Identification of Tetrahymena DNA Rearrangement Genes

描述(申请人提供)：纤毛虫嗜热四膜虫在发育过程中从其体细胞核中切除了大约6000个特定的DNA片段。该项目旨在了解这种大规模基因组重组的调节，并最终学习控制染色体结构和稳定性的基本原理。要理解特定的DNA片段，即所谓的内部消除序列(IES)，是如何被选择性地切除的，因为它们几乎没有共同的结构。目前，我们对识别和切除这6000个IES的蛋白质机制知之甚少。虽然有四种蛋白质与这一过程有关，但它们的确切作用尚未完全阐明。我们已经确定了五个编码新蛋白的基因，这些基因可能与DNA重排有关。使用基于绿色荧光蛋白(GFP)的筛选策略来鉴定这些蛋白质，该策略旨在寻找在DNA重排发生的地点和时间专门定位于分化核的发育表达的蛋白质。为了证明这五个候选基因是否需要重排，我们将敲除它们的基因，并分析产生的转基因菌株的表型。此外，我们将测试这些蛋白质在切除过程中是否与先前发现的DNA重排蛋白相互作用和/或与IES相关联。对其中一个基因LIA1的初步鉴定表明，它的蛋白质很可能参与DNA重排。对DNA重排机制的其他组成部分的表征将极大地增强我们对这一过程的理解。这种生物现象提供了一种独特的系统，可以用来发现细胞用来识别整个基因组中单个染色体片段的机制。这种机制对于确保染色体的稳定性可能是至关重要的。鉴于癌细胞通常表现出异常的DNA重排，识别确保忠实维持染色体的细胞机制是理解与遗传不稳定相关的疾病的分子基础的重要一步。

DESCRIPTION (provided by applicant): The ciliate Tetrahymena thermophila excises approximately 6000 specific DNA segments from its somatic nucleus during development. This project aims to understand the regulation of this massive genome reorganization and ultimately learn fundamental principles governing chromosome structure and stability. Understanding how particular DNA segments, called internal eliminated sequences (IES), are selectively excised is challenged by the fact that they share little common structure. Currently, we have limited knowledge of the protein machinery that recognizes and excises these 6000 IES. While four proteins have been linked to this process, their exact roles have yet to be fully elucidated. We have identified five genes encoding novel proteins putatively involved in DNA rearrangement. These were identified using a Green Fluorescence Protein (GFP)-based screening strategy designed to find developmentally expressed proteins that localize specifically to the differentiating nuclei where and when DNA rearrangement occurs. To demonstrate whether these five candidates are required for rearrangement, we will knock out their genes and analyze the phenotype of the resulting transgenic strains. Furthermore, we will test whether these proteins interact with previously identified DNA rearrangement proteins and/or associate with the IES during their excision. Initial characterization of one of these genes, LIA1, indicates that its protein very likely participates in DNA rearrangement. Characterization of additional components of the DNA rearrangement machinery will greatly enhance our understanding of this process. This biological phenomenon provides a unique system with which to discover mechanisms cells use to recognize individual chromosomal segments throughout the genome. Such mechanisms are likely critical for ensuring chromosome stability. Given that cancer cells commonly exhibit aberrant DNA rearrangements, identification of the cellular mechanisms that ensure faithful chromosome maintenance is an important step leading to an understanding of the molecular basis of disease associated with genetic instability.