Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
Mol Cell. 2017 Jan 5;65(1):176-190. doi: 10.1016/j.molcel.2016.11.022. Epub 2016 Dec 22.
To conduct comprehensive characterization of molecular properties in organisms, we established an efficient method to produce knockout (KO)-rescue mice within a single generation. We applied this method to produce 20 strains of almost completely embryonic stem cell (ESC)-derived mice ("ES mice") rescued with wild-type and mutant Cry1 gene under a Cry1:Cry2 background. A series of both phosphorylation-mimetic and non-phosphorylation-mimetic CRY1 mutants revealed that multisite phosphorylation of CRY1 can serve as a cumulative timer in the mammalian circadian clock. KO-rescue ES mice also revealed that CRY1-PER2 interaction confers a robust circadian rhythmicity in mice. Surprisingly, in contrast to theoretical predictions from canonical transcription/translation feedback loops, the residues surrounding the flexible P loop and C-lid domains of CRY1 determine circadian period without changing the degradation rate of CRY1. These results suggest that CRY1 determines circadian period through both its degradation-dependent and -independent pathways.
为了全面描述生物体内的分子特性，我们建立了一种在单个世代内产生基因敲除（KO）-拯救小鼠的有效方法。我们应用该方法生成了 20 种几乎完全由胚胎干细胞（ESC）衍生的小鼠（“ES 小鼠”），这些小鼠在 Cry1:Cry2 背景下通过野生型和突变型 Cry1 基因的拯救得以恢复。一系列磷酸化模拟和非磷酸化模拟的 CRY1 突变体表明，CRY1 的多位点磷酸化可以作为哺乳动物生物钟的累积计时器。KO 拯救的 ES 小鼠还揭示了 CRY1-PER2 相互作用赋予了小鼠强大的昼夜节律性。令人惊讶的是，与经典转录/翻译反馈环的理论预测相反，CRY1 周围的柔性 P 环和 C-盖结构域的残基决定了昼夜节律周期，而不改变 CRY1 的降解速率。这些结果表明，CRY1 通过其降解依赖和非依赖途径来决定昼夜节律周期。
