Forward genetic analysis of lysosome-related organelle formation in Tetrahymena thermophila

嗜热四膜虫溶酶体相关细胞器形成的正向遗传分析

• 批准号: 1613922
• 负责人: Aaron Turkewitz
• 金额: $ 32.64万
• 依托单位: University of Chicago
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1613922&HistoricalAwards=false
• 关键词: Forward; genetic; analysis; lysosome; related

The overall project objective is to develop a deeper understanding of the cellular mechanisms that have evolved to allow cells to interact, in sophisticated ways, with their surroundings. In particular, the project will investigate a feature of eukaryotic cells called lysosome-related organelles (LROs), by using genetic approaches in a single celled organism called Tetrahymena thermophila. The project will significantly enhance the tools that are available to perform advanced genetics using Tetrahymena, and will allow students at several different levels to be trained in using those tools. While the project is focused on using these genetic tools to understand one aspect of cellular organization in Tetrahymena, the findings will be highly relevant to broad questions about how cells in general have become specialized to perform their many functions. In addition, the tools that are being developed can be applied in the future to probing a wide array of cellular features. Findings from this project may lead to patenting and commercialization of genetic modifications to Tetrahymena, to make this organism a more efficient tool for commercial production of proteins and lipids. The project will involve students from a wide variety of backgrounds, including students from groups that are currently under-represented in science. The project will hone a forward genetic approach to dissecting the biosynthesis of LROs. LROs are ubiquitous and heterogeneous eukaryotic organelles that provide a very wide range of functions in cellular physiology and intercellular communication, and whose evolution appears to have been a major driver of diversification in the secretory pathway. The project will enhance the understanding of mechanisms involved in LRO formation, by identifying genes required for this pathway in Tetrahymena. The approach takes advantage of a high-throughput screen to isolate Mendelian mutants, following chemical mutagenesis, that are defective in LRO formation. Those mutants will be characterized using cell biological and classical genetic approaches. Following that analysis, the mutant genomes will be sequenced, after employing an outcross and backcross strategy to generate pools of F2 meiotic segregants from each initial mutant. By taking this strategy, one can greatly simplify the problem of identifying the causative mutation in each mutant, and thereby identifying the gene required for LRO formation in that strain. It is anticipated that approximately 10 mutants will be analyzed in this fashion, to the level of identifying the causative genetic lesions.

整个项目的目标是加深对细胞机制的理解，这些机制已经演变成允许细胞以复杂的方式与周围环境相互作用。特别是，该项目将通过在一种名为嗜热四膜虫的单细胞有机体中使用遗传方法来研究真核细胞的一种称为溶酶体相关细胞器(LROS)的特征。该项目将大大增强可用于使用四膜虫进行高级遗传学研究的工具，并将允许几个不同级别的学生接受使用这些工具的培训。虽然该项目的重点是使用这些遗传工具来了解四膜虫细胞组织的一个方面，但这些发现将与广泛的问题高度相关，这些问题是关于细胞一般是如何变得专门来执行其多种功能的。此外，正在开发的工具可以在未来应用于探索一系列广泛的蜂窝特征。该项目的发现可能导致四膜虫基因修饰的专利和商业化，使这种生物成为更有效的蛋白质和脂类商业生产工具。该项目将涉及来自不同背景的学生，包括来自目前在科学领域代表性不足的群体的学生。该项目将磨练一种正向遗传学方法来剖析LRO的生物合成。LRO是普遍存在的异质真核细胞器，在细胞生理和细胞间通讯中提供广泛的功能，其进化似乎是分泌途径多样化的主要驱动因素。该项目将通过识别四膜虫中这一途径所需的基因，加强对LRO形成机制的理解。该方法利用高通量筛选来分离化学诱变后在LRO形成中有缺陷的孟德尔突变体。这些突变体将使用细胞生物学和经典遗传学方法进行表征。在这一分析之后，将对突变基因组进行测序，然后采用异交和回交策略从每个初始突变产生F2减数分裂分离体库。通过采取这一策略，人们可以极大地简化识别每个突变体中的致病突变的问题，从而识别该菌株中形成LRO所需的基因。预计将以这种方式分析大约10个突变，以识别致病遗传损伤。