GENETIC AND BIOCHEMICAL STUDY OF SILENCING

该提案的目标是确定不同的域 基因的表达是在真核生物中建立和繁殖的。数据来自 几个实验室已经确定了一些蛋白质是 沉默所必需的，如ORC、RAP1和SIR蛋白。这些 研究还揭示了两种成分之间的密切联系 染色质和沉默从而为详细的 细胞SIR蛋白沉默机制的研究 染色质的背景。 我们的研究将集中在(1)沉默区域被 建立并忠实地世代传播使用遗传 和生化手段，以及(2)沉默结构域的结构及其 体外重建。 Sir1p在交配型基因座上沉默的建立将是 通过分析无法促进Sir1p的突变进行检查 稳定的静默域。这些突变体随后将被用来 分离可绕过Sir1p缺陷的额外突变 沉默。这种抑制子与Sir1p突变体的关系 用生物化学的方法确定。 染色质结构的沉默区域和各种不同的作用 维持沉默结构域的蛋白质也将被研究。 沉默的染色质将被提纯，其蛋白质组成和 研究了染色质的结构。一旦染色质结构 沉默染色质的体外重组特性将是 执行，最初使用的模板减少了对 沉默。将对组装的染色质进行测试，以确定其是否具有 模拟静默状态，这个系统将允许生化 对沉默过程的剖析并产生有价值的新见解 基因稳定被抑制的机制。 这项研究在理解方面具有广泛的医学意义 基因的功能如何受到它们在基因组中的位置的影响 在基因替代疗法中的应用以及染色体 重排导致疾病状态，以及X如何失活 而父母对基因的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.