Michael McCormick and James Moroney, Biological Sciences

Generation and Characterization of Insertional Mutants in Chlamydomonas reinhardtii that Cannot Grow on Air Levels of CO2

A basic approach in genomic studies is to identify a mutant strain defective in some process and compare that strain to the wild-type organism. In Chlamydomonas reinhardtii, the process of interest is the Carbon Concentrating Mechanism (CCM). The function of the CCM is to capture and concentrate various forms of inorganic carbon around Rubisco, the main CO₂ fixing enzyme, in preparation for photosynthesis. It has been found that under low CO₂ conditions, the genes responsible for activating the CCM are up-regulated. By utilizing insertional mutagenesis, it is possible to interrupt genes that are involved in the CCM and create mutants that are defective in this process. The phenotypic evidence for such transformants can be seen in colonies that grow well on elevated CO₂ but minimally on low CO2. The insert used in these screenings was the APHVIII gene, which encodes resistance to the antibiotic paromomycin. In the screen employed in this study, mutants that showed a weak growth phenotype under low CO2 conditions and resistance to paromomycin were selected. In some of these mutants, the inserted APHVIII gene has possibly disrupted a CCM gene. Once a promising mutant is obtained, the site of DNA insertion can be identified using iPCR and Adaptor PCR methods. Both methods operate by exploiting insert-specific primers to amplify the region flanking the insert. About 70 transformants were generated that showed continuous poor growth under low CO₂ conditions. Finally, the sequences that have been obtained will be matched up to the Chlamydomonas database in order to determine where the insert landed in the genome. The location of the insert in two mutants has been determined. One insert is in a gene called cia7, which appears to be associated with the CCM and another is in the RbcS2 gene which encodes the Rubisco small subunit.