Investigation of the spontaneous circadian hamster mutation Theta

描述（由申请人提供）：细胞自主分子钟在时间上组织许多基本的生理功能。因此，生物钟的紊乱会对健康产生诸多影响。正向遗传筛选已经确定了多个时钟组件，时钟的核心原理是高度保守的。最近，我们的合作者发现了一只金仓鼠（Mesocricetus auratus），它有一个自发突变（Theta），导致仓鼠的内源性月经大幅延长。这项提议的重点将是阐明这种突变如何破坏核心时钟基因之间已知的关系，并最终确定仓鼠基因组中的突变。我将研究组织中mRNA和蛋白质的表达动态，以确定已知时钟基因之间关系的变化。为了确定周期延长是否是Theta时钟的细胞自主特征，我将在原代成纤维细胞培养中使用基于细胞的实时发光报告器。最后，使用全基因组测序和整体分离分析，我将在仓鼠基因组中确定一个候选区域，并通过细胞测定缩小变异范围，直到确定致病突变。最终，Theta将提供对内源性时钟正确计时机制的深入了解。

DESCRIPTION (provided by applicant): A cell autonomous molecular clock temporally organizes many essential physiological functions. Disruption of the clock thus has numerous consequences for health. Forward genetic screens have identified multiple clock components and the core principles of the clock are highly conserved. Recently, our collaborator discovered a golden hamster (Mesocricetus auratus) with a spontaneous mutation (Theta) that results in a dramatic elongation of the hamster's endogenous period. The focus of this proposal will be to elucidate how this mutation disrupts known relationships between core clock genes and, eventually, to identify the mutation in the hamster genome. I will look at mRNA and protein expression dynamics in tissue to identify alterations in the relationships between known clock genes. To determine whether period elongation is a cell autonomous feature of the Theta clock, I will use cell-based real time luminescence reporters in primary fibroblast cultures. Finally, using whole genome sequencing and bulk segregation analysis, I will identify a candidate region in the hamster genome and narrow the field of variants through cellular assays until the causative mutation has been determined. Ultimately, Theta will provide insight into the mechanisms governing the correct timing of the endogenous clock.