ANALYSES OF A NEW TETRAHYMENA CILIARY DYNEIN

纤毛和鞭毛是普遍存在的复杂的微管细胞器。 在包括人类在内的真核生物中，它们被发现在 呼吸道、女性生殖道和精子细胞。 动力蛋白缺乏引起的睫毛和鞭毛功能障碍 据报道，武器会导致呼吸和生育问题。 人类和其他哺乳动物。这项工作的长期目标是 了解动力蛋白组装成睫状轴丝的机制 并通过使用动态蛋白来了解动力蛋白在纤毛运动中的作用(S) 单细胞原生动物，嗜热四膜虫。作为一个模范系统。 具体地说，角色(S)是一位新发现的睫状肌动力蛋白重 链(天链、动力蛋白、重链)在纤毛运动中起作用 由a)克隆、测序和分析编码 天链动力蛋白重链，b)将突变引入天链 使用靶向基因敲除技术获得动力蛋白重链基因，以及c) 对突变菌株进行分析，从而得出关于 天链动力重链的位置和功能。要克隆 天链基因，天链将被分离并部分测序。 这些序列将被用来设计寡核苷酸引物。底漆 将用于聚合酶链式反应，以扩增编码 天链动力蛋白重链基因的一部分。创建挖空的步骤 载体，则包含可选标记的表达盒将是 插入到天链中间的基因。敲除向量将 在促进生长的条件下转化为共轭四膜虫 转化DNA与靶基因的同源重组 序列。这应该会产生内源性天链的突变株 通过插入表达盒使基因失活，因此， 都错过了天链动力重链。突变菌株将是 进行生化和超微结构分析以确定 新发现的天链动力蛋白重链的位置和功能 链条。这项工作的结果应该会增加我们对动力蛋白的理解 纤毛和鞭毛的组织和功能。

Cilia and flagella are complex microtubular organelles that are ubiquitous among eukaryotes, including humans, where they are found in the respiratory tract, the female reproductive tract and on sperm cells. Ciliary and flagellar dysfunction resulting from the absence of dynein arms has been reported to cause respiratory and fertility problems in humans and other mammals. The long term objectives of this work are to understand the mechanisms by which dyneins assemble into ciliary axonemes and to understand the role(s) of dyneins in ciliary motility by using the unicellular protozoan, Tetrahymena thermophila. as a model system. Specifically, the role(s) that a newly discovered ciliary dynein heavy chain (sky chain dynein heavy chain) plays in ciliary motility will be determined by a) cloning, sequencing and analyzing the gene encoding the sky chain dynein heavy chain, b) introducing mutations into the sky chain dynein heavy chain gene using targeted gene knock out techniques, and c) analyzing the mutant strains to allow inferences to be drawn about the location and function of the sky chain dynein heavy chain. To clone the sky chain gene, the sky chain will be isolated and partially sequenced. The sequences will be used to design oligonucleotide primers. The primers will be used in the polymerase chain reaction to amplify DNA encoding a portion of the sky chain dynein heavy chain gene. To create the knockout vector, an expression cassette containing a selectable marker will be inserted into the middle of the sky chain gene. The knock out vector will be transformed into conjugating Tetrahymena under conditions that promote homologous recombination between the transforming DNA and the target sequence. This should yield mutant strains in which endogenous sky chain genes are inactivated by insertion of the expression cassette and thus, are missing the sky chain dynein heavy chain. Mutant strains will be subjected to biochemical and ultrastructural analyses to determine the location and function of the newly discovered sky chain dynein heavy chain. The results of this work should add to our understanding of dynein organization and function in cilia and flagella.