Abstract

Chlamydomonas reinhardtii Dangeard possesses a CO₂ concentrating mechanism (CCM) that enables it to grow at very low CO₂ concentrations. In previous studies, insertional mutagenesis was successfully used to identify genes required for growth at low CO₂ in C. reinhardtii. These earlier studies used the C. reinhardtii genes, Nit1 and Arg7 to complement nit1^– or arg7^– strains, thereby randomly inserting a second copy of Nit1 or Arg7 into the genome. Because these genes are already present in the C. reinhardtii genome, it was often difficult to identify the location of the inserted DNA and the gene disrupted by the insertion. We have developed a transformation protocol using the Ble^R gene, which confers resistance to the antibiotic Zeocin. The insertion of this gene allows one to use a variety of existing polymerase chain reaction (PCR) methodologies to identify the disrupted gene. In this study the D66 strain (nit2^–, cw15, mt⁺) was transformed by electroporation using a plasmid containing the Ble^R gene. Primary transformants (42 000) were obtained after growth in the dark on acetate plus Zeocin medium. Colonies were then tested for their ability to grow photosynthetically on elevated CO₂ or low levels of CO₂ (100 ppm). About 120 mutants were identified which grew on elevated CO₂ but were unable to grow well at low CO₂ concentrations. About 50% of these mutants had low affinities for inorganic carbon as assessed by K_0.5(CO₂), indicating a potential defect in the CCM. The location of the inserted DNA is being determined using inverse PCR (iPCR) and thermal asymmetric interlaced (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.