A Resource Center for Tetrahymena Thermophila

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EXCEED THE SPACE PROVIDED. This proposal seeks to establish a centralized repository for genetically distinct strains of Tetmhymena thermophila, a ciliated protozoan that has served as a key model for studies of eukaryotic cellular and molecular biology for more than 50 years. The stock center will 1) collect, annotate, and store experimentally useful cell lines; 2) establish a database that will provide essential information about these strains to the community at-large; and, 3) supply actively growing cultures to researchers around the world. This resource will provide a safe haven for the roughly 4000 unique strains developed over the past several decades, and at the same time, serve as an important adjunct to the ongoing Tetrahymena genome sequencing project. Activities spawned by this large-scale, NIH-funded sequencing effort are expected to result in the development of many new strains that will benefit greatly from the existence of a centralized storage and distribution facility. In addition to the establishment of a stock center, the proposal also intends to lay the groundwork for creation of a physical map of the Tetrahymena genome. This second goal is an extension of research currently being undertaken in the Principal Investigator's laboratory, and seeks to adapt the Cre-lox recombinase technology to the Tetrahymena system in order to create a panel of mid-sized (~ 4-5 Mb) overlapping deletions spanning the micronuclear genome. This set of specifically targeted, mid- sized deletions will augment an existing panel of large and small deletions already constructed in our laboratory by random mutagenesis, and will act as a scaffold on which genetic and molecular markers can be quickly aligned. The ability to create viable, homozygous deletions of virtually any size is only possible due to the unique dimorphic nuclear structure of Tetrahymena, and represents a powerful tool that will pave the way for functional genomic studies in this system and enhance the utility of the resource overall. Although a unicellular organism with simple growth requirements, Tetrahymena is endowed with unique genetic properties, and a structural and functional complexity comparable to that of higher metazoa. Not surprisingly, it has contributed enormously to our understanding of a wide range of biological phenomena including many with direct relevance to human health. The resource described in this proposal will ensure the continued use this organism for the benefit of society as a whole.