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HISTONE HYPERACETYLATION IN REPLICATING SV40 CHROMOSOMES

SV40 染色体复制中的组蛋白高度乙酰化

基本信息

批准号：
7686763
负责人：
Barry Ira Milavetz
金额：
$ 6.75万
依托单位：
UNIVERSITY OF NORTH DAKOTA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-15 至 2012-01-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Using the Simian Virus 40 (SV40) chromosome as a model for eukaryotic chromatin, the proposed research will address the role of histone hyperacetylation at an active replication fork and during post-replication chromatin maturation within the regulatory region of a chromosome whose biological function changes over the course of infection. The studies will answer fundamental questions concerning the mechanism of eukaryotic replication and the relationship between eukaryotic replication and gene regulation which will be potentially applicable to the therapy of a wide variety of diseases including viral infections and cancer. The specific aims of the application are 1. Characterization of histone hyperacetylation in the vicinity of a replication fork and 2. Characterization of histone hyperacetylation associated with post-replicative chromatin remodeling within the SV40 promoter/regulatory region. Histone hyperacetylation in the vicinity of a replication fork will be characterized in replicating SV40 chromosomes either immune selected with antibodies which recognize proteins found only at the fork or labeled with biotin conjugated deoxyribonucleotides. In both strategies the status of histone hyperacetylation at various sites on the pre- and post-replicative sides of the replication fork will be determined using a combination of biophysical separation, inhibition of replication by chemicals and siRNAs, and chromatin fragmentation in combination with chromatin immunoprecipitation analyses including two which we have developed: Immune Selection and Fragmentation (ISF) and Immune Selection Fragmentation and Immunoprecipitation (ISFIP). Post-replication chromatin maturation within the regulatory region will be characterized in SV40 chromosomes and confirmed in replication-competent plasmids carrying portions of the SV40 regulatory region. Maturation occurring during replication will be determined in replication intermediates, while maturation occurring after replication will be determined by comparing histone hyperacetylation present in terminating replicating chromosomes to hyperacetylation present in chromosomes competent for late transcription and encapsidation. Immune selection with antibodies specific for replicating, transcribing, and encapsidating chromosomes will be used in conjunction with various ChIP techniques to determine the status of histone hyperacetylation in the chromosomes and plasmids. PUBLIC HEALTH RELEVANCE: The proposed studies will be the first detailed characterization of histone hyperacetylation during the eukaryotic replication process and will directly link changes in histone hyperacetylation to specific events occurring during the replication process. The results obtained will significantly add to our knowledge of the role of replication in eukaryotic gene regulation and be potentially useful in the development of therapeutics for viral infections and cancers.

描述（由申请人提供）：使用猿猴病毒40（SV 40）染色体作为真核染色质的模型，拟议的研究将解决组蛋白超乙酰化在活跃复制叉和复制后染色质成熟过程中的作用，该染色体的调控区的生物学功能在感染过程中发生变化。这些研究将回答有关真核生物复制机制以及真核生物复制与基因调控之间关系的基本问题，这些问题可能适用于治疗各种疾病，包括病毒感染和癌症。该应用程序的具体目标是1。在复制叉附近组蛋白超乙酰化的表征和2。SV 40启动子/调控区内与复制后染色质重塑相关的组蛋白超乙酰化的表征。在复制叉附近的组蛋白超乙酰化将在复制SV 40染色体中表征，其用识别仅在叉处发现的蛋白质的抗体免疫选择或用生物素缀合的脱氧核糖核苷酸标记。在这两种策略中，将使用生物物理分离、通过化学品和siRNA抑制复制以及染色质片段化与染色质免疫沉淀分析的组合来确定复制叉的复制前和复制后侧上的各个位点处的组蛋白超乙酰化的状态，所述染色质免疫沉淀分析包括我们开发的两种：免疫选择和片段化（ISF）和免疫选择片段化和免疫沉淀（ISFIP）。将在SV 40染色体中表征调控区内的复制后染色质成熟，并在携带SV 40调控区部分的可复制质粒中确认。复制期间发生的成熟将在复制中间体中确定，而复制后发生的成熟将通过比较终止复制染色体中存在的组蛋白超乙酰化与能够进行晚期转录和转录的染色体中存在的超乙酰化来确定。使用对染色体复制、转录和重排具有特异性的抗体进行免疫选择，将与各种ChIP技术结合使用，以确定染色体和质粒中组蛋白超乙酰化的状态。公共卫生相关性：拟议的研究将是真核生物复制过程中组蛋白超乙酰化的第一个详细表征，并将组蛋白超乙酰化的变化与复制过程中发生的特定事件直接联系起来。所获得的结果将显着增加我们的知识的复制在真核基因调控的作用，并有可能用于病毒感染和癌症的治疗方法的发展。