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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（Synechococcus elongatus）PCC 7942是一种生物钟分析的模式蓝藻。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。通过遗传分析发现，蓝藻的生物钟基本上只包括三个时钟基因：kaiA、kaiB和kaiC。在这些基因座中，特别是在kaiC中，鉴定了改变从16小时到超过60小时的周期长度的各种突变。最近，人们发现，混合纯化的蛋白质和ATP在试管中带来了强大的循环磷酸化/去磷酸化KaiC与昼夜节律周期。也就是说，蓝藻的生物钟是一种化学振荡器，不需要生物过程/网络，如转录/翻译。我们已经尝试在实验室中评估这种简单系统对三个kai基因座突变的遗传鲁棒性。使用一组在这些基因座中随机引入氨基酸取代的1000个单突变，我们开始尝试评估这些突变在野生型背景下对周期长度表型的影响。我们发现kaiA突变对昼夜节律的影响比kaiB和kaiC小得多。这表明三个时钟基因之间的遗传鲁棒性是发散的。此外，发现超过90%的kaiB和kaiC突变在节律中表现出表型。因此，表明与其他遗传基因座相比，这些基因座的遗传稳健性显著较低。还使用体外振荡系统分析了耐用性的分子基础。结果表明，稳健的周期性可以通过三个时钟组件，这一结论是一致的，只有在这些位点的突变显示表型上的事实。