Forward Genetics in Tetrahymena: Cloning by Complementation

描述(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做出了太多重要的发现。最近，四膜虫基因组序列的测定达到了很高的准确性和完整性。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，其中许多基因具有人类同源基因(包括58个与人类疾病有关的基因)，而其他著名的单细胞模式生物，如酵母，并不共享这些基因。四膜虫非常适合于许多遗传、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性遗传互补来克隆与突变表型有关的野生型基因。这一实验工具已经为真菌遗传学家提供了20多年，并为它们作为了解对人类健康至关重要的过程的工具做出了巨大贡献。这项提议的目的是开发一种资源，供所有感兴趣的研究人员使用，允许在四膜虫中通过互补进行克隆。一个大插入的基因组文库将被构建在能够进行四膜虫遗传转化的载体中。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，将组装一组覆盖整个基因组的克隆。该资源的用途将通过识别野生型基因来证明，这些基因挽救了之前研究中提供的几个有趣的突变。将举办一次讲习班，向所有感兴趣的研究人员介绍所需的方法。与公共卫生相关：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；这项特定的研究推进了对衰老和癌症的研究。对人类健康具有深远影响的许多其他基础研究将因提供拟议的免费资源而得到便利。

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.