Chromatin Structure and DNA Processing in Euplotes Crassus

克拉苏幼虫的染色质结构和 DNA 加工

• 批准号: 0078182
• 负责人: Carolyn Jahn
• 金额: $ 30万
• 依托单位: Northwestern University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0078182&HistoricalAwards=false
• 关键词: Chromatin; Structure; DNA; Processing; Euplotes

0078182 JahnThe ciliated protozoan Euplotes crassus is being studied as a model system in order to uncover the mechanisms producing rearrangements of DNA. Ultimately, investigating this system should help answer questions about the structure of chromosomes and the nucleus, and how cells manipulate these structures such that the genome remains functionally stable while providing genome plasticity, which can be evolutionarily and developmentally advantageous. By studying a controlled rearrangement process such as that occurring in the E. crassus during the formation of a macronucleus, it can be determined what chromosomal proteins, DNA sequences and enzymes are involved and how the process is controlled. E. crassus, like other ciliated protozoa, possesses two types of nuclei, macronuclei and micronuclei, that differ in their properties. Most notably, chromosome segregation is absent from the macronucleus and transcription is absent from the micronucleus. Chromatin and chromosome structure and genomic organization differ in these two types of nuclei. In addition, during the developmental process of forming macronuclei from micronuclei, extensive DNA elimination occurs that includes site-specific deletions and chromosome breakage with telomere addition. The abundance of these rearrangements, their developmental programming, and their occurrence in single-celled organisms that can be developmentally synchronized en masse, provide a unique model system for the analysis of chromosome structure and the mechanisms of genome rearrangements. The principal investigator has identified an unusual chromatin structure of the highly abundant Tec element transposons that are undergoing elimination during macronuclear development. In addition, her laboratory has identified a lysine/arginine-rich, histone H1 or HMG-like,'chromosome scaffold' protein (p85) that has the following properties: a) it is only found in the developing macronucleus, b) it is associated with eliminated DNA (including the Tec elements), c) it colocalizes with topoisomerase II, and d) it affects topoisomerase II activities in vitro. This points to a role for topoisomerase II-mediated, chromosome condensation processes that are conserved in all organisms in ciliate genomic rearrangements. This project aims to determine the sequence of p85 to allow identification of any similar proteins in other organisms. In addition, the role of p85 in DNA elimination will be determined by defining what DNA sequences it binds to and what sequence specificity it has, and by determining how this protein affects chromatin structure. Finally, the interaction of p85 with topoisomerase II will be further analyzed.

0078182为了揭示产生脱氧核糖核酸重排的机制，人们正在将纤毛虫原生动物Eplotes Crassus作为一个模型系统进行研究。最终，研究这个系统应该有助于回答关于染色体和核的结构，以及细胞如何操纵这些结构，使基因组在保持功能稳定的同时提供基因组可塑性，这可能对进化和发育有利。通过研究大核形成过程中的受控重排过程，可以确定哪些染色体蛋白质、DNA序列和酶参与其中，以及这个过程是如何控制的。克拉苏斯原生动物和其他纤毛原生动物一样，具有两种不同性质的核：大核和微核。最值得注意的是，大核中没有染色体分离，微核中没有转录。染色质和染色体的结构和基因组组织在这两种类型的核中不同。此外，在由微核形成大核的发育过程中，会发生广泛的DNA消除，包括位点特异性缺失和端粒增加导致的染色体断裂。这些重排的丰富性，它们的发育编程，以及它们在单细胞生物体中的出现，可以共同发育同步，为分析染色体结构和基因组重排的机制提供了一个独特的模型系统。首席研究人员发现了高度丰富的Tec元件转座子的一种不寻常的染色质结构，在大核发育过程中正在消除这种转座子。此外，她的实验室已经确定了一种富含赖氨酸/精氨酸的组蛋白H1或HMG样蛋白(P85)，它具有以下特性：a)它只存在于发育中的大核中，b)它与消除的DNA(包括Tec元件)有关，c)它与拓扑异构酶II共定位，以及d)它在体外影响拓扑异构酶II的活性。这表明拓扑异构酶II介导的染色体凝聚过程在纤毛虫基因组重排中在所有生物中都是保守的。该项目旨在确定P85的序列，以便在其他生物体中识别任何类似的蛋白质。此外，P85在DNA消除中的作用将通过定义它与什么DNA序列结合和具有什么序列特异性，以及通过确定该蛋白质如何影响染色质结构来确定。最后，我们将进一步分析P85与拓扑异构酶II的相互作用。