| The circadian clock is entrained by environmental stimuli such as the daily light dark cycle, involving different photoreceptors. Among them, cryptochromes (CRYs) play an important role in the circadian systems of a variety of model organisms. Commonly, CRYs share major sequence similarity to DNA photolyases, but in general do not comprise activity. In Arabidopsis thaliana and Drosophila melanogaster, CRYs act as blue- and ultraviolet-light photoreceptors to entrain their internal clock (Lin and Todo, Genome Biol, 2005, 6: 220). Contrary, in mammals CRY represents an integral component of the central circadian oscillator. Homology studies in C. reinhardtii revealed both, a plant-like CRY with a carboxyl terminal extension as well as an animal-like CRY (Mittag et al., Plant Physiol, 2005, 137: 399-409). Hence it is challenging to investigate the role of both CRYs in C. reinhardtii. In a first step, we want to analyze the localization of both proteins inside the cell over the circadian cycle. Whether the CRYs are localized throughout the cytoplasm or are translocated to the nucleus will give important hints regarding their functions. Therefore, the animal-like CRY gene was cloned into a vector (kindly provided by: Lechtreck et al., Cell Motil Cytoskeleton, 2009, 66: 469-82) by adding a triple-HA tag to its C-terminus according to the vectors specifications. The construct was then transformed to wild type C. reinhardtii. We are now on our way to characterize the transgenic lines, which will then be subjected to indirect immunofluorescence with anti-HA antibodies to unravel the localization pattern during the circadian cycle. |