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 pools from an indexed cosmid library of wild-type genomic DNA. Transformation of mutant cells with cosmid DNA from two microtiter plates in the library produced colonies that grew phototrophically at atmospheric CO2 levels. Transformations with cosmid DNA from each of the rows and files of the two plates pinpointed one well in each plate with a cosmid bearing the targeted gene. Sequencing of cosmid subclones revealed a gene 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 presence of a mutation in the 5'-half of the gene. Comparison of mutant and wild-type gene sequences in this region revealed a G-to-A substitution in the mutant gene, which produced a nonsense codon. The data presented demonstrate that the carbonic anhydrase produced from the CAH3 gene is essential to the inorganic carbon-concentrating mechanism in C. reinhardtii and that genomic complementation can be a facile and efficient means for isolating genes associated with defects affecting photosynthesis and other physiological processes in this eukaryotic green alga.
Funke
R P
RP
Department of Biochemistry, University of Nebraska, Lincoln 68588-0665, USA.
Kovar
J L
JL
Weeks
D P
DP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002245:Carbon Dioxide / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003360:Cosmids; D016054:DNA, Protozoan / Q000235:genetics; D017125:Genes, Protozoan; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D010788:Photosynthesis / Q000235:genetics / Q000502:physiology*; D014170:Transformation, Genetic
D016054:DNA, Protozoan; D002245:Carbon Dioxide; D002256:Carbonic Anhydrases