Molecular and biochemical analysis of the beta class carbonic anhydrases in Caenorhabditis elegans.
The beta class of the carbonic anhydrase (CA) enzyme family has been found in plants, yeast, bacteria and algae, but not in animals. Also, little is known concerning the CAs of C. elegans. Genes possibly encoding beta-CAs were revealed by in silico analysis of the C. elegans genome. Amino acid sequence and 3D structure analysis revealed a resemblance to both plant and cab-type beta-CAs. Temporal expression patterns of the two genes, as well as changes in expression levels under different atmospheric conditions (stress) were analyzed by real-time RT-PCR. Recombinant enzymes, expressed in E. coli were used for in vitro measurement of CA activity, while a yeast complementation experiment was performed in order to assess their ability to complement the function of S. crevisieae beta-CA (NCE103) in vivo. RNAi by feeding was performed on wild-type populations that were then examined for a visible phenotype under normal or various stress conditions (pH, CO(2)/O(2)). Two genes possibly encoding beta-CAs were revealed (bca-1 and y116a8c.28). Their products contain elements of both plant and cab-type CAs. Both assays showed that Y116a8c.28 is an active CA. Both genes showed significant levels of transcript accumulation during development, while they also responded to the stress conditions. No visible phenotype was scored under normal or stress conditions.
Fasseas
Michael K
MK
Department of Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece.
Tsikou
Daniela
D
Flemetakis
Emmanouil
E
Katinakis
Panagiotis
P
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019295:Computational Biology; D057075:Enzyme Assays; D004926:Escherichia coli / Q000378:metabolism; D005247:Feeding Behavior; D015971:Gene Expression Regulation, Enzymologic; D005816:Genetic Complementation Test; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D016415:Sequence Alignment
D012333:RNA, Messenger; D002256:Carbonic Anhydrases