GLUCOCORTICOID RECEPTOR STRUCTURE SUBUNIT ARCHITECTURE

糖皮质激素受体结构亚基结构

• 批准号: 3235444
• 负责人: ROBERT W HARRISON
• 金额: $ 17.18万
• 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/3235444
• 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.

糖皮质激素调节的基因包含一个或多个特定的 与糖皮质激素受体(GR)结合的分子，以及当 与其他基因的关联使它们对糖皮质激素有反应。 这种特殊的元素被命名为糖皮质激素反应 元素或GRE。这一提议断言，一个问题中的基因 或受GR与GRE相互作用调节的细胞 使用一种新的方法进行识别和表征，而不是 需要预先了解受调控基因的蛋白质产物。 这种方法的基础是发现17,000兆瓦 受体(17-K GR)的胰酶片段与含GRE的DNA结合 比完整的GR具有更高的亲和力和特异性。一个 多糖-蛋白A-单抗基质将用于 固定化17-K GR，从而得到与 包含GRE的基因组结构域的特异性。本专栏将 用于提取基因组DNA中含有GRE的片段(GRE- DNA)。所提出的实验将稳定地利用Att-20/d1细胞。 用LTR杂交体导入产生上调表达的基因 补充下调基因POMC。GRE-DNA片段 将被克隆并通过确定哪些功能基因来选择 克隆是对RNA的补充，RNA的结合通过以下方式改变 糖皮质激素治疗。相应的结构基因将 也被分离出来并用来确定哪个基因最先受到影响 由糖皮质激素引起。基因组和基因的进一步表征 结构DNA将从其基因开始 糖皮质激素治疗最早改变转录。 将对该基因进行亚克隆和测序，以确定这些元素 与其他受调控基因有共同之处。功能表征，备注 在这一授权期内可以完成的所有这些都将 包括功能基因转染法、反义寡核苷酸转染法 正义基因，以及基因的表达和特征 他们的蛋白质(如果有的话)。这些项目的顺利完成 实验将第一次允许明确的 所有受糖皮质激素调控的基因的鉴定。逻辑上的 将这一实验范式扩展到其他组织，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。