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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们项目的最终目标是开发生化和质谱学方法，以揭示蛋白质与酿酒酵母染色体上任何选定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DNA区域结合的蛋白质复合体，在这种方法中，我们从酵母细胞裂解物中亲和纯化感兴趣的DNA序列。我们选择了从酿酒酵母中富集2M质粒作为一个模式案例。两个微米质粒为内源性6.3kbp的环状微染色体，存在于大多数常见的酿酒酵母菌株中，每个细胞的拷贝数约为60个。该质粒含有一个复制起点，细胞复制机制在每个细胞周期内复制一次，且仅复制一次。因此，它是研究DNA复制(启动)的合适模型。 我们正在通过筛选参与DNA复制起始的蛋白质，通过浓缩2？M质粒来优化特定DNA序列的亲和纯化程序。为此目的，通过插入Lac操作符序列重复序列阵列来修饰2？m质粒。将含有Lac操纵子重复序列和2M复制起始点的质粒(PSV1)转化为携带绿色荧光蛋白(GFP)-lac阻遏蛋白融合蛋白表达盒的酿酒酵母菌株。荧光显微镜实验显示细胞内有清晰的荧光点，表明GFP-lac蛋白是特异定位的，可能与pSV1质粒上的lac操纵子重复。 为了通过亲和纯化从细胞裂解物中拉出质粒环，使用了高度特异的磁性动力蛋白抗体。经过大量的珠子洗涤，洗脱质粒，然后对蛋白质进行一维十二烷基硫酸钠-PAGE分析和质谱仪鉴定。典型的蛋白质图谱显示GFP-lac阻遏蛋白，组蛋白在低分子量范围内，以及几条带在高分子量范围内。目前正在对这些高分子量蛋白质进行分析。其中，一个已被鉴定为可能的转录因子蛋白，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乳胶操纵子重复序列标记，被特定的限制性内切酶切割，并按照所述进行亲和纯化。

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.