cDNA cloning, sequence, and expression of carbonic anhydrase in Chlamydomonas reinhardtii: regulation by environmental CO2 concentration.
cDNA clones for the periplasmic carbonic anhydrase (CA; carbonate hydro-lyase, EC 4.2.1.1) of Chlamydomonas reinhardtii cells were isolated and characterized. The fact that the cloned cDNA fragments encoded a 377-amino acid polypeptide (41,626 Da) consisting of an NH2-terminal hydrophobic signal peptide of 20 amino acids, a large (35,603 Da) subunit polypeptide, and a small (4144 Da) subunit polypeptide indicates that the two subunits are cotranslated as a precursor polypeptide. The amino acid sequence of mature subunits deduced from the nucleotide sequence showed 20-22% homology with human CA isozymes (CAI, CAII, and CAIII). Three zinc-liganded histidine residues and those forming a hydrogen-bond network to zinc-bound solvent molecules were highly conserved in the plant and animal enzymes. Three possible asparagine-linked glycosylation sites were found in the large subunit. Northern blot analysis was carried out using the cDNA fragment as a probe. The level of 2.0-kilobase CA mRNA increased within 1 hr when CO2 concentration of the bubbling gas was changed from 5% to 0.04% (air level) in the presence of light. On the other hand, CA mRNA did not accumulate when CO2 concentration was lowered in the dark. Experiments using 3-(3,4-dichlorophenyl)-1,1-dimethylurea showed that photosynthesis is absolutely required for the accumulation of CA mRNA. These results indicate that CA biosynthesis is regulated by changes in environmental CO2 concentration as well as light at the level of mRNA abundance.
Fukuzawa
H
H
Institute of Applied Microbiology, University of Tokyo, Japan.
Fujiwara
S
S
Yamamoto
Y
Y
Dionisio-Sese
M L
ML
Miyachi
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002245:Carbon Dioxide / Q000502:physiology*; D002256:Carbonic Anhydrases / Q000235:genetics*; D002696:Chlamydomonas / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic
D012333:RNA, Messenger; D002245:Carbon Dioxide; D004247:DNA; D002256:Carbonic Anhydrases