GENETIC AND BIOCHEMICAL STUDY OF SILENCING

这项建议的目的是确定基因表达的不同区域是如何在真核生物中建立和传播的。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的成分与沉默之间的密切联系，从而为在染色质的背景下详细研究SIR蛋白的沉默机制奠定了基础。我们的研究将集中在(1)利用遗传和生化手段建立沉默结构域并在世代中忠实地传播的机制，以及(2)沉默结构域的结构及其体外分析。将通过分析Sir1p中无法建立和遗传稳定沉默区域的Sir1p突变来检验Sir1p在交配型基因座上建立沉默的机制。这些突变体随后将被用来分离能够抑制沉默中Sir1p缺陷的额外突变。这些抑制子与Sir1p突变体的关系将通过生物化学来确定。沉默的染色质结构域被限制在基因组的特定区域的机制将被研究。将寻找调节边界功能的特定DNA序列元件。一旦确定了这些元件，就会分离出与这些元件结合以防止异染色质扩散到邻近常染色质的蛋白质，并对其进行鉴定。遗传学研究表明，SIR蛋白的不同组合在转录上抑制了多个基因座。当Sir2p、Sir3p和Sir4p介导HML、HMR和端粒的沉默时，只有Sir2p参与rDNA位点的沉默。因此，确定细胞中是否存在多个SIR蛋白复合体以及这些复合体的组成是有意义的。研究的目的是从酵母细胞中提纯SIR蛋白复合体，然后鉴定和表征单个组分。这些络合物还将被分析各种酶的活性，并将用于进一步的体外研究。这项研究对于理解基因的功能如何受到它们在基因组中的位置的影响具有广泛的相关性。这项研究对基因替代治疗、染色体重排如何导致疾病、X失活和父母基因印记如何形成基因表达的可遗传状态以及理解肿瘤发生和衰老具有重要意义。-酵母，沉默，异染色质，SIR蛋白，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