Use of Antisense Ribosome Mutagenesis to Study Purinergic Reception in Tetrahymena

Since ATP normally exists inside of cells, external ATP is a depolarizing chemorepellent in the free-swimming eukaryotic cell Tetrahymena because it represents a "blood-in-the-water" danger signal. The problem to be studied is that the major genes and gene products necessary to generate ATP responses in this model sensory cell are unknown. The methods used are a combination of "forward genetics", using antisense ribosome mutagenesis to generate behavioral mutants and "reverse genetics", using identified gene knockouts and directed antisense ribosomes. Antisense ribosome mutants were produced by transformation of wild type cells with an antisense ribosome library. Each mutant contains an antisense insert in its rRNA which hybridizes to a specific mRNA and reduces the level of that gene product, producing a functional knockout of that protein. Since Tetrahymena normally "dance" back and forth in ATP, behavioral mutant screens were used to isolate individuals, which either don't respond (swim forward) in external ATP or over-respond (dance longer). Over-responsive mutants (called 413) and under-responsive mutants (SA2 and B6) have already been obtained. Their antisense ribosome inserts sequences have been determined by whole cell PCR but this project will re-transform wild type with these inserts to verify them. The sequence information will also be expanded by PCR with cDNA libraries and final verification will involve gene knockouts. This way, the gene responsible for the phenotype can be identified. The broader significance of this work is to provide a model system to study ATP responses in general. Extracellular ATP is detected by nociceptive (chemical pain) nerves to produce pain signals in vertebrates because it indicates danger in the form of cell lysis (breakage). If the Tetrahymena mechanisms are similar enough to those of vertebrates, this may be a simple, humane model system to study these responses and possibly screen for drugs, which may affect them. Graduate and undergraduate students will be very involved in this project.