O'Connor, Helen Elizabeth; (1995) The structure and function of the psbH gene in 'Chlamydomonas reinhardtii'. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes the analysis of the psbH gene structure and function in the unicellular green alga Chlamydomonas reinhardtii. The psbH gene encodes a small photosynthetic protein which co-purifies with Photosystem II (PS II). It is present in both cyanobacterial and eukaryotic PSII and is phosphorylated in the latter. The function of the phosphoprotein is still a matter of conjecture. Prior to the initiation of this thesis the gene had not been mapped or isolated in Chlamydomonas reinhardtii. This thesis describes the localisation of the psbH gene and its position relative to other genes already mapped. The complete sequence of the psbH gene was determined, the protein has a predicted molecular mass of 9.3 kDa. Northern blot analysis of the gene expression in wild type and nuclear mutants of C.reinhardtii reveal that the gene is co-transcribed with psbB located 3.2kB downstream from psbB and that a nuclear factor is required for stable expression of both genes. The precise location of the psbH transcripts revealed in northern analysis was mapped by primer extension. A series of psbH mutants in C.reinhardtii were obtained using a reverse genetics approach. The psbH gene was cloned into a suitable vector and an aadA cassette conferring resistance to the antibiotics spectinomycin and streptomycin was placed either within the psbH gene hence entirely knocking out gene function or just downstream of the gene. In the latter of the constructs, a site directed change was made within the gene resulting in a threonine to alanine substitution at the predicted phosphorylation site. Wild type C.reinhardtii cells were then transformed with this construct using the biolistic technique. Transformants were selected for by growth in media containing spectinomycin. Initial analysis of the mutants using Electron Paramagnetic Resonance studies, fluorescence analysis, and oxygen evolution analysis reveals that complete loss of the phosphoprotein results in loss of photosynthetic function for the alga but loss of the phosphorylation site alone does not lead to loss of photosynthetic function.

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