In vivo Studies of Vertebrate Circadian Clock Genes

描述（由申请人提供）：生物钟控制着许多对生物体正常功能至关重要的过程，包括行为、生理学和生物化学。这些时钟是内源性的计时装置，已被证明存在于从细菌到人类的生物体中。在人类中，这些生物钟的中断发生在时差反应，轮班工作和某些睡眠障碍中。最近的工作已经鉴定出许多参与中央昼夜节律钟的基因，并且已经清楚的是，这些基因中的许多基因在动物王国中是保守的。虽然已经提出了一个通用的分子钟模型，但该模型中的许多步骤仍然没有得到很好的理解。在这个建议中，实验描述了研究脊椎动物昼夜节律钟的分子机制在非洲爪蟾视网膜。非洲爪蟾视网膜包含许多描述良好的细胞和生化节律，可以在体外操纵。此外，用于产生转基因非洲爪蟾胚胎的新方法允许精确操纵完整视网膜内的基因表达，使其成为用于体内时钟机制研究的非常易于处理的系统。该提案的第一个和第二个目标将集中在隐花色素功能的两个方面的体外研究。这些蛋白质是时钟负反馈回路的关键组成部分，我们将分析隐花色素如何从细胞质移动到细胞核（目的1），以及一旦它们进入细胞核（目的2），它们如何引起转录装置的抑制。第三个目标是通过引入突变形式和/或通过改变这些基因在转基因非洲爪蟾胚胎中的表达水平来测试隐花色素在体内的功能。在第四个目标中，我们将制造特定的“分子损伤”，破坏视网膜内特定细胞类型的生物钟。这将通过在几种不同细胞特异性启动子的控制下过表达突变时钟基因来完成，以解决不同细胞类型中的个体时钟如何协调组织水平的节律性。这些实验利用了爪蟾系统的优势，使得在其他脊椎动物系统中难以进行的机制研究得以完成。由于这些生物钟是保守的，从这些研究中获得的信息将提供对脊椎动物生物钟的总体了解，包括人类的生物钟。

DESCRIPTION (provided by applicant): Circadian clocks control many processes important for normal functioning of living organisms, including behavior, physiology and biochemistry. These clocks are endogenous timekeeping devices and have been shown to be present in organisms ranging from bacteria to humans. In humans, disruptions of these clocks occur during jet lag, shift work and in some sleep disorders. Recent work has resulted in the identification of a number of genes involved in the central circadian clock and it has become clear that many of these genes are conserved within the animal kingdom. Although a general molecular clock model has been proposed, many of the steps within this model are still not well understood. In this proposal, experiments are described to study the molecular mechanism of the vertebrate circadian clock within the retina of Xenopus laevis. The Xenopus retina contains many well-described cellular and biochemical rhythms that can be manipulated in vitro. Furthermore, new methods for generating transgenic Xenopus embryos allow precise manipulation of gene expression within the intact retina, making this an extremely tractable system for studies of clock mechanism in vivo. The first and second aims of this proposal will focus on in vitro studies of two aspects of cryptochrome function. These proteins are critical components of the negative feedback loop of the clock and we will analyze how the cryptochromes move from the cytoplasm to the nucleus (aim 1) and how they cause repression of the transcriptional apparatus once they are in the nucleus (aim 2). The third aim will test the function of the cryptochromes in vivo by introduction of mutant versions and/or by altering expression levels of these genes in transgenic Xenopus embryos. In the fourth aim, we will make specific "molecular lesions" that disrupt the clock in specific cell types within the retina. This will be done by overexpressing mutant clock genes under the control of several different cell-specific promoters in order to address how individual clocks in the different cell types orchestrate tissue-level rhythmicity. These experiments take advantage of the strengths of the Xenopus system which allow mechanistic studies to be done that are difficult to do in other vertebrate systems. Because these clocks are conserved, information gained from these studies will provide insight into vertebrate clocks in general, including those in humans.