The chloroplast of Chlamydomonas

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Table of contents

CHAPTER 1
1. EXPRESSION ANALYSIS OF THE TWO PLASTID ATP OPERONS: THE LARGE ATPI/H/F/A AND THE SMALL ATPB/E OPERON
1.1. Transcriptional analysis of atpI transcripts
1.2. Transcriptional analysis of atpH transcripts
1.2.1. 3’ end mapping of -418 initiated atpH transcripts
1.2.2. 3’ end mapping of -45 processed atpH transcripts
1.2.3. Regulation of the higher stoichiometry of ATPH
1.3. Transcriptional analysis of atpF transcript
1.4. Transcriptional analysis of atpA transcripts
1.5. Transcriptional analysis of atpB transcripts
1.6. Expression analysis of the atpE gene
Conclusion
CHAPTER 2
2. DO LIGHT CONDITIONS INFLUENCE THE EXPRESSION OF SIG3 DEPENDENT GENES?
2.1. Illumination of etiolated plantlets
2.1.1. Macroarray analyses
2.1.2. Action of light on SIG3 dependent gene expression Conclusion on the macroarray results
2.1.3. Primer extension analyses
2.2. Light stress of green plants (photoinhibition of chloroplasts)
Conclusion
CHAPTER 3
3. EXPRESSION ANALYSIS OF THE PSBT SENSE/ANTISENSE RNAS
3.1 Previous results obtained in the laboratory
3.2 Mapping of psbT anti sense RNA extremities
3.3 Mapping of psbT sense RNA extremities
3.4 Putative role of psbN expression on processing of psbB operon
3.5 Existence of an internal psbT promoter within psbB gene
Conclusion
GENERAL DISCUSSION
Expression analysis of the two plastid encoded ATPsynthase operons: the large ATPI/H/F/A and the small ATPB/E operon
Influence of light on the expression of SIG3 dependent transcripts
Expression analysis of the psbT sense/antisense RNAs
MATERIAL AND METHODS
Cultivation of plants in vitro
Cultivation of plants in soil
Extraction of RNA
Treatment of RNAs with DNase
Northern Blot analysis
Principal
Probe preparation
Gel electrophoresis
RNA Transfer
Hybridisation
Primer Extension
Preparation of polyacrylamide gel
Sequencing
Oligo labelling
PCR Amplification
Cloning of the DNA Fragment
Principle
Protocol
Table of contents
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Miniprep; Plasmid DNA Extraction
RNA Analysis by RT-PCR
Analysis of plastidial transcript profile expression by cDNA macroarray
cDNA synthesis
Hybridisation of labelled cDNAs on membrane
Analysis of Results
TAP (Tobacco Acid Pyrophosphate) treatment and 5’ RACE
Principle
Protocol
Circular RT- PCR
Principle
Protocol
Western Blot Analysis
Principal
Protein extraction
Protein quantification
Gel preparation
Protein Transfer
Western Blot analysis for smaller proteins
Procedure