PURIFICATION OF THE 2-MICRON PLASMID PARTITIONING COMPLEX

2 微米质粒分配复合物的纯化

• 批准号: 8365896
• 负责人: Makkuni JAYARAM
• 金额: $ 1.28万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365896
• 关键词: Biochemical; Biological Assay; Biology; CDKN1B gene; Cell Cycle; Centromere; Chromatin; Chromatin Remodeling Factor; Chromosomes; Cis-Acting Sequence; Code; Competence; Complex; Data; Elements; Funding; Fungal Genome; Genetic; Grant; Histone H3; Integration Host Factors; Motor; National Center for Research Resources; Nuclear; Plasmids; Principal Investigator; Proteins; Research; Research Infrastructure; Resources; Saccharomyces cerevisiae; Saccharomycetales; Source; United States National Institutes of Health; Variant; chromatin immunoprecipitation; cohesin; cost; functional status; segregation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The multi-copy 2 micron Saccharomyces cerevisiae plasmid, a selfish extra-chromosomal element, displays nearly chromosome-like segregation efficiency. The plasmid coded proteins Rep1 and Rep2 orchestrate the recruitment of host factors to the cis-acting locus STB for assembly of a high-order partitioning complex. Despite obvious distinctions, STB and centromeres share similarities in that the RSC chromatin remodeling complex, the nuclear motor Kip1, the histone H3 variant Cse4, and the cohesin complex contribute to their functional competence. Mutational analyses and chromatin immunoprecipitation assays reveal functional and temporal hierarchies in the assembly and disassembly of the plasmid partitioning complex. Consistent with genetic and biochemical data, TAP-analyses using Rep1 and Rep2 as baits have identified several host factors as potential constituents of the plasmid partitioning complex. In particular, they include components of the RSC chromatin remodeling complex. The functional status of the STB chromatin is established during each cell cycle during the de novo assembly of the partitioning complex. The normal segregation of the 2 micron plasmid is compromised by the absence of Rsc2 as well as thermal inactivation of Rsc8 and Rsc58 functions. The cumulative results suggest that the point centromere of budding yeast may have originated from the partitioning locus of an ancestral plasmid from which the extant 2 micron circle has descended.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 多拷贝2微米糖酿氏菌质粒（一种自私的外染色体元素）显示几乎类似染色体的隔离效率。质粒编码的蛋白REP1和REP2协调了宿主因子募集到顺式作用基因座STB，以组装高阶分配复合物。尽管有明显的区别，但STB和Centromeres具有相似之处，因为RSC染色质重塑络合物，核运动KIP1，组蛋白H3变体CSE4和粘着蛋白复合蛋白复合物有助于其功能能力。突变分析和染色质免疫沉淀测定法揭示了质粒分配复合物的组装和分解中的功能和时间层次结构。与遗传和生化数据一致，使用REP1和REP2作为诱饵的TAP-ANALYSES已将几种宿主因子确定为质粒分配复合物的潜在成分。特别是，它们包括RSC染色质重塑复合物的成分。在分配复合物的从头组装过程中，在每个细胞周期中建立了STB染色质的功能状态。由于缺乏RSC2以及RSC8和RSC58功能的热灭活而损害了2微米质粒的正常分离。累积结果表明，萌芽酵母的点丝粒可能起源于祖先质粒的分配基因座，现存的2微米圆从中下降。