In the fruit fly, Drosophila melanogaster, circadian (~24 hour) rhythms of eclosion and rest:activity are controlled by an endogenous clock that is generated through the coordinated action of specific genes. Dominant among these clock genes are the period (per) and timeless (tim) genes, both of which encode RNAs and proteins that cycle with a circadian rhythm. The two proteins (PER and TIM) interact directly and feed back to negatively regulate synthesis of their own mRNAs. The feedback loop thus generated is thought to constitute the molecular basis of the clock. Entarinemnt of this molecular clock to the day:night cycle is achieved through light-induced degradation of the TIM protein. We recently showed that TIM is degraded by the proteasome in response to light (1). Prior to its degradation by the proteasome, TIM is phosphorylated on tyrosine residues and ubiquitinated. In order to determine the mechanisms that transduce light to TIM, we investigated the role of cryptochrome (CRY) in TIM degradation. Cryptochrome is a flavin-binding molecule that is thought to function as a circadian photoreceptor in Drosophila. In support of this, flies carrying a mutation in the flavin-binding site of CRY show deficits in entrainment (2). In addition, TIM oscillations in the presence of light:dark cycles are abolished in these flies such that TIM levels remain high during the day (2). This indicates that light-induced TIM degradation is affected. We used a cell culture system to investigate the role of CRY in TIM degradation. We found that CRY, but not CRY b increases TIM ubiquitination in a dose-dependent manner. Overexpression of CRY, however, downregulates TIM ubiquitination perhaps because CRY acts as a dominanat negative under these conditions. After transducing the photic signal which leads to TIM ubiquitination, CRY itself is degraded by the proteasome. These data and other relevant recent studies will be discussed.

Index terms: Drosophila melanogaster, circadian rhythms, TIM, CRY, proteasome.

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