MOLECULAR ANALYSIS OF CIRCADIAN RHYTHMS IN SINGLE CELLS

单细胞昼夜节律的分子分析

• 批准号: 2250847
• 负责人: STEVE A KAY
• 金额: $ 11.23万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-30 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2250847
• 关键词: Arabidopsis; Drosophilidae; alternatives to animals in research; biological clocks; cell biology; circadian rhythms; gene expression; genetic promoter element; genetic regulatory element; luciferin monooxygenase; photostimulus; tissue /cell culture

This proposal addresses the molecular control of biological clocks in individual organisms and single cells. Higher plants comprise one system for these studies, in part because these organisms provide cis-acting DNA elements that mediate clock-controlled gene expression. To exploit this property, a non-invasive assay has been developed for clock-regulated gene expression in intact whole plants: the promoter of a clock-regulated gene (cab2) from Arabidopsis has been fused to a cDNA encoding firefly luciferase and transgenic plants were generated containing this construct. Preliminary data demonstrate that when plants transformed by the cab2- luciferase gene fusion are sprayed with luciferin, the plants f"glow" rhythmically, indicating that circadian-regulated transcription can be monitored in intact tissue. We propose to extend this technology to the analysis of dock-regulated transcription in single plant cells. The hypothesis is that individual cells of metazoan organisms can contain circadian clocks. The plant system will provide a powerful cell-level assay to address this issue. We will also identify biochemically the signal transduction pathways between the oscillator and the cab gene promoter in a plant suspension cell culture, to test the hypothesis that calmodulin-dependent pathways are important in regulating cab gene transcription. In another system, Drosophila, cis-acting elements of a 'clock locus' called period have been shown to be involved in mediating cyclic variations in per gene product levels. It is proposed to apply the luciferase reporter system to analysis of the molecular rhythm at the whole-fly level. The specific experiments will involve per-luciferase fusions and transgenic strains analogous to those in Arabidopsis. If individual flies can be shown to glow rhythmically, this will permit several important questions to be asked: Are per-related molecular cyclings actually correlated with behavioral circadian rhythms of Drosophila? The latter are controlled (in part) by per's action, and the flies rest/activity cycles persist for many days in constant conditions: so how long does the molecular rhythm persist? Can light-induced phase shifts of Drosophila's biological rhythmicity be shown to correlate with phase alterations of per-controlled protein cyclings? It is further suggested that the per-luciferase fusion system could eventually be applied to experiments aimed at asking at the molecular level if individual animal cells contain circadian clocks and if these pacemakers share common properties with those of plants? Given the ubiquity of circadian-regulated physiology, the identification of common clock components will have an impact on understanding the pacemaker mechanism and malfunctions associated with known features of human well-being.

该建议涉及对生物钟的分子控制 单个生物和单细胞。较高的植物包括一个系统 对于这些研究，部分原因是这些生物提供了顺式作用DNA 介导时钟控制基因表达的元素。利用这一点 属性是针对时钟调节基因开发的非侵入性测定 完整植物中的表达：时钟调节基因的启动子 （CAB2）来自拟南芥的cab2已融合到编码萤火虫的cDNA 荧光素酶和转基因植物是包含该构建体的。 初步数据表明，当植物被CAB2-转化时 荧光素酶基因融合用荧光素喷洒，植物F“发光” 有节奏地表明昼夜节律的转录可以是 在完整的组织中监测。我们建议将这项技术扩展到 分析单个植物细胞中码头调节的转录。这 假设是，后生生物的个别细胞可以包含 昼夜节律。植物系统将提供强大的单元格级 分析以解决此问题。我们还将在生化上确定 振荡器和CAB基因之间的信号转导途径 植物悬浮细胞培养中的启动子，以检验以下假设。 钙调蛋白依赖性途径在调节驾驶室基因方面很重要 转录。 在另一个系统中，果蝇，顺式作用元素 已显示“时钟基因座”被证明参与了中介 每个基因产物水平的环状变化。建议应用 荧光素酶报告基因分析分子节奏的系统 全面级别。特定的实验将涉及每酸性酶 融合和转基因菌株类似于拟南芥。如果 个体苍蝇可以以节奏的方式显示出来，这将允许 要问的几个重要问题：是每个相关的分子 骑自行车实际上与行为昼夜节律相关 果蝇？后者是由Per的行动控制的，而 在恒定条件下，苍蝇的休息/活动周期持续了很多天： 那么分子节奏持续多长时间？可以光引起的相 果蝇生物节奏的转移与 人均蛋白质自行车的相位改变？进一步 建议每个葡聚糖酶融合系统最终可能是 应用于旨在询问分子水平的实验 单个动物细胞包含昼夜节律时钟，如果这些起搏器 与植物的共同特性？考虑到无处不在 昼夜节律的生理学，公共时钟的识别 组件将对了解起搏器机制有影响 以及与人类福祉的已知特征相关的故障。