Abstract

This thesis initiated a long term project that uses the green alga Chlamydomonas reinhardtii to identify new mechanisms of defence to toxic trace metals and pathways that regulate these responses. Strains of C. reinhardtii that express reporter genes under the control of alga's metal response were generated and analysed. The reporter gene, an Arg7 cDNA minigene, restores arginine prototrophy following its insertion into random sites of the C. reinhardtii genome where it is expressed from resident regulatory sequences. Cells were selected and screened by for rare insertions that express the reporter gene only in the presence of bioavailable metal. For all eight transformants induction of the arg+ phenotype is not specific to particular metal ions. Southern analysis showed that two strains, Ni70 and Cd3600, have only one insertion of the reporter gene. Northern analysis showed that Ni70 and Cd3600 activate expression of the introduced Arg7 cDNA, at the RNA level by nickel and cadmium. Initial results, from a real time RT-PCR assay on strain Ni70, demonstrated that increasing concentrations of nickel or cadmium results in an increase of the reporter gene expression. Therefore, Ni70 indeed appears to have the reporter gene inserted under the influence of promoter/enhancer which is activated by metal exposure.