MODULATION OF ETS FUNCTION BY PROTEIN-PROTEIN INTERACTIONS

人类基因组包含几个ETS相关基因(ETS1、ETS2、ERG、 ERGB、ELK1、ELK2、SAP1、ELF、SPI1、SPI2)。其他与ETS相关的基因有 在哺乳动物(GABPalpha，PU.1/Spi-1，Fli-1，PEA3)和 无脊椎动物(D-ELG，E74)，表明额外的人类ETS相关 基因可能还存在。除了羧基末端的DNA结合 结构域是ETS家族高度保守的螺旋-环-螺旋基序 成员，提示它可能在ETS的功能中发挥重要作用 蛋白质。这个基序被认为与蛋白质有关- 蛋白质的相互作用。要了解其功能和机制 负责ETS相关基因之间的功能差异， 其产物能够与ETS蛋白相互作用的基因将是 已确认身份。为了实现这一目标，包含已定义段的构造 人类ETS1和ETS2基因的开放阅读框(ORF)中 被放入原核载体中，允许过度表达和 快速纯化每个构建体所产生的融合蛋白。 快速纯化是通过高亲和力的相互作用实现的 载体衍生组氨酸残基和镍螯合树脂。ETS/向量 构造保留了这种亲和力，提供了一种快速获得ETS的方法 地缘柱。来自相互作用的标记细胞提取物的蛋白质 利用这些亲和性柱可以被识别。这些向量还包含 确定的蛋白质切割位点，允许分离未融合的 蛋白。这些蛋白质将被标记并用作探针 确定与之相关的基因产物(和适当的基因) 能够互动。这一方法将应用于其他基因和 将使我们能够开始剖析ETS基因在人类进化中的作用(S) 基因调控和细胞信号转导的复杂网络。

The human genome contains several ets-related genes (ETS1, ETS2, ERG, ERGB, ELK1, ELK2, SAP1, ELF, SPI1, SPI2). Other ets-related genes have been found in mammals (GABPalpha, PU.1/Spi-1, Fli-1, PEA3) and invertebrates (D-elg, E74), suggesting that additional human ets-related genes may yet exist. In addition to the carboxy terminal DNA-binding domain, a helix-loop-helix motif is highly conserved among ETS family members, suggesting it may play an important role in the function of ETS proteins. This motif has been postulated to be involved in protein- protein interactions. To understand the functions and mechanisms responsible for the functional differences between the ets-related genes, genes whose products are able to interact with ETS proteins will be identified. To achieve this goal, constructs containing defined segments of the open reading frames (ORF) from the human ETS1 and ETS2 genes have been placed into prokaryotic vectors that allow for overexpression and rapid purification of the fusion protein produced by each construct. Rapid purification is accomplished by high-affinity interaction between vector-derived histidine residues and nickel-chelated resins. Ets/vector constructs retain this affinity, affording a rapid means of obtaining Ets affinity columns. Proteins from labelled cell extracts which interact with these affinity columns can be identified. These vectors also contain defined protein cleavage sites that allow for isolation of the unfused protein. These proteins will be labelled and utilized as probes to identify gene products (and the appropriate genes) with which they are able to interact. This methodology will be applied to other genes and will allow us to begin to dissect the role(s) of the ETS genes in the complex network of gene regulation and cellular signal transduction.