MOLECULAR MECHANISMS OF GLUCOCORTICOID HORMONE ACTION

了解基因表达的调控机制是至关重要的。 对发展和分化的理解，因此， 当这些过程出错时出现的紊乱。类固醇激素 受调控的基因表达提供了一个很好的模型系统，用来 调查监管机制。我选择了利用一个 由糖皮质激素调节的部分特征系统，即 小鼠乳腺肿瘤病毒(MMTV)的超微结构研究 糖皮质激素受体与血管内皮生长因子的分子相互作用 启动子区域的调控元件。(I)我将利用一个新的和 一种新的寡核苷酸定向诱变方法 突变DNA调控元件文库。(Ii)这些要素将是 使用快速、灵敏的DNA介导的基因检测生物活性 转移试验和我专门为此目的构建的载体。 (3)将对克隆的元素进行检测，以确定它们是否有能力 由糖皮质激素受体特异性识别。更精细的结构 将使用甲基化保护实验进行映射，以 确定野生型中受体-DNA相互作用的特异点 突变序列。(四)第(二)和(三)项的结果将用于 组装对受体结合和激素至关重要的残基图谱 回应。这些数据将解决以下问题：具体而言 受体结合是细胞生物活性的必要条件和充分条件。 激素反应元件。(V)我会确定是否有任何变种人 元素表现出不同的结合糖皮质激素受体Vis的能力 A与孕激素受体的关系。孕激素受体最近被认为是 据报道，在体外结合到荷尔蒙反应的相同区域 作为糖皮质激素受体的元素，尽管孕酮是 作为MMTV转录的直接诱导剂，生物活性不强。(Vi)i 将确定组成功能元件的最小序列。 这些相同的实验也将检验这样一种假设，即 激素反应元件受附近其他转录的影响 控制元素。

Understanding the mechanisms of regulation of gene expression is essential to a comprehension of development and differentiation, and thus, the disorders that arise when these processes go awry. Steroid hormone regulated gene expression provides an excellent model system with which to investigate regulatory mechanisms. I have chosen to take advantage of a partially characterized system regulated by glucocorticoid hormones, that of the mouse mammary tumor virus (MMTV), to investigate the fine structure details of the molecular interaction of glucocorticoid receptor with the regulatory elements of the promoter region. (i) I will utilize a new and novel approach to oligonucleotide-directed mutagenesis to generate a library of mutant DNA regulatory elements. (ii) These elements will be examined for biological activity using a rapid, sensitive DNA-mediated gene transfer assay and a vector I have constructed expressly for this purpose. (iii) The cloned elements will be assayed for their ability to be specifically recognized by glucocorticoid receptor. Further fine structure mapping will be performed employing methylation protection experiments to identify specific points of receptor-DNA interaction in the wild type and mutant sequences. (iv) The results of (ii) and (iii) will be used to assemble maps of the residues critical for receptor binding and for hormone response. These data will address the question of whether specific receptor binding is necessary and sufficient for biological activity of the hormone response element. (v) I will determine if any of the mutant elements exhibit a differential ability to bind glucocorticoid receptor vis a vis the progesterone receptor. Progesterone receptor has recently been reported to bind in vitro to the same region of the hormone response element as the glucocorticoid receptor even though progesterone is biologically inactive as a direct inducer of MMTV transcription. (vi) I will determine the minimal sequence that comprises a functional element. These same experiments will also test the hypothesis that the activity of the hormone response element is influenced by other nearby transcription control elements.