Molecular and biochemical analysis of the α class carbonic anhydrases in Caenorhabditis elegans.
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, CO(2)/O(2)). Silencing cah-3 and cah-4 may reduce the life-span of the worms (at 22 °C).
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
D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D005816:Genetic Complementation Test; D008136:Longevity; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
D029742:Caenorhabditis elegans Proteins; D012333:RNA, Messenger; D002256:Carbonic Anhydrases