Isolation and characterization of plant insulators

绝缘体是能够阻止不适当或不想要的增强子-启动子通信(增强子阻断剂活性)和/或阻止抑制染色质扩散到活性染色质(屏障活性)的DNA元件。它们的作用是由识别它们的蛋白质(S)介导的，导致许多蛋白质的招募，并最终组装成“绝缘体”，即拟议的“功能单位”。因此，绝缘体代表了转录控制的额外水平，他们的研究提供了一个了解基本的、普遍的基因调控机制的机会。 已经确定了许多物种的绝缘体，包括真菌、酵母、果蝇、鸡肉和人类，但不包括植物。我们的工作表明，在拟南芥中，两个绝缘体UASrpg和BEAD-1C可以作为绝缘体发挥作用-首次发表了绝缘体在植物中的活性演示。鉴于绝缘子在植物基因表达中的潜在重要性，以及在植物生物技术中控制组织特异性转基因表达的可能关键作用，我们开始了一项分离和研究植物绝缘子的研究计划。 我们的方法是避免使用候选序列，并通过采用我们的转基因系统来解决这个问题，以直接选择具有绝缘活性的DNA元件。这样分离的序列(S)可以克隆到其他载体中，以消除在各种启动子/增强子组合、定向和一系列不同物种中不起作用的绝缘子。有了这样的序列，我们就可以设计出分离识别它们的蛋白质的方法，并测试为动物系统提出的绝缘体活性模型。我们的长期目标是了解这些相互作用如何调节植物中的基因表达及其进化。此外，功能和通用的植物绝缘体将对新一代植物转化载体的设计产生影响。

Insulators are DNA elements that are able to block inappropriate or unwanted enhancer-promoter communication (enhancer blocker activity) and/or prevent the spread of repressive chromatin into active chromatin (barrier activity). Their action is mediated by protein(s) that recognize them leading to the recruitment of many proteins and ultimately the assembly "insulator bodies" the proposed "functional unit". Thus insulators represent an additional level of transcriptional control and their study represents an opportunity to understand a basic, universal mechanism of gene regulation. Insulators from number of species have been identified including fungi, yeast, fruit fly, chicken and human but not plant. Our work indicated that in Arabidopsis two insulators, UASrpg and BEAD-1C can function as insulators-first published demonstration of insulator activity in a plant. Given the potential importance of insulators in plant gene expression and possible key roles in plant biotechnology for the control of tissue-specific transgene expression, we have embarked on a research programme to isolate and study plant insulators. Our approach is to avoid the use of candidate sequences and to tackle the problem by adapting our transgenic system for the direct selection of DNA elements with insulating activity. Sequence(s) thus isolated can be cloned into other vectors to eliminate insulators that are not functional in a variety of promoter/enhancer combinations, orientation and a range of different species. With such sequences in hand we can devise means to isolate the proteins that recognize them and test models of insulator activity proposed for animal systems. Our long term goal is to understand how these interactions regulate gene expression in plants and their evolution. In addition a function and universal plant insulator would have an impact on the design of a new generation of plant transformation vectors.