Circadian Rhythms of Gene Expression in Cyanobacteria

摘要：黄金，MCB 9982852一种内源计时机制，称为生物钟，允许从动物到蓝藻的生物在时间上调节它们的生理过程，并使这些过程与环境协调。昼夜节律控制过程的特点是，在没有环境提示的情况下，这些节律表现出大约24小时的周期性，它们的相位可以通过明/暗信号来重置，并且它们是温度补偿的。生物钟的这些固有特性使新陈代谢和行为事件与地球的每日周期同步，与白天长度的季节性变化和每日温度的变化相适应。确定引起昼夜节律特性的分子组成和生化机制是昼夜节律研究的中心目标，特别是这个项目。蓝藻是唯一已被证明具有昼夜节律的原核生物。聚球藻基因操作的简便性。PCC7942菌株在理解蓝藻生物钟方面取得了快速进展。通过使用荧光素酶基因融合(哈维氏弧菌，或萤火虫Luc)，蓝藻基因表达的昼夜节律的周期、幅度和相位可以很容易地从任何聚球藻启动子中监测，从而光产生实时报告转录。以前使用这一策略的研究发现了一个对蓝藻昼夜节律至关重要的三基因座(KaIA，Kaib，KaiC)。KAI基因中的任何一个突变都会导致昼夜节律的变化，其中任何一个基因的失活都会导致心律失常。能够很容易地产生突变，在很短的世代时间内培养蓝藻，以及处理大量的细胞群体，使得一些重要的问题能够非常直接地得到解决。例如，用聚球藻进行的实验表明，即使细胞以比每天一次快得多的速度加倍，生物钟也是有效的。野生型和突变型混合细胞的种群被用来明确地表明，其内在周期与环境中的光/暗周期密切匹配的生物钟改善了蓝藻细胞的适合性。这个项目有两个目标。第一个是验证这样的假设，即昼夜节律依赖于转录反馈环，该环涉及KAI基因作为计时机制的一个组成部分。这个模型与提出的动物和真菌时钟机制是一致的。然而，来自突变体的表型使KaIA和kaiBC的正常相变失去同步性，这表明预期的模型可能不正确。异源启动子将被用来绕过KAI基因的自然转录调节，并将对菌株进行监测，以确定计时是否仍然有效。第二个目标是了解输出路径，这些路径允许生物钟协调生物体中基因表达的时间。这将通过以下方式完成：分析允许某些基因(如PurF和OPCA)在特殊阶段(与生物体中的大多数基因不同，在黎明前达到表达高峰)表达的顺式元件；从基因亚群中鉴定影响表达昼夜节律的已知基因的抑制子；以及分离影响启动子昼夜节律控制的新突变体，如kaiBC，其输出途径成分尚未确定。这些实验既涉及时钟本身的机制，也涉及将昼夜节律计时传递给时钟控制的生物过程的方法。

Abstract: Golden, MCB 9982852An endogenous timekeeping mechanism, called the circadian clock, allows organisms from animals to cyanobacteria to regulate their physiological processes temporally and to coordinate these with the environment. The hallmarks of circadian-controlled processes are that these rhythms show a periodicity of approximately 24 h in the absence of environmental cues, their phasing can be reset by light/dark signals, and they are temperature compensated. These intrinsic properties of circadian clocks allow metabolic and behavioral events to be synchronized with the earth's daily cycles, acommodating seasonal changes in day length and daily changes in temperature. Defining the molecular components and biochemical mechanisms that give rise to the properties of circadian clocks is the central goal of circadian research in general, and this project specifically. Cyanobacteria are the only prokaryotes that have been shown to exhibit circadian rhythms. The ease of genetic manipulation in Synechococcus sp. strain PCC 7942 has resulted in rapid progress toward understanding the cyanobacterial circadian clock. The period, amplitude, and phasing of the cyanobacterial circadian rhythm of gene expression can be readily monitored from any Synechococcus promoter by using luciferase gene fusions (Vibrio harveyi luxAB, or firefly luc), such that light production reports transcription in real time. Previous research using this strategy identified a three-gene locus (kaiA, kaiB, kaiC) that is fundamentally important for cyanobacterial circadian rhythms. Mutations in any of the kai genes can cause a change in circadian period, and inactivation of any of them results in arrhythmia. The ability to generate mutants easily, to grow cyanobacteria with a short generation time, and to work with large populations of cells has allowed some important questions to be addressed very directly. For example, experiments with Synechococcus demonstrated that the circadian clock is operative even when cells are doubling much faster than once per day. Populations of mixed wild-type and mutant cells were used to show definitively that a circadian clock whose intrinsic period closely matches the light/dark cycle in the environment improves the fitness of cyanobacterial cells. This project has two aims. The first is to test the hypothesis that circadian rhythmicity depends on a transcriptional feedback loop involving the kai genes as a component of the timekeeping mechanism. This model is consistent with proposed mechanisms for the clocks of animals and fungi. However, phenotypes from mutants that desynchronize the normal phasing of kaiA and kaiBC suggest that the expected model may not be correct. Heterologous promoters will be used to bypass the natural transcriptional regulation of the kai genes and the strains will be monitored to determine whether timekeeping is still operational. The second aim is to understand output pathways that allow the circadian clock to orchestrate the timing of gene expression in the organism. This will be accomplished by: analysis of cis elements that allow some genes (such as purF and opcA) to be expressed in an exceptional phase (expression peaking near dawn, unlike most genes in the organism); identification of suppressors of known genes that affect the circadian timing of expression from subsets of genes; and isolation of new mutants that affect the circadian control of promoters such as that of kaiBC, for which no output pathway components have yet been identified. These experiments address both the mechanism of the clock itself, and the means by which circadian timekeeping is conveyed to the biological processes that the clock controls.