A mutation (K38R) which specifically eliminates kinase activity was created in the Drosophila melanogaster ckI gene (doubletime [dbt]). In vitro, DBT protein carrying the K38R mutation (DBT K/R ) interacted with Period protein (PER) but lacked kinase activity. In cell culture and in flies, DBT K/R antagonized the phosphorylation and degradation of PER, and it damped the oscillation of PER in vivo. Overexpression of short-period, long-period, or wild-type DBT in flies produced the same circadian periods produced by the corresponding alleles of the endogenous gene. These mutations therefore dictate an altered "set point" for period length that is not altered by overexpression. Overexpression of the DBT K/R produced effects proportional to the titration of endogenous DBT, with long circadian periods at lower expression levels and arrhythmicity at higher levels. This first analysis of adult flies with a virtual lack of DBT activity demonstrates that DBT's kinase activity is necessary for normal circadian rhythms and that a general reduction of DBT kinase activity does not produce short periods.Circadian rhythms are molecular, physiological, or behavioral processes which occur with a periodic oscillation of approximately 24 h. These rhythms can be entrained by environmental cues such as light/dark cycles, but they persist in the absence of such cues, thus demonstrating the presence of an endogenous clock (reviewed in reference 40). Drosophila melanogaster has been widely used as a model organism for a genetic analysis of circadian rhythms, and many of the gene products comprising the endogenous clock mechanism were identified in this organism, including the first discovered clock gene, period (per) (reviewed in reference 42). Molecular genetic analysis of these rhythms has elucidated a basic clock mechanism consisting of oscillating clock gene products, which regulate their own expression through positive and negative feedback loops. During the night, PER and Timeless protein (TIM) levels rise in the cytoplasm, where the proteins heterodimerize, and then translocate into the nucleus to negatively regulate the transcription of their own genes and other genes. Negative regulation is effected by interactions with the transcription factors dClock (dCLK) and Cycle (CYC), which activate transcription of the per and tim genes and indirectly repress transcription of dClk in the absence of PER and TIM. Throughout the day, TIM levels do not accumulate in the nucleus because of TIM's degradation via a light-and cryptochrome-mediated degradation pathway (reviewed in reference 42).DBT, an ortholog of mammalian casein kinase Iε and casein kinase I␦ (CKIε/␦), regulates PER cytoplasmic and nuclear accumulation by triggering PER's degradation and regulating the timing of its nuclear accumulation (3,8,9,22,23,43,46,49). DBT's activity on PER is supplemented by the activity of CKII (2, 27, 28) and SGG (31) and is antagonized by a rhythmically expressed protein phosphatase (47). DBT may regulate other aspects of PER function (35) and ot...

Mutations lowering the kinase activity of Drosophila Doubletime (DBT) and vertebrate casein kinase Ie/d (CKIe/d) produce long-period, short-period, and arrhythmic circadian rhythms. Since most ckI shortperiod mutants have been isolated in mammals, while the long-period mutants have been found mostly in Drosophila, lowered kinase activity may have opposite consequences in flies and vertebrates, because of differences between the kinases or their circadian mechanisms. However, the results of this article establish that the Drosophila dbt mutations have similar effects on period (PER) protein phosphorylation by the fly and vertebrate enzymes in vitro and that Drosophila DBT has an inhibitory C-terminal domain and exhibits autophosphorylation, as does vertebrate CKIe/d. Moreover, expression of either Drosophila DBT or the vertebrate CKId kinase carrying the Drosophila dbt S or vertebrate tau mutations in all circadian cells leads to short-period circadian rhythms. By contrast, vertebrate CKId carrying the dbt L mutation does not lengthen circadian rhythms, while Drosophila DBT L does. Different effects of the dbt S and tau mutations on the oscillations of PER phosphorylation suggest that the mutations shorten the circadian period differently. The results demonstrate a high degree of evolutionary conservation of fly and vertebrate CKId and of the functions affected by their period-shortening mutations.

Circadian rhythms are produced by a biological clock that is synchronized (or entrained) by cycles of light and temperature. In Drosophila, light triggers the interaction of the photoreceptor cryptochrome (CRY) with the circadian clock protein timeless (TIM). The absence of this interaction in cryb mutants eliminates this entrainment mechanism. The abundance of TIM and period (PER) oscillate throughout the day, and they form a complex that moves to the nucleus to rhythmically repress transcription of the per and tim genes. Because the CRY:TIM interaction triggers rapid degradation of TIM, the phase of these molecular oscillations is reset by light, which thereby entrains the circadian clock. A study now shows that heat pulses trigger an association between CRY and PER:TIM, which suggests that CRY:PER:TIM also contributes to entrainment by temperature. In wild-type flies, CRY:PER:TIM formation requires high temperatures and is only triggered by heat pulses in the early night, but in per(L) mutants, which exhibit a temperature-sensitive lengthening of circadian periods, CRY:PER(L:)TIM formation is triggered by lower temperatures and throughout the night. Because CRY:PER:TIM is formed under the same conditions that entrain circadian behavior, formation of the complex is likely to mediate entrainment by heat pulses. Whereas per(L )flies exhibit longer periods at higher temperatures, per(L);cry(b) flies exhibit similar periods at different temperatures, which suggests that an altered interaction between CRY and PER(L):TIM contributes to a lack of temperature compensation. Future work should determine how the interaction between CRY and PER:TIM entrains rhythms to temperature and affects temperature compensation.