Abstract
An auto-feedback loop of clock gene transcription and translation is believed to generate the circadian oscillation. Two clock genes, Per2 and Bmal1 are involved in interlocking two molecular feedback loops, and expressed anti-phasically. To investigate the dynamics of interlocked feedback loops, we developed a dual bioluminescence reporter system with two different Phrixothrix luciferases (PTGR/RED). We established stable transformants in fibroblast cell lines transfecting two plasmids, which consisted of Per2 and Bmal1 promoter fragment (390bp/915bp) with PTGR and RED luciferase of green-light emitting and red-light emitting wavelength, respectively. These clones showed anti-phasic circadian rhythm in Per2 and Bmal1 gene expressions after dexamethasone treatment. To find out critical steps in the feedback loop, we perturbed the feedback loop by changing the concentration of constitutional components, and determined the circadian period. When Cry1,2 gene expressions were enhanced, the circadian period was not changed significantly, but the expression of Per2 was reduced by 80%. The result suggests that its suppression of Per2 gene expression is not a critical step to determine the lapse of loop rotation, or that the critical amount of Per2 expression is enough to maintain the auto-feedback loop. We'll also examine the effects of other loop elements such as Bmal-Clock complex, Per1,Per2, and orphan nuclear receptor RORα. [J Physiol Sci. 2007;57 Suppl:S139]
