Forward Genetics in Tetrahymena: Cloning by Complementation

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): The ciliated protozoan Tetrahymena thermophila is a model organism for cellular and molecular biology that has contributed too many discoveries of fundamental importance in basic and biomedical research. Recently, the genome sequence of Tetrahymena was determined to a high level of accuracy and completeness. Annotation of the genome sequence resulted in predictions of over 27,000 protein-coding genes, many of which have human homologs (including 58 corresponding to genes implicated in human disease) that are not also shared by other prominent single-celled model organisms, such as yeast. Tetrahymena is well-suited to many genetic, biochemical, and molecular experimental methods, but has not reached its full potential use as a tool for forward genetics primarily for one reason - the inability to clone the wild type genes responsible for mutant phenotypes by functional genetic complementation. This experimental tool has been available to fungal geneticists for over twenty years and has contributed to their enormous utility as tools for understanding processes fundamental to human health. The aim of this proposal is to develop a resource, available to all interested researchers, that will allow cloning by complementation in Tetrahymena. A large-insert genomic library will be constructed in a vector capable of Tetrahymena genetic transformation. By end-sequencing the clones and mapping them to their genomic locations, a tiled set of clones will be assembled that covers the entire genome. The utility of the resource will be demonstrated by identifying the wild type genes that rescue several interesting mutations available from previous study. A workshop will be held to introduce the required methodology to all interested researchers. Relevance to Public Health: Research with the simple model organism Tetrahymena has contributed to our understanding of several fundamental processes, such as how chromosome ends replicate; that particular study advanced studies of aging and cancer. Numerous other basic studies with far-reaching relevance to human health would be facilitated by the availability of the proposed freely available resource.