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

• Published in: Molecular biology reports 2011-03, Vol.38 (3), p.1777-1785
• Main Authors: Fasseas, Michael K, Tsikou, Daniela, Flemetakis, Emmanouil, Katinakis, Panagiotis
• Format: Article
• Language: English
• Subjects: Animal Anatomy; Animal Biochemistry; Animals; Biomedical and Life Sciences; C. elegans; Caenorhabditis elegans - enzymology; Caenorhabditis elegans - genetics; Caenorhabditis elegans - growth & development; Caenorhabditis elegans Proteins - chemistry; Caenorhabditis elegans Proteins - genetics; Caenorhabditis elegans Proteins - metabolism; carbonate dehydratase; Carbonic Anhydrases - chemistry; Carbonic Anhydrases - genetics; Carbonic Anhydrases - metabolism; Electrophoresis, Polyacrylamide Gel; Enzyme Assays; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Genetic Complementation Test; Histology; Life Sciences; Longevity; Morphology; Nematoda; RNA Interference; RNA, Messenger - genetics; RNA, Messenger - metabolism; Saccharomyces cerevisiae - genetics; Sequence Alignment; Sequence Analysis, Protein; Stress; α Class
• ISSN: 0301-4851; 1573-4978
• DOI: 10.1007/s11033-010-0292-y

In this study, in silico analysis of the Caenorhabditis elegans genome revealed six genes (cah-1, cah-2, cah-3, cah-4, cah-5, and cah-6) possibly encoding α class CAs (carbonic anhydrase). Real-time RT-PCR analysis revealed the temporal expression pattern of each gene, as well as changes in expression levels under different atmospheric conditions (stress). Cah-3 and cah-4 showed the highest levels of transcript accumulation, while most genes responded to the stress conditions. Yeast complementation showed that cah-3 was able to complement the function of Saccharomyces cerevisiae CA (NCE103) in vivo. Recombinant CAH-3, CAH-4a and CAH-5 enzymes, expressed in Escherichia coli were used for in vitro measurement of CA activity. However, in vitro activity was only detectable for CAH-4a. RNAi by feeding was performed on wild-type C. elegans for all genes. The worms were examined for a visible phenotype under normal and stress conditions (pH, CO₂/O₂). Silencing cah-3 and cah-4 may reduce the life-span of the worms (at 22°C).