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.

Synechococcus Elongatus PCC 7942是用于分析昼夜节律的模型蓝细菌。生物发光记者系统可用于有效监测该菌株蓝细菌菌落的昼夜节律。通过遗传分析，发现蓝细菌的昼夜节律基本仅包括三个时钟基因：Kaia，Kaib和Kaic。在这些局部，尤其是在KAIC中，发现了各种突变，这些突变改变了16到60小时以上的周期长度。最近，发现将它们的纯化蛋白质和ATP混合在试管中，带来了KAIC的稳健循环磷酸化/去磷酸化与昼夜节律时期。也就是说，蓝细菌的昼夜节律是一种化学振荡器，不需要生物过程/网络，例如转录/翻译。我们试图通过实验评估实验室三个KAI基因座突变的简单系统的遗传鲁棒性。使用在这些基因座中随机引入氨基酸取代的一组〜300个单个突变，我们开始尝试评估野生型背景中这些突变对周期长度表型的影响。我们发现，KAIA中的突变对昼夜节律的影响要比Kaib和Kaic中的节奏小得多。这表明三个时钟基因之间的遗传鲁棒性有所不同。此外，在KAIB中发现了超过90％的突变，而KAIC在节奏中表现出表型。这表明，与其他遗传基因座相比，这些基因座的遗传鲁棒性非常低。还使用体外振荡系统分析了鲁棒性的分子基础。结果表明，鲁棒的周期性可以由三个时钟组件附加，并且该结论与仅在这些基因座中显示的表型在其上显示的表型的事实是一致的。