CONTROL OF GENE ACTIVITY AND ORGANELLE BIOGENESIS

纤毛和鞭毛是复杂的微管细胞器， 在真核生物中普遍存在。 在人类中，它们存在于呼吸道中。 生殖道和女性生殖道组织中发生率较高 癌症。 此外，在这些组织中，纤毛功能障碍可引起 呼吸和生育问题。 本提案的长期目标 是了解生物起源的分子机制， 纤毛的功能 低等真核生物，嗜热四膜虫， 做模特 该系统的优点包括我们最近的发现， α微管蛋白和β微管蛋白各编码一种类型， 由一个阿尔法和两个贝塔基因，加上我们新开发的 高频转化、基因置换和基因转移的方法 使用电穿孔破坏。 这些方法将允许广泛的 α-微管蛋白和β-微管蛋白的功能分析及其 纤毛中的次级修饰。 利用新开发的载体， 编码纤毛形成和/或纤毛运动所必需的蛋白质 将通过突变体功能互补克隆并测序; 蛋白质产物将被表征和定位， 免疫化学方法。 因为我们最初的三个纤毛突变体中的两个 也有一个细胞周期表型，这些研究不仅应该摆脱 光正常纤毛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，对正常发育至关重要的事件， 在肿瘤细胞中经常异常。

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.