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许多重要的基础性发现。近年来，对四膜虫基因组序列的测定已达到较高的准确性和完整性。基因组序列的注释导致超过27,000个蛋白质编码基因的预测，其中许多具有人类同源基因（包括58个与人类疾病相关的基因相对应的基因），而其他主要的单细胞模式生物，如酵母，也没有共享这些基因。四膜虫非常适合许多遗传、生化和分子实验方法，但尚未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遗传互补克隆负责突变表型的野生型基因。这个实验工具已经为真菌遗传学家提供了20多年，并为他们作为理解人类健康基本过程的工具做出了巨大的贡献。本提案的目的是开发一种资源，可供所有感兴趣的研究人员使用，这将允许在四膜虫中通过互补进行克隆。一个大插入基因组文库将构建在一个载体能够四膜虫遗传转化。通过对克隆进行末端测序，并将它们映射到它们的基因组位置，一组覆盖整个基因组的克隆将被组装起来。资源的效用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。将举行一个讲习班，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对几个基本过程的理解，例如染色体末端如何复制；这项特别的研究推进了衰老和癌症的研究。拟议免费提供的资源将促进与人类健康有深远关系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.