takeout, a Novel DrosophilaGene under Circadian Clock Transcriptional Regulation

Abstract

We report the identification and characterization of a new Drosophila clock-regulated gene, takeout (to). to is a member of a novel gene family and is implicated in circadian control of feeding behavior. Its gene expression is down-regulated in all of the clock mutants tested. In wild-type flies, to mRNA exhibits daily cycling expression but with a novel phase, delayed relative to those of the better-characterized clock mRNAs, period and timeless. The E-box-containing sequence in the to promoter shows impressive similarities with those of period and timeless. However, our results suggest that the E box is not involved in the amplitude and phase of the transcriptional cycling of to. The circadian delayed transcriptional phase is therefore most likely the result of indirect regulation through unknown transcription factors.Circadian (ϳ24-h) behavioral and physiological rhythms are manifest in virtually all organisms. Our understanding of the underlying molecular rhythms comes largely from genetic investigations of five different classes of organisms: plants (28), photosynthetic bacteria (17), Neurospora (8), Drosophila (32), and mice (44,47). Recent progress has reinforced the negative feedback regulation of transcription, originally proposed for Drosophila (14, 14a, 15a, 50), as a central theme of circadian rhythms in these organisms (9). In particular, Drosophila clocks display conservation with mammalian clocks. At the sequence level, many Drosophila clock components have one or more mammalian homologs, which are suggested to play similar roles in mammalian rhythms. This further validates Drosophila as an animal model system for the study of circadian rhythms.The first Drosophila clock component identified was the period (per) gene (3,20,31). Biochemical and genetic data suggested a transcriptional autoregulatory feedback loop involving PER (14, 14a, 15a, 50). The second essential pacemaker component, timeless (tim), was subsequently identified, and both per and tim reciprocally autoregulate at the transcriptional level (29,39). TIM dimerizes with PER (10,24,51), and the interaction is suggested to be important for the posttranscriptional regulation and nuclear entry of both proteins (35,48). Although their precise biochemical functions are not certain, PER and TIM probably function directly in the negative regulation of transcription (7,22). In contrast, the biochemical functions of the recently identified clock genes Clock (Clk) and cycle (cyc) are apparent from their primary sequences (1,7,34). Both CLK and CYC belong to the basic helix-loop-helix (bHLH)-PAS (Per-Arnt-Sim) transcription factor family, members of which are involved in a wide range of other life processes. For example, the mammalian ARNT-AHR heterodimer is involved in xenobiotic resistance (37), and the Drosophila SIM-TANGO heterodimer is involved in embryonic development of the central nervous system midline cells (41).In the Drosophila mutants Clk jrk and cyc 01 (1, 34), the rate of transcription of the two major clock components, per and ti...

The circadian output gene takeout is regulated by Pdp1ε

“…In contrast to what has been described for Clk mRNA, however, takeout levels were found to be down-regulated in Clk JRK , cyc o1 , and tim 01 circadian mutants (25). The simplest way to explain takeout down-regulation in Clk and cyc mutants was to assume that its transcription is regulated by CLK.…”

“…The takeout (to) gene has been consistently identified in these screens. Its RNA and protein have been shown to cycle with a circadian rhythm, with a peak at late night/early morning and a trough in the late morning, closely resembling the cycling pattern described for Clk mRNA (24,25).In contrast to what has been described for Clk mRNA, however, takeout levels were found to be down-regulated in Clk JRK , cyc o1 , and tim 01 circadian mutants (25). The simplest way to explain takeout down-regulation in Clk and cyc mutants was to assume that its transcription is regulated by CLK.…”

“…The takeout (to) gene has been consistently identified in these screens. Its RNA and protein have been shown to cycle with a circadian rhythm, with a peak at late night/early morning and a trough in the late morning, closely resembling the cycling pattern described for Clk mRNA (24,25).…”

“…This poses the question of the nature of the output genes that mediate these processes. Not surprisingly, because many of the main regulators are transcription factors, molecular screens for transcripts that are under circadian control, and change in mutants that affect the core clock, have revealed a large number of such transcripts (20)(21)(22)(23)(24)(25)(26)(27). However, very little is known about the function of these potential output genes and the processes they regulate.…”

Precise RNAi-mediated silencing of metabolically active proteins in the defence secretions of juvenile leaf beetles

“…In silico analyses predicted in the P. cochleariae sequence seven N-glycosylation sites and only one disulphide bond. Along with identifying the two To-specific motives [45] (figure 4) in the C-terminal region, we conclude that our protein can be attributed to the To family. Therefore, we named it PcTo-like.…”