CONTROL OF CILIA BIOGENESIS IN TETRAHYMENA THERMOPHILA

该项目的长期目标是了解分子 纤毛生物发生的机制。 为此，两个四膜虫 在两种纤毛中具有温度敏感缺陷的嗜热突变体 将分析生物发生和细胞周期的某些方面。 当 其中一个变异体的纤毛脱落后会再生瘫痪的纤毛。 孵育时 在生长培养基中不能完成胞质分裂。 的分子基础 纤毛麻痹将通过比较运动的和麻痹的纤毛来确定 通过二维聚丙烯酰胺凝胶电泳和电子显微镜观察。 野生型和突变体纤毛将通过二维聚丙烯酰胺凝胶进行比较 电泳以鉴定由缺陷基因编码的基因产物。 基因产物在生长细胞中的细胞定位将是 测定 另一个突变体不能启动纤毛生物合成， 在生长中孵育时， 介质 为了确定突变体中的缺陷基因是否编码一种 纤毛生物发生和细胞周期进程所需的激酶， 在允许和限制条件下的基底体磷酸化状态 将确定沉降前后的温度。 一 温度敏感性激酶活性将在 突变细胞 为了确定参与纤毛生物发生的其他基因 和/或细胞周期，这两种突变的基因外抑制因子将被 与世隔绝 纤毛和鞭毛在真核生物中几乎无处不在， 在人类生殖和呼吸道的功能，以及功能障碍 细胞器的变化与几种疾病有关。 一个 了解纤毛和鞭毛组装的分子机制 以及组装与细胞周期协调的方式将有助于 了解所有微管组装的机制， 监管. 这一点很重要，因为细胞骨架的异常是 通常在肿瘤细胞中观察到。

The long term goals of this project are to understand the molecular mechanisms of cilia biogenesis. Towards that end two Tetrahymena thermophila mutants with temperature-sensitive defects in both cilia biogenesis and some aspect of the cell cycle will be analyzed. When deciliated one of the mutants regenerates paralyzed cilia. When incubated in growth medium it fails to complete cytokinesis. The molecular basis for cilia paralysis will be determined by comparing motile and paralyzed cilia by 2-D polyacrylamide gel electrophoresis and by electron microscopy. Wild-type and mutant cilia will be compared by 2-D polyacrylamide gel electrophoresis to identify the gene product encoded by the defective gene. The cellular location of the gene product in growing cells will be determined. The other mutant fails to initiate cilia biogenesis following deciliation and arrests in late micronuclear G2 when incubated in growth medium. To determine whether the defective gene in that mutant encodes a kinase required for cilia biogenesis and for cell cycle progression, the phosphorylation state of basal bodies at permissive and restrictive temperatures before and following deciliation will be determined. A temperature-sensitive kinase activity will be identified in extracts of mutant cells. To identify additional genes involved in cilia biogenesis and/or the cell cycle, extragenic suppressors of the two mutations will be isolated. Cilia and flagella are nearly ubiquitous in eukaryotes and function in the human reproductive and respiratory tracts, and dysfunction of the organelles has been implicated in several disease states. An understanding of the molecular mechanisms of cilia and flagella assembly and the way assembly is coordinated with the cell cycle will contribute to an understanding of the mechanisms by which assembly of all microtubules is regulated. This is important because abnormalities of the cytoskeleton are often observed in neoplastic cells.