Cloning and expression of cryptochrome2 in the bullfrog (Rana catesbeiana)

Cryptochromes (CRYs) are non-opsin photoactive pigments that have recently been implicated in circadian photo-entrainment in humans, mice and Drosophila. In order to study the mechanism of circadian rhythm in amphibians, we have cloned and characterized a Rana cryptochrome in the bullfrog. We isolated a cDNA of about 2.1 kb from a bullfrog brain cDNA library by screening with a partial cry2 cDNA probe obtained by RT-PCR using degenerate primers. The cloned Rana cry2 cDNA has a complete single open-reading frame consisting of 323 amino acids, and its deduced amino acid sequence has a high degree of homology with human and mouse CRY2. Interestingly, we also isolated two other cry2 cDNAs, which may be splicing variants. Rana cry2 is expressed in all tissues as a 2.2 kb transcript. It is particularly highly expressed in the brain and ovaries, and also showed seasonal variation in expression in ovarian tissue. To examine its involvement in circadian rhythm, we tested whether expression in brain tissue followed a light-dark cycle, and found that expression was higher in the dark than in the light. Rana cry2 should therefore, be useful for studying circadian rhythms in seasonally breeding wild animals.