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.
