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A tidal clock

潮汐钟

基本信息

批准号：
BB/R01776X/1
负责人：
Charalambos Kyriacou
金额：
$ 93.32万
依托单位：
University of Leicester
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2018
资助国家：
英国
起止时间：
2018 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FR01776X%2F1
关键词：
tidal clock

项目摘要

The molecular basis of 24 hour circadian rhythms in terrestrial organisms is well understood and represents one of the major advances in the study of gene regulation of complex characters. However, the predominant rhythms in marine species that live on the coast in the intertidal zone is 12. 4 hours, reflecting the ebb and flow of the tides which are determined by the gravitational pull of the Moon and Sun on the Earth. For decades, scientists have speculated whether tidal rhythms are also related to circadian rhythms and whether they share some or all of the underlying molecular components of the 24 hour clock. We have been studying the specked sea louse, Eurydice pulchra, which shows both circadian rhythms in pigment dispersion and clear tidal rhythms in its swimming behaviour. We have identified all the main circadian clock genes in Eurydice and we can divide them up into their function. There are the genes that encode the positive regulators CLOCK and BMAL1, and these activate the genes for the negative regulators TIM, CRY2 and PER, which then feed back in a loop to deactivate the function of the positive regulators in a 24 hour cycle. This is called the negative feedback loop and explains how rhythms in gene transcription and translation of clock gene products can generate 24 hour molecular cycles. We have discovered that tidal rhythms share the positive factors but not the negative factors of the circadian clock. Furthermore we have identified putative circadian neurons and putative tidal cells in the Eurydice brain. These are major insights into how tidal clocks work.We have assembled a draft genome for Eurydice and it contains several additional genes whose products act to regulate the positive factors and the negative factors. We would expect to find these in the corresponding tidal and circadian cells, so we shall localise the expression of these additional clock genes in the brain to see whether they are found in tidal or circadian cells, or both, or even other neurons, using a very sensitive technique called RNAscope. We shall also knock down the expression of these genes in Eurydice and examine whether they show changes in tidal or circadian behaviour thereby associating specific clock genes with specific types of rhythmic behaviour, tidal or circadian, or both. One of the positive factors that is important for tidal rhythms is BMAL1. This protein is known as the circadian transcription factor and with its partner CLOCK, binds to genes and activates them (see above). Consequently whatever DNA sequence BMAL1 binds, is potentially a control region for a circadian or tidal gene. We shall use a technique called ChIPseq to identify the DNA sequences and corresponding genes to which BMAL1 binds by referring to our Eurydice draft genome. Some of the genes under BMAL1 control may be switched on rhythmically with a tidal period and we shall compare these genes to those we already know are activated in 12 h cycles. Any that cross-match are candidates for being tidal output genes or the tidal regulators. We imagine that the crucial tidal regulators will also physically interact with BMAL1 in the same way that the circadian regulators like PER-TIM-CRY2 interact with the positive factors to generate circadian rhythms, so we shall compare the identity of proteins that interact with BMAL1 (using two techniques called co-IP and yeast-two hybrid) and again crossmatch any interactors with the genes we know bind BMAL1 or cycle with 12 h periods. In this way we hope to generate candidate genes for the elusive tidal regulators. When we have these candidate genes, we shall study where they are expressed in the brain and also knock down their expression levels to see whether they disrupt tidal behaviour. Our strategies will converge on the important genes that generate tidal rhythms and will perhaps provide a general model as to how these lunar-related rhythms are regulated in the animal kingdom.

陆地生物24小时昼夜节律的分子基础已经被很好地理解，并代表了复杂性状基因调控研究的主要进展之一。然而，生活在潮间带海岸的海洋物种的主导节律是12。4小时，反映了潮汐的涨落，这是由月球和太阳对地球的引力决定的。几十年来，科学家们一直在猜测潮汐节律是否也与昼夜节律有关，以及它们是否共享24小时生物钟的部分或全部潜在分子成分。我们一直在研究斑点海虱，Eurydice pulchra，它既显示了昼夜节律的色素分散和明确的潮汐节奏在其游泳行为。我们已经确定了欧律狄刻所有主要的生物钟基因，我们可以将它们分为它们的功能。有编码正调节因子CLOCK和BMAL 1的基因，这些基因激活负调节因子TIM、BMAL 2和PER的基因，然后这些基因在24小时循环中反馈回正调节因子的功能。这被称为负反馈回路，解释了基因转录和时钟基因产物翻译的节奏如何产生24小时的分子周期。我们发现潮汐节律具有生物钟的正因子，但不具有负因子。此外，我们已经确定了假定的昼夜神经元和假定的潮汐细胞在Eurydice的大脑。这些都是关于潮汐钟如何工作的重要见解。我们已经组装了欧律狄刻的基因组草图，它包含了几个额外的基因，其产物起着调节正因子和负因子的作用。我们希望在相应的潮汐和昼夜节律细胞中找到这些基因，因此我们将使用一种非常灵敏的称为RNAscope的技术，定位这些额外的时钟基因在大脑中的表达，看看它们是否存在于潮汐或昼夜节律细胞中，或者两者兼而有之，甚至是其他神经元。我们还将敲除Eurydice中这些基因的表达，并检查它们是否显示潮汐或昼夜节律行为的变化，从而将特定的时钟基因与特定类型的节律行为（潮汐或昼夜节律）或两者联系起来。对潮汐节律很重要的积极因素之一是BMAL 1。这种蛋白质被称为昼夜节律转录因子，与其伴侣CLOCK结合并激活基因（见上文）。因此，无论BMAL 1结合的DNA序列是什么，都可能是昼夜节律或潮汐基因的控制区。我们将使用一种名为ChIPseq的技术，通过参考我们的Eurydice基因组草图来识别BMAL 1结合的DNA序列和相应的基因。BMAL 1控制下的一些基因可能与潮汐周期有节奏地开启，我们将这些基因与我们已经知道的在12小时周期内激活的基因进行比较。任何交叉匹配的基因都有可能成为潮汐输出基因或潮汐调节基因。我们想象，关键的潮汐调节器也会与BMAL 1发生物理相互作用，就像PER-TIM-BMAL 2等昼夜节律调节器与积极因素相互作用以产生昼夜节律一样，因此我们将比较与BMAL 1相互作用的蛋白质的身份（使用称为co-IP和酵母双杂交的两种技术），并再次交叉匹配任何与我们已知的结合BMAL 1或具有12小时周期的周期的基因的相互作用物。通过这种方式，我们希望产生难以捉摸的潮汐调节因子的候选基因。当我们有了这些候选基因后，我们将研究它们在大脑中的表达位置，并降低它们的表达水平，看看它们是否会扰乱潮汐行为。我们的策略将集中在产生潮汐节律的重要基因上，并可能提供一个关于这些与月球有关的节律在动物王国中如何调节的一般模型。