A key temporal delay in the circadian cycle of Drosophila is mediated by a nuclear localization signal in the timeless protein

Regulated nuclear entry of the Period (PER) and Timeless (TIM) proteins, two components of the Drosophila circadian clock, is essential for the generation and maintenance of circadian behavior. PER and TIM shift from the cytoplasm to the nucleus daily, and the length of time that PER and TIM reside in the cytoplasm is an important determinant of the period length of the circadian rhythm. Here we identify a TIM nuclear localization signal (NLS) that is required for appropriately timed nuclear accumulation of both TIM and PER. Transgenic flies with a mutated TIM NLS produced circadian rhythms with a period of ～30 hr. In pacemaker cells of the brain, PER and TIM proteins rise to abnormally high levels in the cytoplasm of tim(ΔNLS) mutants, but show substantially reduced nuclear accumulation. In cultured S2 cells, the mutant TIM(ΔNLS) protein significantly delays nuclear accumulation of both TIM and wild-type PER proteins. These studies confirm that TIM is required for the nuclear localization of PER and point to a key role for the TIM NLS in the regulated nuclear accumulation of both proteins.     

The clock gene period of the housefly,Musca domestica,rescues behavioral rhythmicity in Drosophila melanogaster. Evidence for intermolecular coevolution?

In Drosophila, the clock gene period (per), is an integral component of the circadian clock and acts via a negative autoregulatory feedback loop. Comparative analyses of per genes in insects and mammals have revealed that they may function in similar ways. However in the giant silkmoth, Antheraea pernyi, per expression and that of the partner gene, tim, is not consistent with the negative feedback role. As an initial step in developing an alternative dipteran model to Drosophila, we have identified the per orthologue in the housefly, Musca domestica. The Musca per sequence highlights a pattern of conservation and divergence similar to other insect per genes. The PAS dimerization domain shows an unexpected phylogenetic relationship in comparison with the corresponding region of other Drosophila species, and this appears to correlate with a functional assay of the Musca per transgene in Drosophila melanogaster per-mutant hosts. A simple hypothesis based on the coevolution of the PERIOD and TIMELESS proteins with respect to the PER PAS domain can explain the behavioral data gathered from transformants.