Regulation of Genes Encoding Flagellar Proteins in Chlamydomonas reinhardtii

描述（由申请人提供）：纤毛和鞭毛从人体细胞表面延伸，具有运动和信号传导功能。许多疾病，包括多囊肾病和原发性纤毛运动障碍，都是由纤毛丢失或功能障碍引起的。纤毛的结构和功能在动物和许多单细胞真核生物中是高度保守的，包括莱茵衣藻，它是研究纤毛和鞭毛结构和功能的首要模式生物。在人和衣单胞细胞中，纤毛和鞭毛的组装都伴随着编码鞭毛蛋白的基因的上调。虽然对诱导机制知之甚少，但衣藻有一个独特的实验工具组合来解决这个问题。编码衣藻鞭毛蛋白的基因在鞭毛脱落后在大培养或多孔板中同步诱导，使生化分析和大规模突变体筛选成为可能。实验引入的DNA整合在整个衣藻基因组中，使得使用可选择的标记来创建突变和识别突变基因成为可能。本研究的长期目标是利用衣藻作为模式生物，更好地了解纤毛和鞭毛组装过程中的基因诱导机制。已确定的转录调节因子将成为寻找致病突变的候选者。本提案的工作将验证衣藻中鞭毛特异性转录调控因子的激活是鞭毛诱导基因表达所必需的假设。在脱毛后上调荧光素酶/鞭毛动力蛋白轻链（LC8）启动子结构的菌株将通过选择性标记进行转化诱变，并筛选表达上调缺陷的转化子。突变基因将被鉴定和预测转录调控因子将进一步表征。在一项独立的研究中，LC8启动子的功能特征将是在LC8/荧光素酶结构中产生突变，并将这些突变结构重新引入野生型细胞。启动子的重要功能区域将用于从组装鞭毛的细胞中纯化dna结合蛋白。这些蛋白质将通过生物化学和RNA干扰抑制其表达来进一步表征。虽然衣藻已经成为许多研究鞭毛组装过程中基因诱导的主题，但尚未发现参与该调控的蛋白质。这些实验将把衣藻作为模式生物的独特优势与现有的基因调控研究技术相结合，克服这一领域进展的障碍。

DESCRIPTION (provided by applicant): Cilia and flagella extend from human cell surfaces and function in motility and signaling. Many disorders, including polycystic kidney diseases and primary ciliary diskinesia, result from loss or malfunction of cilia. Ciliary structure and function are highly conserved among animals and many unicellular eukaryotes, including the green alga Chlamydomonas reinhardtii, the premier model organism for the study of ciliary and flagellar structure and function. In both human and Chlamydomonas cells, assembly of cilia and flagella is accompanied by up regulation of genes encoding flagellar proteins. While little is understood about the mechanism of induction, Chlamydomonas has a unique combination of experimental tools for addressing this problem. Genes encoding Chlamydomonas flagellar proteins are synchronously induced following flagellar detachment either in large cultures or in multi-well plates, making biochemical analysis and large- scale mutant screens possible. Experimentally introduced DNA integrates throughout the Chlamydomonas genome making it possible to use selectable markers both to create mutations and to identify the mutated genes. The long-term goal of this research is to develop a better understanding of the mechanism of gene induction during ciliary and flagellar assembly using Chlamydomonas as a model organism. Identified transcriptional regulators will become candidates in the search for disease-causing mutations. The work in this proposal will test the hypothesis that activation of flagella-specific transcriptional regulators is required for deflagellation-induced gene expression in Chlamydomonas. A strain that upregulates a luciferase/flagellar dynein light chain (LC8) promoter construct following deflagellation will be mutagenized by transformation with a selectable marker and the transformants will be screened for defects in upregulation. The mutant genes will be identified and predicted transcriptional regulators will be further characterized. In an independent line of investigation, the LC8 promoter will be functionally characterized by generating mutations in the LC8/luciferase construct and reintroducing these mutant constructs into wild- type cells. The functionally important regions of the promoter will be used to purify DNA-binding proteins from cells assembling flagella. These proteins will be further characterized both biochemically and by knocking down their expression using RNA interference. Although Chlamydomonas has been the subject of many studies on gene induction during flagellar assembly, no proteins involved in this regulation have been identified. These experiments will combine the unique advantages of Chlamydomonas as a model organism with established techniques for studying gene regulation to overcome this barrier to progress in the field. PUBLIC HEALTH RELEVANCE: Cilia extend from the surfaces of cells to receive signals and facilitate movement. Since defective cilia cause human disorders affecting millions of people, the potential of this research to determine how genes are regulated to allow normal growth of cilia makes the proposed work of great importance to public health.