GENETIC AND BIOCHEMICAL STUDY OF SILENCING

这个建议的目的是确定如何在真核生物中建立和繁殖不同的基因表达域。来自几个实验室的数据已经确定了一些沉默所需的蛋白质，如Orc，Rap1和Sir蛋白质。这些研究还揭示了染色质组分与沉默之间的密切联系，从而为详细研究染色质背景下Sir蛋白的沉默机制奠定了基础。我们的研究将集中在（1）沉默结构域的建立和忠实地通过世代使用遗传和生化手段繁殖的机制和（2）沉默结构域的结构及其体外分析。将通过分析Sir1p中不能建立和继承稳定沉默结构域的突变来检查Sir1p在交配型基因座处沉默的建立。这些突变体随后将用于分离可以抑制Sir1p沉默缺陷的其他突变。这种抑制因子与Sir1p突变体的关系将通过生物化学方法确定。将研究沉默的染色质结构域被限制在基因组特定区域的机制。将寻找介导边界功能的特定DNA序列元件。一旦这些元件被鉴定，结合这些元件以防止异染色质扩散到相邻的常染色质中的蛋白质将被分离和表征。遗传学研究表明，不同的组合的爵士蛋白质转录抑制多个基因座。虽然Sir2p、Sir3p和Sir4p介导HML、HMR和端粒处的沉默，但只有Sir2p参与rDNA基因座处的沉默。因此，确定细胞中是否存在多种Sir蛋白复合物以及这些复合物的组成可能是什么是有意义的。研究的目的是从酵母细胞中纯化Sir蛋白复合物，然后对各个组分进行鉴定和表征。还将分析复合物的各种酶活性，并将用于进一步的体外研究。这项研究对于了解基因在基因组中的位置如何影响基因的功能具有广泛的相关性。这项研究在基因替代治疗，染色体重排如何导致疾病，X失活和基因的父母印记如何形成基因表达的遗传状态以及理解肿瘤发生和衰老方面具有意义。- 酵母，沉默，异染色质，爵士蛋白质，遗传学，生物化学，边界元素

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 analysis 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 establish and inherit stable silent domains. These mutants will be subsequently employed to isolate additional mutations that can suppress the Sir1p defect in silencing. The relationship of such suppressors with Sir1p mutants will be determined biochemically. The mechanism by which the silenced chromatin domain is restricted to a specific region of the genome will be investigated. Specific DNA sequence elements that mediate boundary function will be sought. Once such elements are identified, proteins that bind these elements to prevent the spread of heterochromatin into neighboring euchromatin will then be isolated and characterized. Genetic studies have revealed that distinct combinations of the Sir proteins transcriptionally repress multiple loci. While Sir2p, Sir3p and Sir4p mediate silencing at HML, HMR and at the telomeres, only Sir2p is involved in silencing at the rDNA locus. It is therefore of interest to determine whether there are multiple Sir protein complexes in the cell and what the composition of these complexes might be. Studies will aim at purifying the Sir protein complexes from yeast cells followed by the identification and characterization of the individual components. The complexes will also be analyzed for various enzyme activities and will be uilized for further in vitro studies. This research has broad relevance with respect to understanding how the function of genes is influenced by their position in the genome. This research has implications in gene replacement therapy, how chromosome rearrangements can lead to diseases, how X-inactivation and parental imprinting of genes form heritable states of gene expression as well as in understanding oncogenesis and aging. - Yeast, Silencing, Heterochromatin, Sir Proteins, Genetics, Biochemistry, Boundary Elements