DNA Rearrangements and Macronuclear Development in Cilated Protozoa

基因组重塑发生在整个真核生物王国中，包括众所周知的例子，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，大量的DNA被消除并重新排列，形成转录活跃的大核，作为有性繁殖的正常部分。在草履虫中，估计有50,000个被称为内部消除元件(IESS)的短DNA元件通过位点特异性重组从基因组中移除。先前的研究导致假设草履虫Iess与转座因子有关。这个项目将确定草履虫IES的28个碱基的区域，这些区域是准确的DNA剪接所需的，并将这些区域与那些对切除转座元件至关重要的区域进行比较。其中一系列实验将涉及在克隆的IES中构建特定突变。每个突变的IESS都将通过注射到正在进行大核发育的细胞中进行准确剪接测试。第二种方法将涉及对来自不同草履虫菌株的几个进化上相关的IESS样本进行测序。这些序列的比较将确定保守的核苷酸，这些核苷酸被认为是重要的，因为它们在进化上是保守的。这一假设将通过在IES中构建适当的突变来检验。对相关的嗜热四膜虫的研究将集中在最近发现的一个发育调节的翻译延长因子-2上。基因中断和替换实验将确定该基因及其相应的蛋白质是否参与了基因组重塑过程所需的翻译控制机制。这项研究将有助于更广泛地理解纤毛虫DNA消除与转座元件插入-切除之间的关系，并可能揭示这种生物中DNA剪接的新的生化特征。对发育调节的延伸因子2的研究可能揭示控制真核生物蛋白质合成的新机制。

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.