Molecular Biology of the Biological Clock : From Gene to Behavior

在真核生物昼夜节律模型系统中，振荡基因产物向细胞核的移位是产生24小时生物钟周期的关键步骤。我们研究了哺乳动物周期基因家族的时钟蛋白的核输入以及血清休克的影响，血清休克诱导培养细胞中的同步时钟。我们研究了mper蛋白在COS7细胞中的核输入，发现mPER1和mPER2的核转位涉及到与mPER3的物理相互作用。这表明mPER1的核转位也可以在生理条件下发生，在没有CRY蛋白的情况下。最近我们证明了辅助转录环的存在，帮助核心时钟反馈环。DBP是PAR亮氨酸拉链转录因子家族的一员，它的转录水平在SCN中表现出强劲的节律。我们报道，DBP能够通过直接与mPer1启动子结合来激活可能的时钟振荡基因mPer1的启动子。DBP和Clock-BMAL1协同激活mPer1启动子。另一方面，DBP转录被Clock-BMAL1通过E-box激活，并被mper和mCRY蛋白抑制，就像mPer1一样。反相昼夜节律调节的E4BP4是亮氨酸拉链转录因子的另一个成员，没有PAR结构域，拮抗地抑制mPer1的转录。因此，时钟控制的DBP和e4bp4基因可能在中枢时钟振荡中发挥重要作用。最近，我们利用携带mPer1启动子和荧光素酶(mPer1-Luc)基因的转基因小鼠，成功地监测了荧光素酶介导的生物发光，并在脑片和活体动物中检测到SCN的昼夜变化。我们可以记录几天的振荡光子发射，其周期和相位准确地反映了天然mPer1mRNA的表达。基因表达的实时光学成像将成为研究哺乳动物脑功能的新的有力工具。

In the eukaryotic circadian model systems, translocation of the oscillatory gene products into the nucleus is a key step for generation of a 24 hour cycle of the biological clock. We have examined nuclear import of clock proteins of the mammalian period gene family and the effect of serum shock, which induces a synchronous clock in cultured cells. We examined the nuclear import of mPER proteins in COS7 cells and found that nuclear translocation of mPER1 and mPER2 involves physical interactions with mPER3. This indicates that nuclear translocation of mPER1 also can occur under physiological conditions in the absence of CRY proteins.Recently we demonstrated that an accessory transcription loop exists helping to the core clock feedback loop. Transcript levels of DBP, a member of the PAR leucine zipper transcription factor family, exhibit a robust rhythm in the SCN.We report that DBP is able to activate the promoter of a putative clock oscillating gene, mPer1, by directly binding to the mPer1 promoter. DBP and CLOCK-BMAL1 cooperatively activate the mPer1 promoter. On the other hand, dbp transcription is activated by CLOCK-BMAL1 through E-boxes and inhibited by the mPER and mCRY proteins, as is the case for mPer1. Antiphase circadian regulated E4BP4, another member of leucine zipper transcription factor without PAR domain, antagonistically suppresses mPer1 transcription. Thus, a clock-controlled dbp and e4bp4 genes may play an important role in the central clock oscillation.Using transgenic mice carrying the mPer1 promoter fused to the luciferase (mPer1-luc) gene, we recently succeeded to monitor luciferase-mediated bioluminescence with a day-night variation in the SCN in brain slices and in living animals. We can record for several days oscillating photon emission with a periodicity and phase that accurately mirrored native mPer1 mRNA expression. The real-time optical imaging of gene expression will be a new powerful tool to study mammalian brain function.