Use of Antisense Ribosome Mutagenesis to Study Purinergic Reception in Tetrahymena

由于三磷酸腺苷通常存在于细胞内，因此在自由游泳的真核细胞四膜虫中，外部三磷酸腺苷是一种去极化的化学排斥剂，因为它代表着一种“水中有血”的危险信号。需要研究的问题是，在这个模型感觉细胞中产生ATP反应所需的主要基因和基因产物是未知的。使用的方法是“正向遗传学”和“反向遗传学”的结合，前者使用反义核糖体突变来产生行为突变，后者使用已识别的基因敲除和定向反义核糖体。用反义核糖体文库转化野生型细胞，获得反义核糖体突变体。每个突变体都在其rRNA中包含一个反义插入片段，该片段与特定的mRNA杂交，并降低该基因产物的水平，产生该蛋白质的功能性敲除。由于四膜虫通常在ATP中来回“跳舞”，行为突变筛选被用来隔离在外部ATP中没有反应(向前游泳)或过度反应(跳舞更长时间)的个体。已经获得了高反应突变体(称为413)和低反应突变体(SA2和B6)。它们的反义核糖体插入序列已经通过全细胞聚合酶链式反应确定，但本项目将用这些插入重新转化野生型来验证它们。序列信息还将通过使用cDNA文库的聚合酶链式反应进行扩展，最终验证将涉及基因敲除。通过这种方式，可以确定与表型有关的基因。这项工作的更广泛的意义是提供一个总体上研究ATP响应的模型系统。在脊椎动物中，伤害性(化学疼痛)神经检测到细胞外的三磷酸腺苷产生疼痛信号，因为它以细胞裂解(破裂)的形式指示危险。如果四膜虫的机制与脊椎动物的机制足够相似，这可能是一个简单、人道的模型系统来研究这些反应，并可能筛选可能影响它们的药物。研究生和本科生将非常参与这个项目。

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.