
8.7 TRANSCRIPTS ARE NOT STRONGLY REGULATED BY THE CIRCADIAN CLOCK 101
8.8 MODEL 103
8.9 CONCLUSION 103
9 OUTLOOK 104
10 ACKNOWLEDGEMENTS 106
11 BIBLIOGRAPHY 107
12 NOMENCLATURE 124
12.1 GENES, PROTEINS AND MUTANTS 124
5
12.2 STRAINS 124
12.3 EXPERIMENT ABBREVIATIONS 124
12.3.1 COLOUR CODES 125
12.3.2 HEATMAPS 125
12.4 ABBREVIATIONS 126
13 APPENDIX 128
13.1 DIURNAL STARCH ACCUMULATION 128
13.2 SYNTHETIC OLIGONUCLEOTIDES 128
13.2.1 MICROARRAY OLIGOS: PLASTID GENES 128
13.2.2 MICROARRAY OLIGOS: MITOCHONDRIAL GENES 129
13.2.3 QRT-PCR PRIMERS 130
13.3 COCLUSTERING VALIDATION 133
13.4 CELL DENSITIES 134
13.5 METABOLITE DATA 135
13.6 STRAIN EXPRESSION LEVELS 138
13.7 TATAATAT PROMOTER ANALYSIS 139
13.8 SIGMA FACTOR ALIGNMENT 141
13.9 OVERLAP WITH KUCHO ET AL. (2005) 145
13.10CULTURE TEMPERATURE 145
13.11DATA SUPPLEMENT 145
6
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| spelling | Dolbel Idoine, Adam Verfasser (DE-588)1046085913 aut Diurnal regulation of chloroplast gene expression in the green alga Chlamydomonas reinhardtii von Adam Dolbel Idoine Potsdam Univ.-Bibl. 2013 137 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Potsdam, Univ., Diss., 2013 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027028866&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Dolbel Idoine, Adam Diurnal regulation of chloroplast gene expression in the green alga Chlamydomonas reinhardtii |
| subject_GND | (DE-588)4113937-9 |
| title | Diurnal regulation of chloroplast gene expression in the green alga Chlamydomonas reinhardtii |
| title_auth | Diurnal regulation of chloroplast gene expression in the green alga Chlamydomonas reinhardtii |
| title_exact_search | Diurnal regulation of chloroplast gene expression in the green alga Chlamydomonas reinhardtii |
| title_full | Diurnal regulation of chloroplast gene expression in the green alga Chlamydomonas reinhardtii von Adam Dolbel Idoine |
| title_fullStr | Diurnal regulation of chloroplast gene expression in the green alga Chlamydomonas reinhardtii von Adam Dolbel Idoine |
| title_full_unstemmed | Diurnal regulation of chloroplast gene expression in the green alga Chlamydomonas reinhardtii von Adam Dolbel Idoine |
| title_short | Diurnal regulation of chloroplast gene expression in the green alga Chlamydomonas reinhardtii |
| title_sort | diurnal regulation of chloroplast gene expression in the green alga chlamydomonas reinhardtii |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027028866&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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