Chromatin Structure and DNA Processing in Euplotes Crassus

[00:78 . 182]为了揭示产生DNA重排的机制，人们正在把有纤毛的原生动物长尾拟虫（Euplotes crasssus）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细胞核结构的问题，以及细胞如何操纵这些结构，使基因组保持功能稳定，同时提供基因组可塑性，这可能是进化和发展的优势。通过研究一种受控的重排过程，比如在大核形成过程中发生的重排过程，可以确定哪些染色体蛋白质、DNA序列和酶参与其中，以及如何控制这一过程。像其他纤毛原生动物一样，大毛藻也有两种核，即微核和大核，它们的性质不同。最值得注意的是，染色体分离不存在于宏核中，转录不存在于微核中。这两种核的染色质、染色体结构和基因组组织不同。此外，在微核形成宏核的发育过程中，发生了广泛的DNA消除，包括位点特异性缺失和端粒添加的染色体断裂。这些重排的丰富性，它们的发育规划，以及它们在单细胞生物中可以大量同步发育的发生，为染色体结构和基因组重排机制的分析提供了一个独特的模型系统。首席研究员已经确定了一种不寻常的染色质结构，其高度丰富的Tec元素转座子在大核发育过程中正在被消除。此外，她的实验室还发现了一种富含赖氨酸/精氨酸、组蛋白H1或hmg样的“染色体支架”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元素）相关，c)它与拓扑异构酶II共定位，d)它在体外影响拓扑异构酶II的活性。这指出了拓扑异构酶ii介导的染色体凝聚过程的作用，该过程在纤毛虫基因组重排中的所有生物体中都是保守的。该项目旨在确定p85的序列，以便在其他生物体中鉴定任何类似的蛋白质。此外，p85在DNA消除中的作用将通过定义它结合的DNA序列和它具有的序列特异性，以及通过确定该蛋白质如何影响染色质结构来确定。最后，进一步分析p85与拓扑异构酶II的相互作用。

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.