Smith, Annette Clare; (2001) The transcriptional apparatus of Chlamydomonas chloroplasts. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

The transcriptional apparatus of higher plant chloroplasts is well characterised and consists of a plastid-encoded polymerase (PEP) and a nuclear encoded polymerase (NEP). PEP is dispensable to cell viability. The situation in green algal species, however, is less clear. Chloroplast genes encoding subunits of the PEP have been cloned and sequenced in the green alga Chlamydomonas reinhardtii and preliminary reverse-genetic studies suggest that PEP is essential to cell viability, which is in contrast to the situation in higher plants. To investigate this further a series of gene knockouts were constructed using the chloroplast gene rpoC2, encoding the " subunit of PEP. Results indicate that PEP is essential to C. reinhardtii cell viability. In addition, inhibitors of PEP have been used in an in vivo transcription assay to try to identify a second RNA polymerase activity in C. reinhardtii chloroplasts. In all higher plant and red algal species so far studied the PEP factor is encoded in the nuclear genome. A C. reinhardtii nuclear gene (rpoD) encoding a putative PEP factor has been cloned and partially sequenced. This is the first factor cloned from a green algal species. A transcript of ~2.9 kb was detected for the rpoD gene by northern analysis. Finally, epitope tagging technology was developed for chloroplast and bacterial gene products. The rpoC2 gene of C. reinhardtii was modified to produce a 6x-histidine tagged polypeptide and an attempt was made to purify this polypeptide from C. reinhardtii cells using IMAC. In addition, a 3x haemagglutinin (HA) epitope tag was codon optimised for use in C. reinhardtii chloroplasts and this epitope was used to tag -galactosidase in E. coli. The protein was detected in a western blot using anti-HA monoclonal antibodies. This epitope will prove useful as a tool to tag C. reinhardtii chloroplast proteins.

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