Molecular Biology of the Biological Clock : From Gene to Behavior

生物钟的分子生物学：从基因到行为

基本信息

批准号：
11470018
负责人：
OKAMURA Hitoshi
金额：
$ 9.66万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470018/
关键词：
mPer1 clock genes dbp cry e4bp4 circadian rhythm transgenic mice suprachiasmatic nucleus 哺乳類時計遺伝子サーカディアンリズム Cry 単一細胞のリズム

项目摘要

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.

在真核昼夜节律模型系统中，振荡基因产物易位到细胞核中是产生 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时钟蛋白的核输入以及血清休克的影响，血清休克在培养细胞中诱导同步时钟。我们检查了 COS7 细胞中 mPER 蛋白的核输入，发现 mPER1 和 mPER2 的核转位涉及与 mPER3 的物理相互作用。这表明在没有 CRY 蛋白的生理条件下，mPER1 的核易位也可能发生。最近我们证明存在辅助转录环，有助于核心时钟反馈环。 DBP 是 PAR 亮氨酸拉链转录因子家族的成员，其转录水平在 SCN 中表现出强大的节律。我们报道 DBP 能够通过直接与 mPer1 启动子结合来激活假定的时钟振荡基因 mPer1 的启动子。 DBP 和 CLOCK-BMAL1 协同激活 mPer1 启动子。另一方面，dbp 转录由 CLOCK-BMAL1 通过 E-box 激活，并被 mPER 和 mCRY 蛋白抑制，与 mPer1 的情况一样。反相昼夜节律调节的 E4BP4 是不带 PAR 结构域的亮氨酸拉链转录因子的另一个成员，可拮抗抑制 mPer1 转录。因此，时钟控制的 dbp 和 e4bp4 基因可能在中央时钟振荡中发挥重要作用。使用携带与荧光素酶（mPer1-luc）基因融合的 mPer1 启动子的转基因小鼠，我们最近成功地监测了荧光素酶介导的生物发光脑切片和活体动物中 SCN 的昼夜变化。我们可以记录几天的振荡光子发射，其周期性和相位准确地反映了天然 mPer1 mRNA 的表达。基因表达的实时光学成像将成为研究哺乳动物大脑功能的新的强大工具。

项目成果

期刊论文数量（76）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Takumi T, Nagamine Y, Miyake S, Matsubara C, Taguchi K, Takekida S, Sakakida Y, Nishikawa K, Kishimoto T, Niwa S, Okumura K, Okamura H: "A mammalian orthologue of Drosophila timeless, highly expressed in SCN and retina, forms a complex with mPER1"Genes Ce

Takumi T、Nagamine Y、Miyake S、Matsubara C、Taguchi K、Takekida S、Sakakida Y、Nishikawa K、Kishimoto T、Niwa S、Okumura K、Okamura H：“果蝇永恒的哺乳动物直系同源物，在 SCN 和视网膜中高度表达