CHLAMYDOMONAS REINHARDTII MUTANTS THAT REQUIRE ELEVATED CO 2 FOR OPTIMAL GROWTH

Colombo, S. L., Pollock, S. V., Eger, K., Mason, C. B. & Moroney J. V. Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70808 USA Chlamydomonas reinhardtii possesses a CO 2 concentrating mechanism that enables it to grow at very low CO 2 concentrations. In order to identify genes required for growth at low CO 2 , an extensive screening of C. reinhardtii insertional mutants was conducted. The D66 strain (nit2 ‐ , cw15, mt  + ) was transformed by electroporation using a plasmid containing a gene that confers resistance to the antibiotic Zeocin. About 42,000 primary transformants were screened. These transformants were first grown on acetate in the dark. The colonies were then tested for their ability to grow photosynthetically on elevated CO 2 or very low levels of CO 2 (100 ppm). About 80 mutants were identified which were unable to grow well at low CO 2 concentrations. Another 80 mutants were unable to grow photoautotrophically at high or low CO 2 concentrations. The location of the inserted DNA is being determined using a combination of inverse PCR and TAIL PCR. Using these methods, one can rapidly locate the inserted DNA in the genome and identify the gene that has been disrupted by the insertion. One of the mutants has an insertion in the gene that encodes Rubisco Activase. Our evidence suggests that Rubisco Activase is required for growth under low levels of CO 2 . Supported by NSF grant IBN‐09904425 to JVM and a postdoctoral fellowship to SLC from Fund Antorchas.