INTERACTIONS OF PROTEINS WITH ANY CHOSEN REGION OF THE S CEREVISIAE CHROMOSOME

蛋白质与酿酒酵母染色体任何选定区域的相互作用

• 批准号: 8361502
• 负责人: Brian T Chait
• 金额: $ 1.3万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361502
• 关键词: Affinity; Affinity Chromatography; Binding; Biochemical; Cell Cycle; Cells; Chimeric Proteins; Chromosomes; Cleaved cell; DNA; DNA Sequence; DNA biosynthesis; Fluorescence Microscopy; Funding; Fungal Genome; Goals; Grant; Green Fluorescent Proteins; Histones; Lac Operon; Lac Repressors; Magnetism; Mass Spectrum Analysis; Methods; Modeling; Molecular Weight; National Center for Research Resources; Pattern; Plasmids; Play; Principal Investigator; Procedures; Proteins; Replication Initiation; Replication Origin; Repressor Proteins; Research; Research Infrastructure; Resources; Role; Saccharomyces cerevisiae; Screening procedure; Source; Study models; United States National Institutes of Health; Yeasts; antibody conjugate; cost; interest; macromolecule; novel; plasmid DNA; protein complex; research study; restriction enzyme; transcription factor

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 ultimate goal of our project is developing biochemical and mass spectrometric methods that can reveal stable interactions of proteins with any chosen region of the S. cerevisiae chromosome. Our strategy is to develop a novel method to investigate protein complexes that bind to a given region of DNA, in which we affinity-purify the DNA sequence of interest from a yeast cell lysate. We have chosen to use the enrichment of 2 ?m plasmids from S. cerevisiae as a model case. Two micron plasmids are endogenous circular 6.3 kbp minichromosomes, which are present in most common strains of S. cerevisiae at a copy number of approximately 60 per cell. The plasmid contains one origin of replication and is replicated once, and only once, per cell cycle by the cellular replication machinery. Therefore, it serves as an appropriate model for studying (initiation of) DNA replication. We are optimizing affinity-purification procedures of a specific DNA sequence by enrichment of 2 ?m plasmids by screening for proteins that are involved in DNA replication initiation. For this purpose, 2 ?m plasmids have been modified by insertion of an array of lac operator sequence repeats. A S. cerevisiae strain carrying an expression cassette for green fluorescent protein (GFP) ¿ lac repressor fusion protein was transformed with a plasmid containing the lac operator repeat sequence and the 2 ?m plasmid origin of replication (pSV1 plasmid). Fluorescence microscopy experiments showed clear fluorescent dots in the cells and suggest that the GFP-lac protein is specifically localized, probably to the lac operator repeats on the pSV1 plasmids. In order to pull-out plasmid circles from cell lysates by affinity-purification, highly specific ?-GFP antibodies conjugated to magnetic Dynabeads are used. After extensive washing of the beads, the plasmids are eluted and the proteins are subsequently analyzed by 1D SDS-PAGE and identified by mass spectrometry. Typical protein patterns show GFP-lac repressor protein, histones in the low molecular weight range and several bands in the high molecular weight range. Analysis of these high-molecular weight proteins is currently performed. Of those, one has been identified as being a putative transcription factor protein, which may play a role in plasmid DNA replication. Ultimately, in principle, any specific sequence in the entire yeast genome can be tagged with an array of lac operon repeats, cleaved off by specific restriction enzymes and affinity-purified as described.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供 该子项目的主要支持。 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源的子项目可能列出的总成本。 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 我们项目的最终目标是开发生物化学和质谱方法，以揭示蛋白质与酿酒酵母染色体任何选定区域的稳定相互作用。我们的策略是开发一种新方法来研究与给定区域结合的蛋白质复合物。 DNA，其中我们从酵母细胞裂解物中亲和纯化感兴趣的 DNA 序列，我们选择使用来自酿酒酵母的 2μm 质粒作为模型案例。是内源性环状 6.3 kbp 微型染色体，存在于最常见的酿酒酵母菌株中，每个细胞的拷贝数约为 60 个。该质粒包含一个复制起点，并且每个细胞周期仅复制一次。因此，它是研究 DNA 复制（起始）的合适模型。 我们正在通过筛选参与 DNA 复制起始的蛋白质来富集 2 µm 质粒，从而优化特定 DNA 序列的亲和纯化程序。为此，通过插入 lac 操纵子阵列对 2 µm 质粒进行了修饰。带有绿色荧光蛋白 (GFP) 表达盒的酿酒酵母菌株 ¿ lac 阻遏物融合蛋白用含有 lac 操纵子重复序列和 2 μm 质粒复制起点（pSV1 质粒）的质粒转化。荧光显微镜实验显示细胞中有清晰的荧光点，表明 GFP-lac 蛋白是特异性定位的。 ，可能是 pSV1 质粒上的 lac 操作符重复序列。 为了通过亲和纯化从细胞裂解物中提取质粒环，使用与磁性 Dynabeads 缀合的高度特异性的 α-GFP 抗体。在对磁珠进行大量洗涤后，洗脱质粒，随后通过 1D SDS- 分析蛋白质。 PAGE 和质谱鉴定典型的蛋白质模式显示 GFP-lac 阻遏蛋白、低分子量范围内的组蛋白和高分子量范围内的几个条带。目前正在进行高分子量蛋白质的研究，其中一种已被鉴定为假定的转录因子蛋白质，它可能在质粒 DNA 复制中发挥作用。 最终，原则上，整个酵母基因组中的任何特定序列都可以用一系列 lac 操纵子重复序列进行标记，通过特定的限制性酶切割并按所述进行亲和纯化。