INTRACELLULAR CARBONIC-ANHYDRASE IS ESSENTIAL TO PHOTOSYNTHESIS IN CHLAMYDOMONAS-REINHARDTII AT ATMOSPHERIC LEVELS OF CO2 - DEMONSTRATION VIA GENOMIC COMPLEMENTATION OF THE HIGH-CO2-REQUIRING MUTANT CA-1

Genomic complementation of the high-CO2-requiring mutant ca-1-12-1C of Chlamydomonas reinhardtii was achieved by transformation with DNA poo ls from an indexed cosmid library of wildtype genomic DNA. Transformat ion of mutant cells with cosmid DNA from two microtiter plates in the library produced colonies that grew phototrophically at atmospheric CO 2 levels. Transformations with cosmid DNA from each of the rows and fi les of the two plates pinpointed one well in each plate with a cosmid bearing the targeted gene. Sequencing of cosmid subclones revealed a g ene encoding a recently identified C. reinhardtii chloroplast carbonic anhydrase (CAH3). Transformations with chimeric constructs combining different portions of the wild-type and mutant genes indicated the pre sence of a mutation in the 5'-half of the gene. Comparison of mutant a nd wild-type gene sequences in this region revealed a C-to-A substitut ion in the mutant gene, which produced a nonsense codon. The data pres ented demonstrate that the carbonic anhydrase produced from the CAH3 g ene is essential to the inorganic carbon-concentrating mechanism in C. reinhardtii and that genomic complementation can be a facile and effi cient means for isolating genes associated with defects affecting phot osynthesis and other physiological processes in this eukaryotic green alga.