GLUCOCORTICOID RECEPTOR STRUCTURE SUBUNIT ARCHITECTURE

糖皮质激素受体结构亚基结构

• 批准号: 3235441
• 负责人: ROBERT W HARRISON
• 金额: $ 17.39万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3235441
• 关键词: DNA binding protein; DNA footprinting; affinity chromatography; chemical binding; complementary DNA; deoxyribonuclease I; dexamethasone; genetic manipulation; genetic transcription; glucocorticoids; hormone binding protein; hormone receptor; hormone regulation /control mechanism; molecular cloning; monoclonal antibody; protein sequence; tissue /cell culture; transfection

Glucocorticoid-regulated genes contain one or more specific elements that bind glucocorticoid receptor (GR) and, when associated with other genes make them glucocorticoid responsive. This particular element has teen named the glucocorticoid response element or GRE. This proposal asserts that are genes in a issue or cell that are regulated by GR interaction with a GRE can be identified and characterized using a novel approach which does not require foreknowledge of the regulated gene's protein products. The basis for this approach is the discovery that a 17,000 MW tryptic fragment of the receptor (17-K GR) binds GRE-containing DNA with higher affinity and specificity than does the intact GR. A spharose-protein A-monoclonal antibody matrix will be used to immobilize the 17-K GR thus resulting in an affinity column with specificity for genomic domains containing a GRE. This column will be used to extract GRE-containing fragments of genomic DNA (GRE- DNA). The proposed experiments will utilize AtT-20/D1 cells stably transfected with an LTR hybrid to yield an up-regulated gene to complement the down-regulated gene, POMC. The GRE-DNA fragments will be cloned and functional genes selected by determining which clones are complementary to RNA whose bundance is changed by glucocorticoid treatment. The corresponding structural genes will also be isolated and used to determine which gene is first affected by glucocorticoids. Further characterization of the genomic and structural DNAs will be done starting with the gene whose transcription is modified earliest by glucocorticoid treatment. The gene will be subcloned and sequenced to determine the elements common to other regulated genes. Functional characterization, not all of which can be accomplished in this granting period will include transfection of functional genes, transfection of anti- sense genes, and expression of the genes and characterization of their proteins (if any). The successful completion of these experiments will, for the first time, allow the unambiguous identification of all genes regulated by glucocorticoids. Logical extensions of this experimental paradigm to other tissues, such as, for example, lymphocytes will lead to a fundamental understanding of the mechanism of glucocorticoid-induced cell death.

糖皮质激素调节基因含有一个或多个特异性的 结合糖皮质激素受体（GR）的元件，当 与其他基因相关使他们对糖皮质激素有反应。 这种特殊的元素被称为糖皮质激素反应 元素或GRE。 这个提议断言，基因在一个问题中 或受GR与GRE相互作用调节的细胞， 使用一种新的方法进行鉴定和表征， 需要预先知道受调控基因的蛋白质产物。 这种方法的基础是发现一个17,000兆瓦的 受体的胰蛋白酶片段（17-KGR）结合含GR的DNA 具有比完整GR更高的亲和力和特异性。 将使用球蛋白-蛋白A-单克隆抗体基质 将17-K GR纯化，从而得到具有以下的亲和柱： 对含有GRE的基因组结构域的特异性。 本专栏将 用于提取含有GRE的基因组DNA片段（GRE- DNA）。 所提出的实验将稳定地利用AtT-20/D1细胞 用LTR杂合体转染以产生上调的基因， 补充下调基因POMC。 GRE-DNA片段 将被克隆，功能基因的选择，通过确定 克隆与RNA互补，RNA的Bundance被改变， 糖皮质激素治疗 相应的结构基因将 也可以被分离并用于确定哪个基因首先受到影响 糖皮质激素。 进一步表征基因组和 结构DNA将从基因开始， 糖皮质激素治疗最早改变转录。 该基因将被亚克隆和测序，以确定元件 与其他受调控的基因相同。 功能表征，而不是 所有这些都可以在这个授予期内完成， 包括功能基因转染、抗- 有义基因，以及基因的表达和 蛋白质（如果有的话） 成功完成这些 实验将首次允许明确的 鉴定所有受糖皮质激素调节的基因。 逻辑 将该实验范例扩展到其他组织，例如， 例如，淋巴细胞将导致一个基本的理解 糖皮质激素诱导细胞死亡的机制。