Regulated DNA Deletion in Tetrahymena thermophila

纤毛原生动物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控，并最终了解控制染色体结构和遗传稳定性的基本原理。特定的DNA片段（称为删除元件）是如何被选择性切除的，这一事实对它们在大小和序列上的多样性提出了挑战。PI了解细胞如何识别这些不同序列的方法是详细了解两个DNA片段（称为M和R删除元件）的删除机制，从而确定共同的调节特性。在这项工作中，这些元件将被诱变，以确定针对这些DNA片段的特定序列，以便消除。突变缺失元件将被整合到基因组中，以便在此过程中可以监测它们的切除。这将允许在已鉴定的顺式作用序列中确定突变，这些突变消除了元件与参与切除的蛋白质之间的关联。这些突变分析的一个主要目标是确定激活缺失元件的非基因转录的序列，并确定DNA缺失是否需要转录。为了进一步探索这些DNA重排的表观遗传调控的分子基础，将定义这种调控的最低要求。先前的一些遗传学研究支持一种模型，即从亲本细胞核产生的元件的RNA拷贝作为抑制信号，阻止同源元件的DNA删除。这种解释和其他可能的解释都将受到检验。结合遗传和表观遗传调控成分的研究将加快对这一过程的全面理解。本项目研究了纤毛原生动物嗜热四膜虫发育过程中发生的受调节的DNA重排过程。精确重排四膜虫基因组的细胞机制可能涉及许多确保该生物体适当遗传稳定性的成分。这项研究的目的是了解特定的DNA片段是如何被清除的。其长期目标是了解这些DNA片段的移除如何影响体细胞基因组的正常遗传稳定性。对四膜虫中这一过程的研究将为了解所有生物的遗传机制提供基本的见解。

The ciliated protozoan Tetrahymena thermophila excises an estimated 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 genetic stability. Understanding how particular DNA segments, called deletion elements, are selectively excised is challenged by the fact that they are quite diverse in size and sequence. The PI's approach toward understanding how the cell recognizes these diverse sequences has been to understand in detail the mechanism of deletion of two DNA segments, called the M and R deletion elements, and thus identify common regulatory properties. In this work, these elements will be mutagenized in order to identify the specific sequences that target these DNA segments for elimination. Mutant deletion elements will be integrated into the genome such that their excision can be monitored during the process. This will allow the determination of mutations in the identified cis-acting sequences that abolish the association between the element and proteins involved in excision. A major goal of these mutational analyses is to delimit the sequences that activate the non-genic transcription of deletion elements and determine whether transcription is required for DNA deletion. To further explore the molecular basis of epigenetic regulation of these DNA rearrangements, the minimal requirements for this regulation will be defined. Several prior genetic studies support a model that RNA copies of the element produced from parental nuclei act as inhibitory signals that block DNA deletion of the homologous element. This and other possible explanations will be tested. Combining studies that address both genetic and epigenetic regulatory components will expedite a comprehensive understanding of this process. This project investigates a process of regulated DNA rearrangement that occurs during development of the ciliated protozoan Tetrahymena thermophila. The cellular machinery that accurately rearranges the Tetrahymena genome likely involves many of the components that ensure the proper genetic stability of this organism. This research aims to understand how particular DNA segments are targeted for elimination. Its long-term goal is to learn how removal of these DNA segments affects the normal genetic stability of the somatic genome. Investigation of this process in Tetrahymena should provide fundamental insight into genetic mechanisms that operate in all organisms.