DNA elimination mechanisms in Tetrahymena

许多生物体的基因组都含有未知功能的DNA。在一些生物体中，这种所谓的垃圾DNA在发育过程中通过复杂的基因组重排被消除。然而，目前尚不清楚细胞如何分辨哪些是垃圾，哪些不是。这项研究将研究纤毛虫——嗜热四膜虫（Tetrahymena thermophila）如何确定基因组的哪些部分应该作为垃圾扔掉，哪些应该作为重要的功能保留下来。由于垃圾DNA存在于许多生物体的基因组中，这项工作对于理解细胞如何处理外来的、非必需的DNA具有广泛的科学意义。为了完成这项研究，将创建一个可视化基因组相关数据集的工具，并使其可供更广泛的科学界使用，从而改善当前的科学基础设施。该项目将通过从本科到博士后水平的受训者参与假设驱动的研究，增强未来的科学队伍。研究工作将与主要研究者的课程开发活动相协调，从而通过将真实的研究带入大学生物课堂来促进最佳教学实践。参与课堂研究的学生反过来将获得科学发现实践的第一手经验，并增加不断增长的科学知识体系。该项目将阐明真核细胞利用模型实验生物嗜热四膜虫（Tetrahymena thermophila）在基因组的功能不同区域之间建立边界的机制，该生物从其基因组的体细胞拷贝中消除了大部分非编码DNA。利用遗传和生化方法的结合，这项工作将揭示四膜虫细胞用来定义在其发育过程中被消除的DNA片段边界的机制。事实上，四膜虫消除了这些序列，这使得人们可以通过比较重组的体细胞基因组和完整的种系基因组的内容，明确地识别那些被包装为垃圾的序列。该项目将使用现有的四膜虫基因组序列来生成一个基因组浏览器，该浏览器将使用完整的基因组作为参考，绘制保留和消除的区域。先前的研究表明，一种被称为Lia3的调节蛋白对于准确去除被淘汰序列的子集至关重要。基因组浏览器将用于比较正常细胞的DNA与缺乏LIA3的细胞的DNA，以识别当该蛋白缺失时消除边界不准确定义的所有序列。初步实验表明，由Lia3控制的序列含有富含鸟嘌呤(G)的序列，距离每个边界约50个碱基对。当这些序列形成g -四链DNA（一种四链DNA结构）时，Lia3就会与它们结合。这代表了一种新的DNA结合活性，该项目将彻底表征Lia3结合这种非标准DNA结构的能力，并阐明这种结构如何用于组织基因组。此外，四膜虫基因组编码另外三种在序列和基因表达上与Lia3相似的蛋白质，该项目将验证这样一个假设，即这些蛋白质中的每一种都能识别被淘汰DNA的不同子集的边界。总之，这些方法将描述一个以前未知的真核细胞用来定义基因和非编码DNA之间界限的机制，并提供明确的证据表明g -四重体结构具有重要的调节作用。

The genomes of many organisms contain DNA with no known function. In some organisms, this so-called junk DNA is eliminated during development by a complex rearrangement of the genome. However, it is not clear how the cell can tell what is junk and what is not. This research will study how the ciliate, Tetrahymena thermophila, establishes what parts of the genome should be thrown away as junk and what should be retained as functionally important. Because junk DNA is found in the genomes of many organisms, this work could have broad scientific significance on understanding how cells deal with extraneous, non-essential DNA. To accomplish the research, a tool for visualizing genome-related datasets will be created and made accessible for use by the broader scientific community, thereby improving the current scientific infrastructure. The project will enhance the future scientific workforce by engaging trainees from the undergraduate to post-doctoral level in hypothesis-driven research. The research efforts will be coordinated with curricular development activities of the principle investigator, and thus will facilitate best teaching practice by bringing authentic research into the university biology classroom. Students engaged in the classroom research, in turn, will gain first-hand experience in the practice of scientific discovery and add to a growing body of scientific knowledge. This project will elucidate mechanisms that eukaryotic cells use to establish boundaries between functionally distinct regions of the genome by exploiting the model experimental organism, Tetrahymena thermophila, which eliminates much of its non-coding DNA from the somatic copy of its genome. Using a combination of genetic and biochemical approaches, the work will uncover mechanisms that Tetrahymena cells use to define the boundaries of the DNA segments that are eliminated during its development. The fact that Tetrahymena eliminates these sequences allows one to unambiguously identify those that get packaged as junk by comparing the content of the reorganized somatic genome and the intact germline genome. The project will use available Tetrahymena genome sequences to generate a genome browser that will map the retained and eliminated regions, using the intact genome as a reference. Previous research revealed that a regulatory protein called Lia3 is critical for the accurate removal of a subset of the eliminated sequences. The genome browser will be used to compare the DNA of normal cells to DNA from cells lacking LIA3 to identify all the sequences for which elimination boundaries are inaccurately defined when this protein is absent. Preliminary experiments have revealed that the sequences controlled by Lia3 contain guanine (G)-rich sequences positioned about 50 base pairs from each boundary. Lia3 binds these sequences when they form G-quadruplex DNA, a four-stranded DNA structure. This represents a novel DNA binding activity, and the project will thoroughly characterize both the ability of Lia3 to bind this non-standard DNA structure and elucidate how this structure can serve to organize the genome. In addition, the Tetrahymena genome encodes three other proteins that are similar to Lia3 in sequence and gene expression, and the project will test the hypothesis that each of these proteins identifies the boundaries of a distinct subset of eliminated DNA. Together, these approaches will characterize a previously unknown mechanism that eukaryotic cells use to define boundaries between genes and non-coding DNA and provide clear evidence that G-quadruplex structures have important regulatory roles.