Phosphorylation of period is influenced by cycling physical associations of double-time, period, and timeless in the Drosophila clock.

The clock gene double-time (dbt) encodes an ortholog of casein kinase Iepsilon that promotes phosphorylation and turnover of the PERIOD protein. Whereas the period (per), timeless (tim), and dClock (dClk) genes of Drosophila each contribute cycling mRNA and protein to a circadian clock, dbt RNA and DBT protein are constitutively expressed. Robust circadian changes in DBT subcellular localization are nevertheless observed in clock-containing cells of the fly head. These localization rhythms accompany formation of protein complexes that include PER, TIM, and DBT, and reflect periodic redistribution between the nucleus and the cytoplasm. Nuclear phosphorylation of PER is strongly enhanced when TIM is removed from PER/TIM/DBT complexes. The varying associations of PER, DBT and TIM appear to determine the onset and duration of nuclear PER function within the Drosophila clock.     

A temperature-dependent timing mechanism is involved in the circadian system that drives locomotor rhythms in the fruit fly Drosophila melanogaster

The circadian clock of Drosophila melanogaster is thought to include rhythmic expression of period gene. Recent studies suggested, however, that a per-less oscillation is also involved in the regulation of circadian locomotor rhythms. In the present study, we examined the existence and the property of the possible per-less oscillation using arrhythmic clock mutant flies carrying per (01), tim(01), dClk(Jrk) or cyc(01), which lack rhythmic per expression. When temperature cycles consisting of 25 degrees C and 30 degrees C with various periods (T=8-32 hr) were given, wild-type (Canton-S) flies showed locomotor rhythms entrained to temperature cycles over a wide range of period (T=8-32 hr) in constant light (LL) while only to T=24 hr in constant darkness (DD). The mutant flies showed rhythms synchronizing with the given cycle both under LL and DD. In per(01) and tim(01) flies, the phase of a major peak slightly changed dependent on Ts in DD, while it did not in dClk(Jrk) and cyc(01) flies. When they were transferred from a constant temperature to a temperature cycle under DD, several cycles were necessary to establish a clear temperature entrainment in per(01) and tim (01) flies. These results suggest that per(01) and tim(01) flies have a temperature-entrainable weak oscillatory mechanism and that the per-less oscillatory mechanism may require dClk and cyc. In addition, per (01) and tim(01) flies changed from thermoactive in DD to cryoactive in LL, while dClk(Jrk) and cyc(01) flies did not. It is thus suggested that dClk and cyc are also involved in determining the light-associated temperature preference in per(01) and tim(01) flies.     

A novel circadianly expressed Drosophila melanogaster gene dependent on the period gene for its rhythmic expression.

The Drosophila melanogaster period (per) gene is required for expression of endogenous circadian rhythms of locomotion and eclosion. per mRNA is expressed with a circadian rhythm that is dependent on Per protein; this feedback loop has been proposed to be essential to the central circadian pacemaker. This model would suggest the Per protein also controls the circadian expression of other genetic loci to generate circadian behavior and physiology. In this paper we describe Dreg-5, a gene whose mRNA is expressed in fly heads with a circadian rhythm nearly identical to that of the per gene. Dreg-5 mRNA continues to cycle in phase with that of per mRNA in conditions of total darkness and also when the daily feeding time is altered. Like per mRNA, Dreg-5 mRNA is not expressed rhythmically in per null mutant flies. Dreg-5 encodes a novel 298 residue protein and Dreg-5 protein isoforms also oscillate in abundance with a circadian rhythm. The phase of Dreg-5 protein oscillation, however, is different from that of Per protein expression, suggesting that Dreg-5 and per have common translational but different post-translational control mechanisms. These results demonstrate that the per gene is capable of modulating the rhythmic expression of other genes; this activity may form the basis of the output of circadian rhythmicity in Drosophila.     

Robust oscillations within the interlocked feedback model of Drosophila circadian rhythm

A mechanism for generating circadian rhythms has been of major interest in recent years. After the discovery of per and tim, a model with a simple feedback loop involving per and tim has been proposed. However, it is recognized that the simple feedback model cannot account for phenotypes generated by various mutants. A recent report by Glossop, Lyons & Hardin [Science286, 766 (1999)] on Drosophila suggests involvement of another feedback loop by dClk that is interlocked with per-tim feedback loop. In order to examine whether interlocked feedback loops can be a basic mechanism for circadian rhythms, a mathematical model was created and examined. Through extensive simulation and mathematical analysis, it was revealed that the interlocked feedback model accounts for the observations that are not explained by the simple feedback model. Moreover, the interlocked feedback model has robust properties in oscillations.     

New short period mutations of the Drosophila clock gene per.

Earlier work has indicated that the period length of Drosophila circadian behavioral rhythms is dependent on the abundance of the period (per) gene product. Increased expression of this gene has been associated with period shortening for both the circadian eclosion (pupal hatching) rhythm and circadian locomotor activity rhythms of adult Drosophila. In this study it is shown that a wide variety of missense mutations, affecting a region of the per protein consisting of approximately 20 aa, predominantly generate short period phenotypes. The prevalence of such mutations suggests that short period phenotypes may result from loss or depression of function in this domain of the per protein. Possibly mutations in the region eliminate a regulatory function provided by this segment, or substantially increase stability of the mutant protein.     

Isolation of a cloned DNA segment containing a ribosomal protein gene of Drosophila melanogaster

A Drosophila genomic DNA library in the vector Charon 4 was screened using cDNA derived from the small (6S-12S) poly(A)+ mRNA of 2-6-h-old Drosophila embryos. This fraction of mRNA is enriched for ribosomal protein-coding sequences. The selected recombinants were hybridized to total mRNA under conditions which allowed for isolation of homologous mRNAs. The mRNA from these RNA/DNA hybrids was eluted and translated in vitro. The translation products were analyzed by one- and two-dimensional electrophoresis with authentic ribosomal proteins as standards. One cloned DNA segment was found to contain a ribosomal protein gene, and a sequence which hybridizes strongly to at least 5 other ribosomal protein mRNAs.