Molecular analysis of the light regulation of Drosophila cryptochrome

Drosophila CRYPTOCHROME (dCRY) is the light receptor in the Drosophila central clock. In response to a light signal dCRY interacts with the clock component TIMELESS (TIM), targeting TIM for degradation thus delaying the progress of the molecular oscillations that underlie the clock, until dark-associated repression of dCRY function allows TIM to accumulate. The mechanism by which dCRY is regulated by light is unknown, though recent evidence indicates the important of the unique C-terminal domain. To explore the manner by which the C-terminus regulates dCRY, chimerics were created with the mouse CRYFTOCHROME 1 (mCRYl) whereby the unique C-terminuses were exchanged. mCRYl is a component of the central clock and has not been found to be a photoreceptor or light regulated. Yeast assays using dCRY/mCRY variants against the clock components Drosophila PERIOD (PER) and TIM reveal that the mCRY C-terminal domain interacts with PER light-independently, and that the dCRY photolyase domain can mediate a light signal to regulate this interaction when expressed as a chimeric construct. Extending the study to transgenic fly lines expressing dCRY with a mCRYl C-terminus, lines demonstrate a behavioral profile and TIM abundance profile similar to that of flies in constant light. This raises further questions over the light regulation of dCRY but provides no definitive answers. In a concurrent study, putative sites for post translation modification in the C-terminal domain of dCRY were determined using a predictive algorithm and altered through site-directed mutagenesis. The dCRY variants were tested as bait against PER and TIM in yeast assays and in a semi-quantitative yeast assay. The assays revealed that residue position 530 may be important in the light regulation of dCRY. Finally, an anti-body against dCRY was created by expressing the protein as three fragments that together represented the whole protein in E. coli. Two of the fragments representing the first and last third of the dCRY protein were successfully expressed, purified and sent to a commercial company used to successfully generate an antibody.