Mutants of Tetrahymena With Altered Responses to External ATP and GTP

外源ATP和GTP在四膜虫中是去极化的趋化剂。这些驱避剂可能作为细胞质指示剂，使这些自由游动的真核细胞能够避免裂解（破裂）细胞和任何导致这些细胞裂解的物质，就像“水中有血”的信号。嘌呤能受体参与检测许多脊椎动物细胞类型的细胞裂解，其中ATP作为细胞裂解的主要细胞质指标，通过离子性P2X和代谢性P2Y型ATP受体。需要验证的假设是，在四膜虫中嘌呤能的接受有两种不同的途径，一种需要代谢性ATP受体途径，另一种需要一种新型的嗜离子性GTP受体。如果ATP反应通路的任何部分是单独的通路，则敲除突变不应影响GTP反应。目的是使用组合的正向和反向遗传学方法来确定这些反应的新途径和特征成员的作用。对于正向遗传学方法，游泳试验将用于行为突变体筛选，以获得对ATP或GTP反应改变的突变体。诱变过程将利用一种新的反义核糖体文库，它提供了功能基因产物的随机破坏和识别rDNA中负责表型的基因序列的能力。这已被用于鉴定许多有趣的突变体和负责其表型的基因产物。四膜虫至少有两种不同的行为突变体（B6和413）的基因序列已经通过这种方法被识别出来，它们都改变了对外部GTP的游泳反应。反向遗传学方法将涉及使用这些（和其他）反义突变序列在四膜虫基因组数据库中识别全长序列，然后使用该序列作为设计载体以产生稳定的基因敲除。其他被怀疑参与ATP或GTP反应的基因（如蛋白激酶C、蛋白激酶A、外ATP酶、酪氨酸激酶和假定的ATP受体，我们称之为TP2Y）也会发生敲除突变，并测试突变体对ATP和GTP的反应。这项工作将为嘌呤能接受的新机制提供见解，并为理解化学感觉转导和适应途径提供简单的模型系统。该项目将为研究生和本科生以及感兴趣的高中学生、他们的老师和小型文理学院的合作者提供假设驱动的研究经验。

External ATP and GTP are depolarizing chemorepellents in Tetrahymena. These repellents may serve as cytoplasmic indicators to enable these free swimming eukaryotic cells to avoid lysed (broken open) cells and whatever caused those cells to lyse, acting like a "blood in the water" signal. Purinergic receptors are known to be involved in detecting cell lysis in many vertebrate cell types, with ATP acting as the primary cytoplasmic indicator for cell lysis by way of both ionotropic P2X and metabotropic P2Y type ATP receptors. The hypothesis to be tested is that there are two distinct pathways for purinergic reception in Tetrahymena, one requires a metabotropic ATP receptor pathway and the other uses a novel ionotropic GTP receptor. Knockout mutants in any part of the ATP response pathway should not affect GTP responses if they are separate pathways. The objectives are to use a combined forward and reverse genetics approach to identify the roles of both the novel and the characterized members of the proposed pathways for these responses. For the forward genetics approach, swimming assays will be used in behavioral mutant screens to obtain mutants with alterations in their responses to ATP or GTP. The mutagenesis procedure will take advantage of a novel antisense ribosome library, which provides for both random disruption of functional gene products and the ability to identify the gene sequence in the rDNA which is responsible for the phenotype. This has been used to identify many interesting mutants and the gene products responsible for their phenotypes. Gene sequences for at least 2 different behavioral mutants of Tetrahymena have already been identified by this approach (called B6 and 413) and both have altered swimming responses to external GTP. A reverse genetics approach will involve using these (and other) antisense mutant sequences to identify the full-length sequences in the Tetrahymena Genome Database and then using that sequence to a design vectors for producing stable gene knockouts. Knockout mutations will also be made in other genes suspected to be involved in ATP or GTP responses (like protein kinase C, protein kinase A, the ecto-ATPase, tyrosine kinase and the putative ATP receptor we call TP2Y) and the mutants tested for their responses to ATP and GTP. This work will provide insights into novel mechanisms involved in purinergic reception and a simple model system for understanding chemosensory transduction and adaptation pathways. The project will provide hypothesis-driven research experience to graduate and undergraduate students as well as to interested high scholl students, their teachers and collaborators in small liberal arts colleges.