Molecular genetic studies on kai gene : mechanism of circadian oscillator

自1999年启动本课题“蓝藻昼夜节律振荡器的分子基础研究”以来，取得了丰硕的研究成果。部分研究结果已发表在Proc. NAS（2000），Cell（2000），Science（2000）等期刊上，并得到了同行的高度评价。1）KaiC与ATP/GTP的结合与昼夜节律KaiC蛋白携带两个ATP/GTP结合基序。我们证实了KaiC与ATP结合，并在基序上引入突变完全取消了ATP结合和昼夜节律，表明这种活性对振荡至关重要。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出与mRNA节律相比延迟8 h的昼夜节律。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。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的表达而发挥昼夜节律振荡器放大器的作用。4）KaiA在体内和体外均促进KaiC蛋白的磷酸化。该结果表明RaiC激活kaiBC基因的机制。5）与Golden的实验室（Texas A&M）合作克隆了光敏色素样蛋白CikA。CikA的破坏通过5 h暗脉冲降低相移，并表明CikA将光信号传递到振荡器以重置相位。

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.