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， 当这些过程出错时产生的紊乱。 类固醇激素 调节基因表达提供了一个极好的模型系统， 研究监管机制。 我选择利用一个 部分特征系统由糖皮质激素调节， 的小鼠乳腺肿瘤病毒（MMTV），研究其精细结构 糖皮质激素受体的分子相互作用的细节， 启动子区的调控元件。 (i)我将使用一个新的， 一种新的方法，以阿托伐他汀定向诱变，以产生一个 突变体DNA调控元件文库。 (ii)这些要素将 使用快速、灵敏的DNA介导基因检测生物活性 转移试验和载体，我已经构建了明确的这一目的。 (iii)将测定克隆的元件的能力， 糖皮质激素受体特异性识别。 进一步的精细结构 将使用甲基化保护实验进行作图， 鉴定野生型中受体-DNA相互作用的特定点， 突变序列 (iv)（ii）和（iii）的结果将用于 组装对受体结合和激素结合至关重要的残基的图谱， 反应 这些数据将解决是否具体的问题 受体结合是必需的，并且对于所述受体的生物活性是足够的。 激素反应元件 (v)我会确定是否有变种人 元件显示出与糖皮质激素受体结合的不同能力维斯 孕酮受体。 孕激素受体最近被 据报道，在体外与激素反应的相同区域结合， 元素作为糖皮质激素受体，即使孕酮是 作为MMTV转录的直接诱导剂是生物学上无活性的。 (vi)我 将确定包含功能元素的最小序列。 这些相同的实验也将检验这样的假设， 激素反应元件受到附近其它转录的影响 控制元件。

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.