Evaluation for genetic robustness in the machinery of period determination of the cyanobacterial circadian clock

蓝藻生物钟周期确定机制的遗传鲁棒性评估

基本信息

批准号：
17370088
负责人：
OYAMA Tokitaka
金额：
$ 9.09万
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：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17370088/
关键词：
circadian clock cyanobacteria genetic robustness evolution mutation suppressor

项目摘要

Synechococcus elongatus PCC 7942 is a model cyanobacterium for analysis of circadian clock. A bioluminescent reporter system can be used for effective monitoring of circadian rhythms of cyanobacterial colonies of this strain. Through a genetic analysis, it was found that the circadian clock of cyanobacteria basically comprises only three clock genes : kaiA, kaiB, and kaiC. In these loci, especially in kaiC, various mutations that altered period lengths ranging from 16 to over 60 hours were identified. Recently, it was found that mixing purified proteins of them and ATP in a test tube brought about a robust cyclic phosphorylation/dephosphorylation of KaiC with a circadian period. Namely, the circadian clock of cyanobacteria is a chemical oscillator that does not require biological processes/networks such as transcription/translation. We have tried to experimentally evaluate the genetic robustness of this simple system against mutations in the three kai loci in the laboratory. Using a set of〜300 single mutations with randomly introduced amino acid substitutions in these loci, we began trying evaluation of effects of those mutations in wildtype background on the phenotype of period lengths. We found that mutations in kaiA had much smaller effects on the circadian rhythm than those in kaiB and kaiC. This suggested that the genetic robustness among the three clock genes is diverged. Further, it was found more than 90% of mutations in kaiB and kaiC showed phenotypes in the rhythm. Thus, it was shown that the genetic robustness of these loci are remarkably low compared to other genetic loci. The molecular basis for the robustness was also analyzed using the in vitro oscillation system. It was shown that the robust periodicity can be attained by the three clock components and this conclusion is consistent with the fact that mutations only in these loci show phenotypes on it.

细长聚球藻 PCC 7942 是一种用于生物钟分析的蓝藻模型，生物发光报告系统可用于有效监测该菌株的蓝藻菌落的昼夜节律。这些基因座中只有三个时钟基因：kaiA、kaiB 和 kaiC。最近，人们发现了改变周期长度从 16 小时到超过 60 小时不等的各种突变。最近，人们发现，在试管中混合纯化的蛋白质和 ATP 会导致 KaiC 随昼夜周期发生强烈的循环磷酸化/去磷酸化。也就是说，蓝细菌的生物钟是一种化学振荡器，不需要转录/翻译等生物过程/网络。我们尝试通过实验评估遗传。这个简单系统对实验室中三个 kai 位点突变的稳健性使用一组约 300 个单突变，并在这些位点中随机引入氨基酸取代，我们开始尝试评估野生型背景中的这些突变对表型的影响。我们发现 kaiA 的突变对昼夜节律的影响比 kaiB 和 kaiC 的影响小得多，这表明三个时钟基因之间的遗传稳健性存在差异。 kaiB 和 kaiC 中 90% 的突变表现出节律表型，因此，与其他基因位点相比，这些基因座的遗传稳健性明显较低。还使用体外振荡系统分析了稳健性的分子基础。结果表明，三个时钟组件可以实现稳健的周期性，这一结论与仅在这些基因座中的突变显示表型的事实是一致的。