DNA Rearrangements and Macronuclear Development in Cilated Protozoa

纤毛原生动物的 DNA 重排和大核发育

• 批准号: 0112260
• 负责人: James Forney
• 金额: $ 64万
• 依托单位: Purdue University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0112260&HistoricalAwards=false
• 关键词: DNA; Rearrangements; Macronuclear; Development; Cilated

Genome remodeling occurs throughout the eukaryotic kingdom and includes well known examples such as mating type switching in yeast and recombination of immunoglobulin genes in mammals. The ciliated protozoa present an extreme example in which large amounts of DNA are eliminated and rearranged to form the transcriptionally active macronucleus as a normal part of sexual reproduction. In Paramecium tetraurelia, an estimated 50,000 short DNA elements called internal eliminated elements (IESs) are removed from the genome via site specific recombination. Previous research led to the hypothesis that Paramecium IESs are related to transposable elements. This project will define the regions of a 28 bp Paramecium IES that are required for accurate DNA splicing and compare these regions with those critical for excision of transposable elements. One series of experiments will involve the construction of specific mutations in the cloned IES. Each mutant IESs will be tested for accurate splicing by injection into cells that are undergoing macronuclear development. A second approach will involve sequencing several examples of evolutionarily related IESs from different Paramecium strains. Comparison of these sequences will identify conserved nucleotides that are presumed to be important due to their evolutionary conservation. This assumption will be tested by constructing appropriate mutations in the IES. Investigations in the related ciliate Tetrahymena thermophila will focus on a recently identified developmentally regulated translation elongation factor-2. Gene disruption and replacement experiments will determine whether this gene and its corresponding protein are involved in a translational control mechanism that is required for the genome remodeling process. The research will contribute to a broader understanding of the relationship between ciliate DNA elimination and transposable element insertion-excision, and perhaps reveal novel biochemical features of DNA splicing in this organism. The study of a developmentally regulated elongation factor 2 may reveal a novel mechanism for the control of protein synthesis in eukaryotes.

基因组重塑发生在整个真核生物界，包括众所周知的例子，例如酵母中的交配类型转换和哺乳动物中免疫球蛋白基因的重组。纤毛原生动物是一个极端的例子，其中大量的 DNA 被消除并重新排列，形成转录活性大核，作为有性生殖的正常部分。在草履虫中，估计有 50,000 个称为内部消除元件 (IES) 的短 DNA 元件通过位点特异性重组从基因组中去除。先前的研究提出了草履虫 IES 与转座因子相关的假设。该项目将定义精确 DNA 剪接所需的 28 bp 草履虫 IES 区域，并将这些区域与转座元件切除的关键区域进行比较。一系列实验将涉及在克隆 IES 中构建特定突变。将通过注射到正在经历大核发育的细胞中来测试每种突变体 IES 的精确剪接。第二种方法将涉及对来自不同草履虫菌株的进化相关 IES 的几个例子进行测序。这些序列的比较将鉴定出由于其进化保守性而被认为很重要的保守核苷酸。这一假设将通过在 IES 中构建适当的突变来测试。对相关纤毛虫嗜热四膜虫的研究将集中于最近发现的发育调节翻译延伸因子 2。基因破坏和替换实验将确定该基因及其相应的蛋白质是否参与基因组重塑过程所需的翻译控制机制。该研究将有助于更广泛地了解纤毛虫 DNA 消除与转座元件插入-切除之间的关系，并可能揭示该生物体中 DNA 剪接的新生化特征。对发育调节延伸因子 2 的研究可能揭示真核生物中控制蛋白质合成的新机制。