| Cryptochromes are found in many organisms including plants, insects, amphibians, birds, and mammals. Studies show that all cryptochromes function in circadian clock. However, their functions in these organisms are different. In Drosophila and plants, cryptochromes are responsible for circadian photoreception. In mammals, they are part of the oscillator. To study why these conserved protein function differently, I analyzed the roles of cryptochromes in Xenopus laevis, as the organism provides an evolutionary intermediate between flies and mice.; In addition to the similarity to cryptochromes from other species, Xenopus cryptochromes are also very closely related to Xenopus 6-4 photolyase which repairs UV light damaged DNA. Although the only extended difference in sequence between cryptochrome and photolyase is the presence of an extra carboxyl tail in cryptochrome, the cryptochrome and photolyase have mutually exclusive functions.; In this study, I cloned cryptochromes and characterized their expression profiles in Xenopus laevis retina (Chapter II). I examined the roles of conserved functional domains between cryptochrome and photolyase and between cryptochromes from different organisms. My results show that most of these domains are crucial for cryptochrome function (Chapter III). Therefore, despite vastly different functions, how cryptochromes and photolyases function biochemically appear to be quite similar. I also tested the role of cryptochrome's extended carboxyl terminal sequence. I found the tail can regulate cryptochrome subcellular localization possibly through specific protein interaction (Chapter IV).; In total, this work provides insights on how Xenopus cryptochromes function in the retinal clock. Overall, Xenopus and mouse clocks utilize similar proteins. CLOCK/BMAL1 from both species can activate the E-box containing promoters. And both mCRYs and xCRYs can suppress this activation. Since these activation and suppression steps are believed to comprise the central oscillator of the vertebrate animal, this indicates that molecular aspect of the clock is similar in mouse and Xenopus laevis. However, there are some marked differences in their gene expression profiles which suggest that there might be some subtle differences between the Xenopus retinal clock and mouse SCN clock. My mutagenesis analyses also indicate that they have some distinct ways to utilize conserved functional domains. |
