Cadmium (Cd) is a serious environmental heavy metal pollutant due to its toxicity and bioaccumulation. The goal of study was to identify and characterize genes that contribute to the Cd response in the unicellular green alga, Chlamydomonas reinhardtii, a model species with its entire genome sequence determined. Using the linear plasmid pSP124S DNA containing the selectable marker ble gene as a mutagen, I generated over 40,000 transformants with random DNA insertion and isolated many mutants with altered Cd response compared to the wild type. One Cd sensitive mutant, Cds18, and one Cd resistant mutant, CdrB2, each containing a single copy of inserted DNA were selected for further characterization. I located the insertion site at putative CrMRP2 and CrPMA2 in mutant Cds18 and CdrB2 respec...[ Read more ]

Cadmium (Cd) is a serious environmental heavy metal pollutant due to its toxicity and bioaccumulation. The goal of study was to identify and characterize genes that contribute to the Cd response in the unicellular green alga, Chlamydomonas reinhardtii, a model species with its entire genome sequence determined. Using the linear plasmid pSP124S DNA containing the selectable marker ble gene as a mutagen, I generated over 40,000 transformants with random DNA insertion and isolated many mutants with altered Cd response compared to the wild type. One Cd sensitive mutant, Cds18, and one Cd resistant mutant, CdrB2, each containing a single copy of inserted DNA were selected for further characterization. I located the insertion site at putative CrMRP2 and CrPMA2 in mutant Cds18 and CdrB2 respectively. For the putative CrMRP2 and CrPMA2 gene, I cloned and sequenced the genomic region, demonstrated their transcription and isolated the corresponding full length cDNAs. My study showed that the transcription of CrMRP2 was silenced in mutant Cds18 and a truncated transcript for CrPMA2 was generated in mutant CdrB2. The vacuolar location for CrMRP2 was determined by constructing a transformant expressing the CrMRP2-GFP fusion protein. The transcription of each gene and the accumulation of different types of phytochelatin (PC) - Cd complexes in algal cells subjected to Cd treatment were investigated. The involvement of these genes in altered Cd response was confirmed by various complementation tests including heterologous expression of CrMRP2 in a Saccharomyces cerevisiae ΔScYCF1 mutant strain showing Cd sensitivity. I also participated in a collaboration project with scientists at the University of Liege, Belgium for the characterization of a Cd sensitive mutant, Cd34, isolated in their lab and demonstrated the involvement of CrCds1, a gene coded for a half size ABC transporter. Therefore, I have demonstrated at the molecular level the contribution of genes coding for two ABC transporters: CrCds1 and CrMRP2; one P type ATPase: CrPMA2, to the accumulation of high molecular weight PC-Cd complex, a stable Cd sequestration form, in C. reinhardtii. I also compared the predicted structure of C. reinhardtii gene with the corresponding homologs in yeast, the land plant Arabidopsis, and humans. This information has contributed to the understanding and evolution of genes involved in metal homeostasis and tolerance.