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: 8169119
• 负责人: Brian T Chait
• 金额: $ 1.16万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169119
• 关键词: Affinity; Affinity Chromatography; Binding; Biochemical; Cell Cycle; Cells; Chimeric Proteins; Chromosomes; Cleaved cell; Computer Retrieval of Information on Scientific Projects Database; DNA; DNA Sequence; DNA biosynthesis; Fluorescence Microscopy; Funding; Fungal Genome; Goals; Grant; Green Fluorescent Proteins; Histones; Institution; Lac Operon; Lac Repressors; Magnetism; Mass Spectrum Analysis; Methods; Modeling; Molecular Weight; Pattern; Plasmids; Play; Procedures; Proteins; Replication Initiation; Replication Origin; Repressor Proteins; Research; Research Personnel; Resources; Role; Saccharomyces cerevisiae; Screening procedure; Source; Study models; United States National Institutes of Health; Yeasts; antibody conjugate; interest; 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. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 我们项目的最终目标是开发生化和质谱方法，这些方法可以揭示蛋白质与酿酒酵母染色体的任何选择区域的稳定相互作用。我们的策略是开发一种新的方法来研究与给定的DNA区域结合的蛋白质复合物，在这种蛋白质复合物中，我们将来自酵母细胞裂解物的DNA感兴趣序列相关联。我们选择以酿酒酵母的2质质粒的富集为模型情况。两个微米的质粒是内源性圆形6.3 kbp小浓度小体，它们以每个细胞约为60的拷贝数中最常见的酿酒酵母菌株中存在。质粒包含复制的一个来源，并且通过细胞复制机制复制一次，并且每个细胞周期仅复制一次。因此，它是研究（启动）DNA复制的合适模型。 我们正在通过筛选参与DNA复制计划的蛋白质来优化特定DNA序列的亲和力 - 纯化程序。为此，通过插入一系列LAC操作员序列重复序列来修改2？m质粒。用含有LAC操作员重复序列的质粒和2？M质粒的复制起源（PSV1质粒）转化了含有绿色荧光蛋白（GFP）表达盒的酿酒酵母菌株。荧光显微镜实验显示了细胞中清晰的荧光点，并表明GFP-LAC蛋白是特异性的，可能是对PSV1质粒上的LAC操作员重复序列。 为了通过亲和力 - 纯化从细胞裂解物中拉出质粒回路，使用了与磁性驱动器结合的高度特异性？-GFP抗体。大量洗涤珠后，洗脱质粒，然后通过1D SDS-PAGE对蛋白质进行分析，并通过质谱法鉴定。典型的蛋白质模式显示GFP-LAC复制蛋白，低分子量范围内的组蛋白以及高分子量范围内的几个条带。目前对这些高分子重量蛋白进行分析。其中，已经被确定为推定的转录因子蛋白，它可能在质粒DNA复制中起作用。 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的lac Operaon重复序列标记，并按照特定的限制酶裂解，并如所述的亲和力纯化。