Bateman, Joseph Matthew; (1999) The maintenance and expression of foreign genes in the chloroplast of Chlamydomonas. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The chloroplast of the unicellular green alga Chlamydomonas reinhardtii is readily amenable to molecular genetic analysis. Using particle-gun-bombardment, exogenous DNA can be introduced into the chloroplast where it will recombine into the organellar genome. Coupled with the high levels of homologous recombination this allows precisely targeted insertion of introduced DNA anywhere into this 196 kbp genome. Several different chloroplast expression vectors have been constructed in order to introduce and ectopically express foreign genes within the Chlamydomonas chloroplast. One of these genes, aphA-6, a eubacterial aminoglycoside antibiotic resistance gene, confers resistance to kanamycin and amikacin in transformed cells and so can be used as a dominant selectable marker for chloroplast transformation. Experiments to demonstrate the utility of this new marker to chloroplast molecular genetics were also carried out. The same expression vector was used in an attempt to express the protochlorophyllide oxidoreductase (POR) gene from Synechocystis sp. PCC 6803 in a chlorophyll-less Chlamydomonas double mutant (pc-ly-7) lacking active POR. In addition, the tufA gene encoding the protein elongation factor EF-Tu, from the plastid of the Apicomplexan Plasmodium falciparum was introduced into the chloroplast. This was carried out in order to attempt a functional replacement of the endogenous chloroplast EF-Tu. The results of the introduction and expression of these two foreign genes are discussed. Finally, an experiment is described which attempted to isolate cells in which chloroplast DNA had transferred to the nuclear genome. It was hoped that this would serve as a model for the process occurring throughout chloroplast evolution by which the majority of plastid genes have become nuclear encoded. To achieve this the 'ble' selectable marker for nuclear transformation in Chlamydomonas, conferring zeomycin resistance, was introduced into the chloroplast genome where it was not functional. Cells in which this marker had moved to the nuclear genome were then selected by their ability to survive on zeomycin containing medium. The results of this screen are discussed.

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