Identification of Tetrahymena DNA Rearrangement Genes

描述（由申请人提供）：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控，并最终了解控制染色体结构和稳定性的基本原理。被称为内部消除序列（IES）的特定DNA片段是如何被选择性切除的，这一事实对它们几乎没有共同的结构这一事实提出了挑战。目前，我们对识别和处理这6000个IES的蛋白质机制了解有限。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基因编码新的蛋白质被认为参与DNA重排。这些都是通过基于绿色荧光蛋白（GFP）的筛选策略进行鉴定的，该筛选策略旨在发现在DNA重排发生时特异性定位于分化细胞核的发育表达蛋白。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，并分析由此产生的转基因菌株的表型。此外，我们将测试这些蛋白是否与先前鉴定的DNA重排蛋白相互作用和/或在切除过程中与IES相关。其中一个基因LIA1的初步鉴定表明，其蛋白很可能参与DNA重排。DNA重排机制的其他组成部分的表征将大大提高我们对这一过程的理解。这种生物学现象提供了一个独特的系统，用它来发现细胞在整个基因组中识别单个染色体片段的机制。这种机制可能对确保染色体稳定性至关重要。鉴于癌细胞通常表现出异常的DNA重排，确定确保染色体忠实维持的细胞机制是导致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.