The role of clockwork orange in the circadian clock of the cricket Gryllus bimaculatus
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RESEARCH ARTICLE
Open Access
The role of clockwork orange in the circadian clock of the cricket Gryllus bimaculatus Yasuaki Tomiyama1, Tsugumichi Shinohara1, Mirai Matsuka1, Tetsuya Bando2, Taro Mito3 and Kenji Tomioka1*
Abstract The circadian clock generates rhythms of approximately 24 h through periodic expression of the clock genes. In insects, the major clock genes period (per) and timeless (tim) are rhythmically expressed upon their transactivation by CLOCK/CYCLE, with peak levels in the early night. In Drosophila, clockwork orange (cwo) is known to inhibit the transcription of per and tim during the daytime to enhance the amplitude of the rhythm, but its function in other insects is largely unknown. In this study, we investigated the role of cwo in the clock mechanism of the cricket Gryllus bimaculatus. The results of quantitative RT-PCR showed that under a light/dark (LD) cycle, cwo is rhythmically expressed in the optic lobe (lamina-medulla complex) and peaks during the night. When cwo was knocked down via RNA interference (RNAi), some crickets lost their locomotor rhythm, while others maintained a rhythm but exhibited a longer free-running period under constant darkness (DD). In cwoRNAi crickets, all clock genes except for cryptochrome 2 (cry2) showed arrhythmic expression under DD; under LD, some of the clock genes showed higher mRNA levels, and tim showed rhythmic expression with a delayed phase. Based on these results, we propose that cwo plays an important role in the cricket circadian clock. Keywords: Circadian clock, Clockwork orange, Clock gene, Cricket, cry2, Molecular oscillation, Locomotor rhythm
Introduction Most insects exhibit daily rhythms in their physiology, including in their locomotor activity. The rhythms are driven by an endogenous oscillatory mechanism called the circadian clock, which generates approximately 24-h rhythms that persist in the absence of environmental cues [1]. The clock is based on the rhythmic expression of clock genes such as per, tim, Clock (Clk), and cycle (cyc). It is generally thought that the CLOCK (CLK)/CYCLE (CYC) heterodimer activates the transcription of per and tim by binding to the Ebox located upstream of their promoter region [1, 2]. The protein products of per and tim accumulate * Correspondence: [email protected] 1 Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan Full list of author information is available at the end of the article
during the night, form PER/TIM heterodimers and enter the nucleus to inhibit the transcriptional activity of CLK/CYC late at night. In the fruit fly Drosophila melanogaster, the transcriptional activator CLK is also rhythmically expressed by a mechanism that includes vrille (vri) and Par domain protein 1ɛ (Pdp1ɛ) [3, 4]. Both vri and Pdp1ɛ are transactivated by CLK/CYC through the E-box in the late day to early night, similar to per and tim. Soon after, the transcribed vri mRNA is translated to its product protein VRI, which suppresses the transcription of Clk durin
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