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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们项目的最终目标是开发生物化学和质谱方法，可以揭示蛋白质与S.酿酒酵母染色体我们的策略是开发一种新的方法来研究结合到给定区域的DNA的蛋白质复合物，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2的浓缩？m质粒。作为一个典型案例。两个微米质粒是内源性环状6.3kbp微型染色体，存在于大多数常见的S.酿酒酵母以每个细胞约60个拷贝数存在。质粒含有一个复制起点，并且通过细胞复制机制在每个细胞周期复制一次且仅复制一次。因此，它是研究DNA复制（起始）的合适模型。 我们正在优化亲和纯化程序的特定DNA序列的富集2？通过筛选参与DNA复制起始的蛋白质来构建质粒。为此，2？已经通过插入一系列lac操纵子序列重复序列修饰了M质粒。色葡萄将携带绿色荧光蛋白（GFP）-lac阻遏物融合蛋白表达盒的酿酒酵母菌株用含有lac操纵子重复序列和2？m质粒复制起点（pSV 1质粒）。荧光显微镜实验显示细胞中有清晰的荧光点，表明GFP-lac蛋白特异性定位于pSV 1质粒上的lac操纵子重复序列。 为了通过亲和纯化从细胞裂解物中拉出质粒环，使用与磁性Dynabeads缀合的GFP抗体。在充分洗涤珠子后，洗脱质粒，随后通过1D SDS-PAGE分析蛋白质，并通过质谱法鉴定。典型的蛋白质模式显示GFP-lac阻遏蛋白，低分子量范围的组蛋白和高分子量范围的几个条带。目前正在对这些高分子量蛋白进行分析。其中，一种已被鉴定为推定的转录因子蛋白，其可能在质粒DNA复制中起作用。 最后，原则上，整个酵母基因组中的任何特定序列都可以用乳糖操纵子重复序列阵列标记，通过特定的限制酶切割并如所述进行亲和纯化。

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.