Altered entrainment and feedback loop function effected by a mutant period protein.

The period (per) and timeless (tim) genes encode interacting components of the circadian clock. Levels and phosphorylation states of both proteins cycle with a circadian rhythm, and the proteins drive cyclic expression of their RNAs through a feedback mechanism that is, at least in part, negative. We report here that a hypophosphorylated mutant PER protein, produced by creating a small internal deletion, displays increased stability and low-amplitude oscillations, consistent with previous reports that phosphorylation is required for protein turnover. In addition, this protein appears to be defective in feedback repression because it is associated with relatively high levels of RNA and high levels of TIM. Transgenic flies carrying the mutant PER protein display a temperature-dependent shortening of circadian period and are impaired in their response to light, particularly to pulses of light in the late night that normally advance the phase of the rhythm. Interestingly, per RNA is induced by light in these flies, most likely because of the removal of the light-sensitive TIM protein, thus implicating a more direct role for TIM in transcriptional inhibition. These data have relevance for mechanisms of feedback repression, and they also address existing models for the differential behavioral response to light at different times of the night.

A role for the proteasome in the light response of the timeless clock protein.

The cyclic expression of the period (PER) and timeless (TIM) proteins is critical for the molecular circadian feedback loop in Drosophila. The entrainment by light of the circadian clock is mediated by a reduction in TIM levels. To elucidate the mechanism of this process, the sensitivity of TIM regulation by light was tested in an in vitro assay with inhibitors of candidate proteolytic pathways. The data suggested that TIM is degraded through a ubiquitin-proteasome mechanism. In addition, in cultures from third-instar larvae, TIM degradation was blocked specifically by inhibitors of proteasome activity. Degradation appeared to be preceded by tyrosine phosphorylation. Finally, TIM was ubiquitinated in response to light in cultured cells.

Alterations of per RNA in noncoding regions affect periodicity of circadian behavioral rhythms.

Circadian rhythms in Drosophila depend on a molecular feedback loop that includes products of the period (per) and timeless (tim) genes. RNA and protein products of both genes cycle with a circadian period and the proteins feedback to inhibit expression of their own mRNAs. While cyclic expression of PER protein appears to be necessary for rhythmic behavior, the function of per RNA cycling is somewhat controversial. Rhythmic transcription accounts, in part, for cycling of per RNA, but it is clear now that posttranscriptional mechanisms also contribute to the cyclic expression of both per RNA and protein. As posttranscriptional mechanisms, such as mRNA stability and translation, are frequently mediated by 3' untranslated regions (UTR) of genes, the authors examined the role of this region of per in the regulation of circadian rhythms. Removal of most of per's 3' UTR had a small effect on the function of a per transgene. However, replacement of per's 3'UTR with corresponding sequences of the tubulin gene led to the rescue of behavioral rhythms in per01 flies with periods that were 3 h shorter than those generated by a wild-type per transgene. The hybrid RNA cycles, but the protein produced by it accumulates earlier in a day-night cycle than the PER protein produced by a control per transgene carrying its own 3'UTR, perhaps because the tubulin sequences counteract the effect of destabilizing elements in the per RNA at earlier points in the circadian cycle. These data indicate that the appropriate regulation of per RNA expression, effected by transcriptional as well as posttranscriptional mechanisms, is critical for the determination of circadian period.

Regulation of the Drosophila protein timeless suggests a mechanism for resetting the circadian clock by light.

Circadian behavioral rhythms in Drosophila depend on the appropriate regulation of at least two genes, period (per) and timeless (tim). Previous studies demonstrated that levels of PER and TIM RNA cycle with the same phase and that the PER and TIM proteins interact directly. Here we show the cyclic expression of TIM protein in adult heads and report that it lags behind peak levels of TIM RNA by several hours. We alsoshow that nuclear expression of TIM depends upon the expression of PER protein. Finally, we report that the expression of TIM, but not PER, is rapidly reduced by light, suggesting that TIM mediates light-induced resetting of the circadian clock. Since both PER and TIM RNA are unaffected by light treatment, the effects of light on TIM appear to be posttranscriptional.

Rhythmic expression of timeless: a basis for promoting circadian cycles in period gene autoregulation.

The clock gene timeless (tim) is required for circadian rhythmicity in Drosophila. The accumulation of tim RNA followed a circadian rhythm, and the phase and period of the tim RNA rhythm were indistinguishable from those that have been reported for per. The tim RNA oscillations were found to be dependent on the presence of PER and TIM proteins, which demonstrates feedback control of tim by a mechanism previously shown to regulate per expression. The cyclic expression of tim appears to dictate the timing of PER protein accumulation and nuclear localization, suggesting that tim promotes circadian rhythms of per and tim transcription by restricting per RNA and PER protein accumulation to separate times of day.

Pharmacologically distinct sodium-dependent L-[3H]glutamate transport processes in rat brain.

The transport of L-[3H]glutamate into crude synaptosomal membrane fractions prepared from cerebellum, brainstem, hippocampus, cortex, striatum, and midbrain was characterized. In all brain regions, greater than 95% of the accumulation of radiolabel was sodium-dependent and the concentration-dependence was consistent with a single high affinity site. Dihydrokainate and L-alpha-aminoadipate were region specific inhibitors of uptake; this inhibition was consistent with a competitive mechanism. In the forebrain regions examined, dihydrokainate inhibited transport with IC50s of approx. 100 microM (range from 80 to 170 microM). Transport in cerebellum was essentially dihydrokainate-insensitive L-alpha-Aminoadipate inhibited transport in forebrain regions with IC50s of approx. 700 microM (range from 590 to 800 microM) and inhibited transport in cerebellum with an IC50 of 40 microM. The inhibition data obtained with forebrain and cerebellar tissues were consistent with nearly homogeneous (greater than 80%) populations of non-interacting sites. Inhibition data obtained with tissue prepared from brainstem were best fit to a mixture of the two sites (35-50% of the type observed in cerebellum). Other previously identified uptake inhibitors, including DL-threo-hydroxyaspartate, L-aspartate-beta-hydroxamate, beta-glutamate, and L-cysteine sulfinate were not selective for the two types of transport. These data demonstrate that there are two pharmacologically distinct sodium-dependent high affinity transport systems with heterogeneous regional distributions.