A tidal clock

陆生生物24小时昼夜节律的分子基础已被充分了解，这是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要节奏是12。4小时，反映了潮汐的涨落，潮汐是由月球和太阳对地球的引力决定的。几十年来，科学家们一直在推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，它在色素分散上显示出昼夜节律，在游泳行为上显示出明显的潮汐节律。我们已经确定了欧律狄刻所有主要的生物钟基因，我们可以根据它们的功能来划分它们。有编码阳性调节因子CLOCK和BMAL1的基因，这些基因激活阴性调节因子TIM， CRY2和PER的基因，然后在一个循环中反馈，在24小时的周期内使阳性调节因子的功能失活。这被称为负反馈循环，并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。我们已经发现，潮汐节律共享生物钟的积极因素，而不是消极因素。此外，我们已经在欧律狄刻大脑中确定了假定的昼夜节律神经元和假定的潮汐细胞。这些是对潮汐钟工作原理的重要见解。我们已经组装了Eurydice的基因组草图，它包含了几个额外的基因，其产物起着调节积极因素和消极因素的作用。我们希望在相应的潮汐细胞和昼夜节律细胞中找到这些，所以我们将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细胞或昼夜节律细胞中，或者两者都存在，甚至其他神经元中，使用一种非常敏感的技术，称为RNAscope。我们还将敲除这些基因在欧律狄刻中的表达，并检查它们是否表现出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联系起来，潮汐或昼夜节律，或两者兼而有之。BMAL1是潮汐节律的一个重要的积极因素。这种蛋白质被称为昼夜节律转录因子，与其伴侣CLOCK结合在基因上并激活它们（见上文）。因此，无论BMAL1结合的DNA序列是什么，它都可能是昼夜节律或潮汐基因的控制区。我们将使用一种称为ChIPseq的技术来识别BMAL1结合的DNA序列和相应的基因，参考我们的Eurydice草图基因组。BMAL1控制下的一些基因可能会随着潮汐周期有节奏地开启，我们将把这些基因与那些我们已经知道的在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是潮汐输出基因或潮汐调节基因的候选者。我们认为，关键的潮汐调节因子也会与BMAL1发生物理相互作用，就像昼夜节律调节因子如per - timm - cry2与积极因子相互作用以产生昼夜节律一样，因此我们将比较与BMAL1相互作用的蛋白质的身份（使用两种称为co-IP和酵母- 2杂交的技术），并再次交叉匹配任何相互作用因子与我们知道结合BMAL1或周期为12小时的基因。通过这种方式，我们希望为难以捉摸的潮汐调节因子产生候选基因。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，并可能为动物王国如何调节这些与月球相关的节律提供一个通用模型。

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.