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Molecular and biochemical analysis of the β class carbonic anhydrases in Caenorhabditis elegans

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Abstract

The β 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 β-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 β-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 β-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, CO2/O2). Two genes possibly encoding β-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.

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Acknowledgments

We thank Prof. Y. Kohara from the Kohara Laboratory (NIG, Japan) for providing the cDNA clones yk832 and yk1390. We also thank Dr. N. Tavernarakis from the Tavernarakis Lab (IMBB, Greece) for his assistance and for providing the C. elegans strain N2, the E. coli strains OP50 and HT115 and the pL4440 vector.

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Authors and Affiliations

  1. Department of Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855, Athens, Greece

    Michael K. Fasseas, Daniela Tsikou, Emmanouil Flemetakis & Panagiotis Katinakis

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  1. Michael K. Fasseas
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  2. Daniela Tsikou
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  3. Emmanouil Flemetakis
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  4. Panagiotis Katinakis
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Correspondence to Panagiotis Katinakis.

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Fasseas, M.K., Tsikou, D., Flemetakis, E. et al. Molecular and biochemical analysis of the β class carbonic anhydrases in Caenorhabditis elegans . Mol Biol Rep 37, 2941–2950 (2010). https://doi.org/10.1007/s11033-009-9857-z

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  • DOI: https://doi.org/10.1007/s11033-009-9857-z

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