TheDrosophila double-time^SMutation Delays the Nuclear Accumulation ofperiodProtein and Affects the Feedback Regulation ofperiodmRNA

Abstract

The Drosophila double-time (dbt) gene, which encodes a protein similar to vertebrate epsilon and delta isoforms of casein kinase I, is essential for circadian rhythmicity because it regulates the phosphorylation and stability of period (per) protein. Here, the circadian phenotype of a short-period dbt mutant allele (dbt(S)) was examined. The circadian period of the dbt(S) locomotor activity rhythm varied little when tested at constant temperatures ranging from 20 to 29 degrees C. However, per(L);dbt(S) flies exhibited a lack of temperature compensation like that of the long-period mutant (per(L)) flies. Light-pulse phase-response curves were obtained for wild-type, the short-period (per(S)), and dbt(S) genotypes. For the per(S) and dbt(S) genotypes, phase changes were larger than those for wild-type flies, the transition period from delays to advances was shorter, and the light-insensitive period was shorter. Immunohistochemical analysis of per protein levels demonstrated that per protein accumulates in photoreceptor nuclei later in dbt(S) than in wild-type and per(S) flies, and that it declines to lower levels in nuclei of dbt(S) flies than in nuclei of wild-type flies. Immunoblot analysis of per protein levels demonstrated that total per protein accumulation in dbt(S) heads is neither delayed nor reduced, whereas RNase protection analysis demonstrated that per mRNA accumulates later and declines sooner in dbt(S) heads than in wild-type heads. These results suggest that dbt can regulate the feedback of per protein on its mRNA by delaying the time at which it is translocated to nuclei and altering the level of nuclear PER during the declining phase of the cycle.

A proposal for robust temperature compensation of circadian rhythms

“…Furthermore, PER translocation is intimately connected to circadian period and temperature compensation. The per L allele encodes a mutant protein PER L with a single amino acid substitution, resulting in long-period rhythms (Ϸ28 h) (32) that are not temperature compensated (14). In mutant cells, nuclear translocation of PER L is delayed (31,33 (14,(34)(35)(36), we favor regulated nuclear import and/or export as a likely candidate for our resetting variable.…”

“…Physiologically, this robustness of the period (T Ϸ 24 h despite genetic variability and environmental fluctuations) is essential to circadian physiology. If the autonomous period of the clock drifts too far from 24 h, then the circadian rhythm would not reliably entrain to the external 24 h light/dark Zeitgeber.(i) Consider first the tight distribution of T [24 Ϯ 1 h, coefficient of variation (CV) ϭ 4%] across populations of fruit flies (13,14). In general, the period of a limit cycle oscillator depends on many rate constants (k i ), and the variability of T as a result of rate constant variations is given by…”

“…(i) Consider first the tight distribution of T [24 Ϯ 1 h, coefficient of variation (CV) ϭ 4%] across populations of fruit flies (13,14). In general, the period of a limit cycle oscillator depends on many rate constants (k i ), and the variability of T as a result of rate constant variations is given by…”

“…As expected, there are mutants with defective temperature compensation in both Neurospora crassa and Drosophila melanogaster (Table 1). However, more intriguingly, geneticists (13,14,(20)(21)(22)(23) have identified many circadian rhythm mutations (aberrant period) that leave temperature compensation intact; indeed, 60-70% of the representative mutants in Table 1 maintain temperature compensation. In order for temperature compensation to survive in the face of a variety of mutations at many different places in the mechanism, many terms in the balance equation must be negligible, i.e., ѨT/Ѩk i ⅐ k i E i Х 0 for many i.…”

Molecular genetics of the fruit-fly circadian clock

“…The delay between per mRNA and protein accumulation is also due to DBT. 49 However, DBT protein does not cycle, but the dephosphorylation of PER does, and is mediated by cycling expression of the relevant phosphatase, PP2A. 50 As PER is translated in the cytoplasm in the early night, it is phosphorylated by DBT as a prelude to degradation, in which the product of slimb (supernumery limbs), a ubiquitin ligase, becomes critical.…”