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.

总体项目目标是对已经进化的细胞机制有更深入的了解，以使细胞以复杂的方式与周围环境相互作用。 特别是，该项目将通过在称为Tetrahymena thermophila的单个细胞生物中使用遗传方法来研究称为溶酶体相关细胞器（LROS）的真核细胞的特征。 该项目将大大增强使用四膜纳州进行高级遗传学的工具，并允许使用这些工具来培训几个不同级别的学生。 虽然该项目的重点是使用这些遗传工具来了解四膜虫中细胞组织的一个方面，但这些发现将与有关细胞如何变得专门执行其许多功能的广泛问题高度相关。 此外，将来可以使用开发的工具来探测各种各样的细胞特征。 该项目的发现可能会导致对四膜虫的遗传修饰的专利和商业化，以使该生物成为蛋白质和脂质商业生产的更有效工具。该项目将涉及来自各种背景的学生，包括目前在科学领域代表性不足的团体的学生。 该项目将磨练一种剖析LRO的生物合成的正向遗传方法。 LROS是普遍存在的和异质的真核细胞器，在细胞生理和细胞间通信中提供了非常广泛的功能，并且其进化似乎是分泌途径中多元化的主要驱动力。该项目将通过鉴定四膜虫中该途径所需的基因来增强对LRO形成的机制的理解。 该方法利用了高通量屏幕来分离孟德尔突变体，在化学诱变后，在LRO形成中有缺陷。这些突变体将使用细胞生物学和经典遗传方法来表征。在进行分析之后，将在采用露出和反向交叉策略来从每个初始突变体中产生F2减数分裂分离剂的池之后，对突变基因组进行测序。通过采用这种策略，人们可以大大简化识别每个突变体中的因果突变的问题，从而识别该菌株中LRO形成所需的基因。预计将以这种方式分析大约10个突变体，以识别病因病变的水平。