Molecular genetic studies on kai gene : mechanism of circadian oscillator

kai基因的分子遗传学研究：昼夜节律振荡器的机制

基本信息

批准号：
11440234
负责人：
KONDO Takao
金额：
$ 2.82万
依托单位：
Nagoya 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-11440234/
关键词：
cyanobacteria circadian clock kai clock genes negative feedback protein interaction ATP GTP binding phosphorylation sensor kinase

项目摘要

Since we have started the current project for molecular basis of cyanobacterial circadian oscillator in 1999, the progress of research has been very fruitful and we obtained following results. Parts of these results have been published in Proc. NAS (2000), Cell (2000), Science (2000) and highly appreciated by colleagues.1) ATP/GTP binding of KaiC and circadian rhythmsKaiC protein carries two ATP/GTP binding motifs. We confirmed that KaiC bound to ATP and introduction of mutations to the motifs completely nullified the ATP binding and the circadian rhythms, suggesting this activity is crucial for the oscillation.2) Circadian rhythms in Kai protein level and KaiC phosphorylationProtein level of three Kai proteins showed circadian rhythms with phases delayed by 8 h from mRNA rhythms. KaiC protein were phosphorylated and the level of phosphorylation also showed circadian rhythms.3) SasA, a histidin kinase that interact with RaiC.A sensory histidine kinase that bound with KaiC was cloned. Disruption of SasA lowered KaiC expression and amplitude of the rhythm. These results indicate that SasA function as amplifier of the circadian oscillator by promoting KaiC expression.4) KaiA enhance phosphorylation of KaiCKaiA protein promotes phosphorylation of KaiC both in vivo and in vitro. This results suggest the mechanism of kaiBC gene activation by RaiC.5) A phytochrome-like protein CikA was cloned with collaboration with Golden's labo (Texas A&M). Disruption of CikA lowered phase shift by 5 h dark pulse and suggest that CikA communicate light signal to the oscillator to reset phase.

我们自1999年启动蓝藻昼夜节律振荡器分子基础研究项目以来，研究工作取得了丰硕的成果，取得了以下成果。这些结果的部分内容已发表在 Proc 上。 NAS (2000)、Cell (2000)、Science (2000) 并受到同事的高度赞赏。1) KaiC 的 ATP/GTP 结合和昼夜节律 KaiC 蛋白携带两个 ATP/GTP 结合基序。我们证实 KaiC 与 ATP 结合，并且在基序中引入突变完全消除了 ATP 结合和昼夜节律，表明这种活性对于振荡至关重要。2) Kai 蛋白水平和 KaiC 磷酸化的昼夜节律三种 Kai 蛋白的蛋白水平显示昼夜节律，相位比 mRNA 节律延迟 8 小时。 KaiC蛋白被磷酸化，磷酸化水平也呈现昼夜节律。3）与RaiC相互作用的组氨酸激酶SasA。克隆了与KaiC结合的感觉组氨酸激酶。 SasA 的破坏降低了 KaiC 的表达和节律的幅度。这些结果表明SasA通过促进KaiC表达发挥昼夜节律振荡器放大器的作用。4)KaiA增强KaiCK的磷酸化。aiA蛋白在体内和体外均促进KaiC的磷酸化。这一结果表明RaiC 激活kaiBC 基因的机制。5) 与Golden's labo (Texas A&M) 合作克隆了光敏色素样蛋白CikA。 CikA 的中断使相移降低了 5 小时暗脉冲，表明 CikA 将光信号传递给振荡器以重置相位。