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Legen, Juliana (2003): Gene expression in plastids of higher plants: evolutionary and functional aspects of different RNA polymerases - coordinated assembly of multiprotein-complexes. Dissertation, LMU München: Fakultät für Biologie



Plastid gene organisation maintains characteristics typical of its prokaryotic ancestry. The regulation of plastid gene expression however strongly deviates from that one its free-living cyanobacteria relatives. This intriguing complication of plastid gene expression characteristics is the result of an integration process of the cellular subgenomes that introduced eukaryotic traits into the formerly prokaryotic compartment (Herrmann et al. 1997). An interesting example for this process is the transcription system, which consists of both prokaryotic (PEP) and eukaryotic (NEP) RNA polymerases. In order to understand, how far the transcriptional apparatus within plastids was adapted to nuclear needs, three approaches have been undertaken. Firstly, the tobacco homologues of the NEP-enzymes known from Arabidopsis were determined and characterised. Secondly, an extensive transcript analysis for all plastid operons was carried out with wild-type and PEP-lacking material in order to assess the contribution of the two systems to transcription. In order to rapidly screen this plant material, an array-based technique was established. As no arrays for the plastid chromosome had been described, the preparation of the filters, optimisation of hybridisation conditions and probe preparation together with the use of the proper controls was one of the main challenges of this work, in particular as macroarrays are interesting for various other applications. Thirdly, to assess the regulation of PEP expression, an in vivo analysis of the promoter for the rpoB operon was performed. According to data from other groups, this operon is controlled by NEP, which - in addition to other data - led to the suggestion that PEP is switched on by NEP (Hajduckiewicz et al., 1997). To test this, point mutations of the described NEP rpoB promoter (Liere and Maliga; 1999) should be prepared and introduced into the plastid compartment by particle transformation. Insight into the regulation of PEP is crucial in order to understand the interplay of the two RNA polymerase systems. Finally, in addition to studies on plastid transcription, later steps in plastid gene expression were examined in the course of this work as well. As a model to study assembly of a multisubunit complex, the cytochrome b6/f complex was analysed by a gene-disruption approach. Deletion and/or insertion mutants of all plastid encoded subunits of the complex were prepared in order to evaluate the assembly strategy for the holocomplex.