Mutants of Tetrahymena With Altered Responses to External ATP and GTP

外源ATP和GTP是四膜虫的去极化化学反应物。这些驱避剂可以作为细胞质指示剂，使这些自由游动的真核细胞避开裂解的(破裂的)细胞和导致这些细胞裂解的任何东西，就像“水中的血”的信号一样。已知在许多脊椎动物细胞中，嘌呤能受体参与细胞裂解的检测，其中ATP通过离子型和代谢型P2Y受体作为细胞裂解的主要细胞质指示物。需要检验的假设是，四膜虫有两条不同的接受嘌呤能的途径，一条需要代谢性ATP受体途径，另一条需要一种新的离子性GTP受体途径。如果三磷酸腺苷反应途径中任何部分的基因敲除突变体是独立的途径，那么它们不应该影响GTP反应。我们的目标是使用正向和反向遗传学相结合的方法来确定这些反应的新的和被描述的路径成员的角色。对于正向遗传学方法，将在行为突变筛选中使用游泳试验，以获得对ATP或GTP反应发生变化的突变。突变过程将利用一个新的反义核糖体文库，该文库既提供了功能基因产物的随机破坏，也提供了识别rDNA中负责表型的基因序列的能力。这已经被用来鉴定许多有趣的突变体和与其表型有关的基因产物。通过这种方法(称为B6和413)已经确定了至少两种四膜虫不同行为突变的基因序列，并且这两种突变都改变了对外部GTP的游泳反应。反向遗传学方法包括使用这些(和其他)反义突变序列来鉴定四膜虫基因组数据库中的全长序列，然后将该序列用于设计载体以产生稳定的基因敲除。被怀疑与ATP或GTP反应有关的其他基因(如蛋白激酶C、蛋白激酶A、胞外ATPase、酪氨酸激酶和我们称为TP2Y的假定的ATP受体)以及突变株也将发生敲除突变，以测试它们对ATP和GTP的反应。这项工作将为理解化学感觉转导和适应途径提供一个新的机制和一个简单的模型系统。该项目将为研究生和本科生以及感兴趣的高中生、他们的老师和小型文科学院的合作者提供假说驱动的研究经验。

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.