GENETIC AND BIOCHEMICAL STUDY OF SILENCING

本提案的目的是确定 在真核生物中建立和繁殖基因表达。数据从 几个实验室已经鉴定出一些蛋白质， 沉默所需的蛋白质，如Orc，Rap 1和Sir蛋白质。这些 研究还表明， 染色质和沉默，从而为详细的 研究Sir蛋白沉默的机制， 染色质的背景。 我们的研究将集中在（1）沉默域的机制， 建立并忠实地通过世代使用遗传 和生物化学手段;（2）沉默结构域的结构及其 体外重构。 Sir 1 p在交配型基因座上沉默的建立将是 通过分析Sir 1 p中不能促进 稳定的沉默域。这些突变体随后将被用于 分离出可以绕过Sir 1 p缺陷的其他突变， 沉默这种抑制子与Sir 1 p突变体的关系将 生物化学决定。 沉默结构域的染色质结构和各种基因的作用 还将研究蛋白质在维持沉默结构域中的作用。 沉默的染色质将被纯化，其蛋白质组成和 染色质结构研究。一旦染色质结构 将在体外表征沉默的染色质的重建， 执行，最初在模板上， 沉默将测试组装的染色质的能力， 模拟沉默状态，这个系统将允许一个生化 剖析沉默的过程，并产生有价值的新见解 基因被稳定抑制的机制。 这项研究具有广泛的医学相关性， 基因在基因组中的位置如何影响基因的功能 与基因替代疗法以及染色体如何 重排导致疾病状态，以及如何X失活 基因的父母印记形成了基因的可遗传状态 表情

The objective of this proposal is to determine how distinct domains of gene expression are established and propagated in eukaryotes. Data from several laboratories have identified some of the proteins that are required for silencing such as Orc, Rap1 and the Sir proteins. These studies have also revealed an intimate association between components of chromatin and silencing thus setting the stage for a detailed investigation of the mechanism of silencing by the Sir proteins in the context of chromatin. Our studies will focus on (1) the mechanisms by which silent domains are established and faithfully propagated through generations using genetical and biochemical means and (2) the structure of the silent domain and its reconstitution in vitro. The establishment of silencing by Sir1p at the mating type loci will be examined by analyzing mutations in Sir1p that are unable to promote stable silent domains. These mutants will be subsequently employed to isolate additional mutations that can bypass the Sir1p defect in silencing. The relationship of such suppressors with Sir1p mutants will be determined biochemically. The chromatin structure of the silenced domain and the role of various proteins in maintaining the silenced domain will also be investigated. Silenced chromatin will be purified and its protein composition and chromatin structure studied. Once the chromatin structure has been characterized in vitro reconstitution of silenced chromatin will be performed, initially on templates that reduce the requirements for silencing. The assembled chromatin will be tested for its ability to mimic the silent state and this system will allow a biochemical dissection of the process of silencing and yield valuable new insights into the mechanisms by which genes are stably repressed. This research has broad medical relevance with respect to understanding how the function of genes is influenced by their position in the genome with implications in gene replacement therapy and how chromosome rearrangements lead to the diseased state, as well as how X inactivation and the parental imprinting of genes forms heritable states of gene expression.