CHROMOSOME STRUCTURE AND GENE EXPRESSION

描述（改编自研究者摘要）： 这项研究是为了了解真核生物染色体的结构域是如何 组装成特定的染色质结构，调节转录。 该建议的重点是S的HMR位点。cerevisiae，一个 通过一种被称为沉默的机制保持转录抑制。 沉默需要建立并随后维持 抑制性染色质结构的限制域。 在HMR基因座a 建立沉默的关键步骤需要Sir 1蛋白 与一种被称为沉默者的DNA元素相关联。 消音器里有 几种普遍存在的蛋白质的结合位点，包括Rap 1 p、Abf 1 p和 ORC，origin recognition complex。 有人提出这样的假设， sir 1 p通过与沉默子结合来识别并与沉默子结合。 沉默子相关蛋白如ORC。 因为ORC被认为是 存在于所有复制起点，而Sir 1致力于沉默， 两个位点，HMR和HML，Sir 1被假定具有非常特异的相互作用 关于ORC 本申请中提出的实验被设计为定义 Sir 1 p识别沉默者的机制。 基因筛选 鉴定了sir 1 srd突变体，其在HMR沉默物识别中存在缺陷 而不是沉默本身。 这些突变体形成了遗传的基础， 分子和生物化学研究，解决有关的几个问题， Sirp在沉默中的作用。 在第一个具体目标中， 将研究具有ORC的Sir 1 p。 将在三个ORC中进行突变 基因，并测试它们抑制非交配缺陷的能力， sir 1 srd等位基因 Sir 1 p和ORC之间的物理相互作用将是 通过使用表位标记的Sir 1 p的免疫共沉淀分析来检查。 这项分析将扩展到Sir 1 psrd蛋白，以确定是否 突变蛋白质显示有缺陷的ORC相互作用。 的可能性 Sir 1 p与其他沉默子结合蛋白如Rap 1 p和Abf 1 p相互作用 也将在免疫共沉淀试验中进行检查， 将探测Sir 1 p免疫沉淀物中的其它蛋白质。 在第二个具体目标中，Sir 1 p的功能结构域是 将识别所需的消音器识别。 sir 1 srd屏幕 将在SIR 1空背景中重复，以确定 Sir 1 p的N-末端以及C-末端含有一个 沉默识别域。 Sir 1 p作为一种 低聚物在消音器也将测试使用遗传和 免疫学方法。 突变将在Sir 1 p中产生 C-末端，其含有推定的沉默物识别结构域，和 测试它们补充sir 1 srd等位基因的能力。 相互作用 Sir 1 C-末端与突变Sir 1 p蛋白也将通过 与表位标记的蛋白质的免疫共沉淀。 在第三个具体目标中，新的蛋白质，帮助Sir 1 p识别 将使用两种不同的基因筛选来鉴定沉默者。 现有 sir 1 srd等位基因的剂量抑制因子将被更充分地表征， 他们在沉默中的角色。 所需基因的功能丧失突变 sir 1 p对沉默者的识别将在alpha的屏幕中被识别 有特殊遗传背景的母亲 在该筛选中鉴定的基因 将被分离并表征它们对沉默的影响。 ORC在DNA复制和沉默中的功能将在 第四个具体目标。 侧翼HMR-E序列在ORC中的作用 在复制和沉默中的功能将通过缺失来确定 分析. HMR-E用作复制起点的效率 体内将通过2D叉移位分析进行测试，并与两个 ORC如何影响复制和沉默的模型。

DESCRIPTION (adapted from investigator's abstract): The long term goal of this research is to understand how domains of eukaryotic chromosomes are assembled into specific chromatin structures that regulate transcription. The proposal is focused on the HMR locus of S. cerevisiae, a locus that is kept transcriptionally repressed by a mechanism known as silencing. Silencing requires the establishment and subsequent maintenance of restricted domains of repressive chromatin structure. At the HMR locus a key step in the establishment of silencing requires that the Sir1 protein associate with a DNA element known as a silencer. The silencer contains binding sites for several ubiquitous proteins, including Rap1p, Abf1p, and ORC, the origin recognition complex. The hypothesis has been presented that Sir1p recognizes and associates with the silencer by binding to silencer-associated proteins such as ORC. Because ORC is presumed to be present at all replication origins, while Sir1 is dedicated to silencing at two loci, HMR and HML, Sir1 is postulated to have very specific interactions with ORC. The experiments proposed in this application are designed to define the mechanisms by which Sir1p recognizes a silencer. A genetic screen has identified sir1srd mutants that are defective in HMR silencer recognition but not in silencing per se. These mutants form the basis of genetic, molecular, and biochemical studies that address several issues relating to Sirp's function in silencing. In the first specific aim, the interaction of Sir1p with ORC will be investigated. Mutations will be made in three ORC genes and tested for their ability to suppress the nonmating defect of sir1srd alleles. Physical interaction between Sir1p and ORC will be examined by co-immunoprecipitation analysis using epitope tagged Sir1p. This analysis will be extended to the Sir1psrd proteins to determine whether the mutant proteins show defective ORC interactions. The possibility that Sir1p interacts with other silencer binding proteins such as Rap1p and Abf1p will also be examined in co-immunoprecipitation assays, and the presence of additional proteins in Sir1p immunoprecipitates will be probed. In the second specific aim, the functional domains of Sir1p that are required for silencer recognition will be identified. The sir1srd screen will be repeated in a sir1 null background to determine whether the N-terminus as well as the C-terminus of Sir1p contains a silencer-recognition domain. The possibility that Sir1p functions as an oligomer at silencers will also be tested using both genetic and immunological approaches. Mutations will be generated in the Sir1p C-terminus, which contains a putative silencer recognition domain, and tested for their ability to complement sir1srd alleles. Interaction of the Sir1 C-terminus with mutant Sir1p proteins will also be tested by co-immunoprecipitation with epitope tagged proteins. In the third specific aim, novel proteins that assist Sir1p to recognize a silencer will be identified using two different genetic screens. Existing dosage suppressors of sir1srd alleles will be characterized more fully for their roles in silencing. Loss-of-function mutations in genes required for Sir1p's recognition of a silencer will be identified in a screen for alpha maters in a specialized genetic background. Genes identified in this screen will be isolated and characterized for their effects on silencing. The functions of ORC in DNA replication and silencing will be examined in the fourth specific aim. The role of flanking HMR-E sequences in ORC function in replication and silencing will be determined through deletion analysis. The efficiency with which HMR-E is used as a replication origin in vivo will be tested by 2D fork migration analysis and related to two models for how ORC might affect both replication and silencing.