EXPRESSION OF PRO ACTH/ENDORPHIN GENE IN VARIOUS TISSUES

其基本目的是阐明涉及的分子机制。 前阿片黑素皮质素(POMC)基因的表达调控 脑垂体和下丘脑组织。使用一系列大鼠POMC特异的 CDNA和基因克隆，我们建议进一步表征 类固醇、多肽和蛋白质对POMC基因的转录调控 儿茶酚胺，既有单独的，也有组合的。一些研究将是 在完整的动物身上进行；但是，某些研究将在初级阶段进行 垂体腺细胞和AtT20肿瘤细胞培养排除间接 对POMC基因表达的影响。POMC基因中涉及的确切位置 在特定激素的调节下，基因表达将通过 缺失分析和定点突变。我们已经这么做了 构建大鼠POMC启动子/大鼠催乳素编码区 为这些研究提供帮助。利用这些区域进行蛋白质/DNA结合研究 在以前的研究中发现了一种重新启动的核转录 化验，我们将开始鉴定和表征交易因素 它们介导了多肽激素、神经递质和类固醇的调节 POMC基因表达的调控。在相关研究中，我们将 糖皮质激素受体编码基因的诱导特性 中叶细胞培养后。除了……之外 了解糖皮质激素受体基因是如何表达的 在完好的动物身上被抑制，这些研究将允许我们开发出一种 相对同质的原代培养体系，我们可以在其中研究 糖皮质激素/CRF调节相互作用。我们将进一步描述 视网膜节周围区POMC基因系统的调控 使用几种生殖模式的下丘脑。研究将会是 通过溶液杂交和原位杂交进行分析 杂交分析，结合几种解剖学技术 在功能上细分POMC神经元。我们将进一步开发这一用途 POMC基因介导性序列特异性DNA探针的研究 原位杂交作为一种间接分析转录的方法 分别在少量组织和单个细胞中的比率。 这些研究应该会对这个复合体有很大的理解 不同类型激素物质在细胞周期调控中的相互作用 在复杂但正常的生理情况下的特定基因。

The basic objective is to elucidate the molecular mechanisms involved in the regulation of expression of the proopiomelanocortin (POMC) gene in pituitary and hypothalamic tissues. Using a series of rat POMC-specific cDNAs and gene clones, we propose to further characterize the transcriptional regulation of the POMC gene by steroids, peptides, and catacholamines, both singlely and in combination. Some studies will be done in the intact animal; however, certain studies will be done in primary cultures of pituitary cells and in AtT20 tumor cells to preclude indirect effects on POMC gene expression. The exact sites in the POMC gene involved with regulation by specific hormones of gene expression will be mapped by deletion analysis and site directed mutagenesis. We have already constructed a rat POMC promoter/rat Prolactin coding region construct to aid in these studies. Using protein/DNA binding studies using the regions identified in the previous studies and a reinitiating nuclear transcription assay, we will begin to identify and characterize the transacting factors which mediate the peptide hormone, neurotransmitter, and steroid mediated modulation of POMC gene expression. In related studies, we will characterize the induction of the glucocorticoid receptor encoding gene after culturing of the intermediate lobe cells. In addition to understanding how expression of the glucocorticoid receptor gene is suppressed in the intact animal, these studies will allow us to develop a relatively homogeneous primary culture system in which we can study the glucocorticoid/CRF regulatory interactions. We will further characterize the regulation of the POMC gene system in the periarcuate region of the hypothalamus using several reproductive paradigms. Studies will be performed both by solution hybridization assays as well as in situ hybridization assays, coupled with several anatomical techniques to functionally subdivide the POMC neurons. We will further develop the use of POMC gene intervening sequence specific DNA probes in solution and in situ hybridization as an indirect method for analyzing transcriptional rates in small amounts of tissues and individual cells, respectively. These studies should yield a great deal of understanding of the complex interaction of various types of hormonal substances in the regulation of a specific gene in a complex but normal physiological situation.