Forward Genetics in Tetrahymena: Cloning by Complementation

描述（由申请人提供）：纤毛原生动物嗜热四膜虫是细胞和分子生物学的模式生物，在基础和生物医学研究中贡献了太多具有根本重要性的发现。最近，四膜虫的基因组序列被确定到一个高水平的准确性和完整性。对基因组序列的注释导致了超过27，000个蛋白质编码基因的预测，其中许多具有人类同源物（包括58个与人类疾病有关的基因），这些同源物也没有被其他突出的单细胞模式生物（如酵母）共享。四膜虫非常适合于许多遗传学、生物化学和分子实验方法，但还没有充分发挥其作为正向遗传学工具的潜力，主要原因之一是无法通过功能性遗传互补克隆导致突变表型的野生型基因。真菌遗传学家已经使用这种实验工具超过20年，并且作为理解人类健康基本过程的工具，为它们的巨大效用做出了贡献。这项建议的目的是开发一个资源，提供给所有感兴趣的研究人员，这将允许克隆的互补在四膜虫。将在能够进行四膜虫遗传转化的载体中构建大插入基因组文库。通过对克隆进行末端测序并将其映射到其基因组位置，将组装覆盖整个基因组的平铺克隆集。该资源的实用性将通过鉴定野生型基因来证明，这些基因拯救了先前研究中可用的几个有趣的突变。将举办一个讲习班，向所有感兴趣的研究人员介绍所需的方法。与公共卫生的相关性：对简单模式生物四膜虫的研究有助于我们理解几个基本过程，例如染色体末端如何复制;这项特别的研究推进了对衰老和癌症的研究。拟议的免费资源的提供将促进对人类健康具有深远意义的许多其他基础研究。

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.