Ralley, Louise; (1996) Nuclear Photosynthetic Mutants of Chlamydomonas reinhardtii. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In a previous study (Gumpel et al., 1995) a collection of nuclear photosynthetic mutants of Chlamydomonas reinhardtii were generated using an insertional mutagenesis technique. Subsequent biophysical and fluorescence analysis identified mutants that were affected in either photosystem II, photosystem I or the cytochrome b6f complex. A detailed genetic analysis of the latter mutants identified three (MΦ11, MΦ3O and MΦ37) in which the mutant phenotype co-segregates with the insertional mutagen. Protein and RNA analysis revealed that MΦ11 fails to accumulate transcript for the chloroplast gene (petA) encoding the apoprotein of cytochrome f, whereas MΦ37 shows a severely reduced accumulation of the transcript for the chloroplast gene (petB) encoding the apoprotein of cytochrome b6. In this study, the collection of mutants have been further characterised: i) The photosystem I mutants have been further characterised by fluorescence and EPR spectroscopy and by Southern and western blot analysis. The results indicate that a functional PSI complex fails to assemble but that none of the nuclear genes encoding known PSI subunits are affected, suggesting that nuclear genes other than those encoding structural components are required for PSI biogenesis. ii) Using DNA probes isolated from the genomic region flanking the insertional mutagen in mutant MΦll, a cosmid wildtype genomic library was screened. A 42kb cosmid (termed B5) was shown to rescue the mutant phenotype when transformed into the nuclear genome. B5 transformants show restored photosynthetic function and were able to grow photoautotrophically on minimal medium. Protein analysis of the transformants showed the presence of the cytochrome f protein. Restriction analysis of B5 combined with complementation studies identified a 18kb genomic fragment that retained the ability to complement the mutant. This fragment was used to screen a wildtype C. reinhardtii cDNA library. Two independent cDNA clones were obtained, named ACV4 and ACV5. Southern analysis showed that the gene encoding ACV4 showed restriction fragment length polymorphisms when comparing the genomes of wildtype cells and MΦ11. In the case of ACV5, the genomic sequence corresponding to this cDNA was completely lacking from the mutant, indicating that insertion of the mutagen has lead to the deletion of genomic DNA. Northern analysis revealed that the ACV5 transcript is also absent in MΦ11, whereas the ACV4 transcript was undetectable in both wildtype and mutant cells. Sequence analysis was initiated on both cDNAs and indicates that both appear not to be full-length. In the case of ACV5, the deduced protein sequence has revealed some homology to known nucleic acid binding protein sequences in the database. iii) Southern analysis was carried out on mutant MΦ37 in order to generate a restriction map covering the site of integration of the mutagen. This enabled the cloning of a flanking genomic fragment and the screening of the wildtype cosmid library. A number of hybridising clones were isolated, however none were able to complement the mutant. Simultaneously, a plasmid rescue strategy was also employed to attempt to isolate flanking genomic DNA. However, this too failed to isolate a complementing cosmid clone. Possible reasons for this failure are discussed.

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