DNA Rearrangements and Macronuclear Development in Cilated Protozoa

基因组重塑发生在整个真核生物界，包括众所周知的例子，如酵母中的交配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，大量的DNA被消除和重排，形成转录活性大核作为有性生殖的正常部分。在四脲草履虫中，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移除。先前的研究导致草履虫IES与转座因子相关的假设。本项目将确定草履虫IES的28 bp区域，这些区域是精确的DNA剪接所需的，并将这些区域与切除转座因子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。将通过注射到正在进行大核发育的细胞中来测试每个突变IES的准确剪接。第二种方法将涉及从不同草履虫株中测序几个进化相关的IES的例子。这些序列的比较将鉴定保守的核苷酸，这些核苷酸由于其进化保守性而被认为是重要的。将通过在IES中构建适当的突变来测试这一假设。在相关的纤毛虫四膜虫嗜热的调查将集中在最近确定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消除和转座因子插入-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。对发育调节的延伸因子2的研究可能揭示真核生物蛋白质合成控制的新机制。

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.