Forward Genetics in Tetrahymena: Cloning by Complementation

四膜虫的正向遗传学：互补克隆

• 批准号: 7298417
• 负责人: Robert Stephen Coyne
• 金额: $ 38.01万
• 依托单位: J. CRAIG VENTER INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7298417
• 关键词: Aging; Animal Model; Biochemical Genetics; Bioinformatics; Biological Models; Biomedical Research; Cell model; Characteristics; Chromosome Mapping; Chromosomes; Cloning; Code; Communities; DNA Fingerprinting; Development; Dynein ATPase; Educational workshop; Engineering; Enzymes; Genes; Genetic; Genetic Markers; Genetic Transformation; Genome; Genomic Library; Genomics; Goals; Health; Histone Acetylation; Homologous Gene; Human; Hylobates Genus; Insertional Mutagenesis; Knock-out; Letters; Libraries; Location; Malignant Neoplasms; Maps; Methodology; Methods; Modeling; Molecular; Molecular and Cellular Biology; Motor; Mutation; Organism; Participant; Personal Satisfaction; Phenotype; Positioning Attribute; Process; Proteins; Public Health; RNA; Research; Research Personnel; Resolution; Resource Development; Resources; Role; Sampling; Small RNA; Structure; Technology; Telomerase; Testing; Tetrahymena; Tetrahymena thermophila; Transcriptional Regulation; United States National Institutes of Health; Whole-Genome Shotgun Sequencing; Work; Yeasts; career; gene cloning; gene complementation; genome sequencing; human disease; interest; mutant; novel; programs; telomere; tool; vector

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.

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为高度准确和完整。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 个对应于与人类疾病有关的基因），而其他著名的单细胞模型生物（例如酵母）也没有共享这些同源物。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，主要有一个原因：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。这种实验工具已经被真菌遗传学家使用了二十多年，并为它们作为理解人类健康基础过程的工具的巨大效用做出了贡献。该提案的目的是开发一种资源，供所有感兴趣的研究人员使用，从而允许通过四膜虫互补进行克隆。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，将组装一组覆盖整个基因组的平铺克隆。该资源的实用性将通过鉴定野生型基因来证明，这些基因挽救了先前研究中可用的几个有趣的突变。将举办一个研讨会，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；这项特别研究对衰老和癌症进行了深入研究。拟议的免费资源的提供将促进与人类健康具有深远相关性的许多其他基础研究。