CONTROL OF GENE ACTIVITY AND ORGANELLE BIOGENESIS

纤毛和鞭毛是复杂的微管细胞器，几乎 在真核生物中无处不在。在人类体内，它们存在于呼吸道中。 在女性生殖道组织中发病率较高 癌症的威胁。此外，在这些组织中，睫状体功能障碍可导致 呼吸系统和生育问题。这项提议的长期目标是 是了解生物发生和发展的分子机制 纤毛的功能。使用的是低等真核生物嗜热四膜虫。 作为一名模特。这个系统的优点包括我们最新的发现 α-微管蛋白和β-微管蛋白各只有一种编码类型 通过一个阿尔法基因和两个贝塔基因，再加上我们新开发的 高频转化、基因置换和基因转化的方法 使用电穿孔进行破坏。这些方法将允许广泛的 α-微管蛋白和β-微管蛋白及其功能的分析 纤毛的二次修饰。利用新开发的载体，基因 编码纤毛形成和/或纤毛运动所必需的蛋白质 将通过突变功能的互补进行克隆和测序；它们的 蛋白质产品将通过原位表征和定位 免疫化学方法。因为我们最初的三个纤毛突变体中有两个 也有细胞周期表型，这些研究不应该只掉落 正常纤毛的生物发生和微管蛋白的功能，但也 细胞骨架功能与细胞周期的相互关系 进展，对正常发展至关重要的事件，以及 肿瘤细胞中经常出现异常。

Cilia and flagella are complex microtubular organelles that are nearly ubiquitous among eukaryotes. In humans they are found in the respiratory tract and in the female reproductive tract tissues with high incidences of cancer. Also in these tissues ciliary dysfunction can cause respiratory and fertility problems. The long-term goals of this proposal are to understand the molecular mechanisms underlying the biogenesis and function of cilia. The lower eukaryote, Tetrahymena thermophila, is used as a model. Advantages of this system include our recent findings that there is only one type each of alpha- and beta- tubulin proteins encoded by a single alpha and two beta genes, coupled with our newly developed methods for high frequency transformation, gene replacement and gene disruption using electroporation. These methods will allow extensive analysis of the function of alpha- and beta- tubulin and of their secondary modifications in cilia. Using newly developed vectors, genes encoding proteins essential for cilia formation and/or ciliary motility will be cloned by complementation of mutant function and sequenced; their protein products will be characterized and localized by in situ immunochemical methods. Because two of our initial three ciliary mutants also have a cell cycle phenotype, these studies should not only shed light on normal cilia biogenesis and the function of tubulins, but also on interrelationships between cytoskeletal functions and cell cycle progression, events that are critical for normal development and that are frequently abnormal in neoplastic cells.