ACTH INDUCTION OF CYTOCHROME P-450 IN ADRENAL CELLS

所有哺乳动物的正常繁殖和生命维持 物种需要复杂的类固醇合成活性模式。在……里面 建议在生物化学中阐明这一更新应用。 类固醇羟基酶基因的多因素调控 在肾上腺皮质中的表达。在过去的批准期内 我们已经研究了三个水平的表达调控 这些基因(维持最佳类固醇合成能力 部分受ACTH通过cAMP、组织特异性和 发育)使用针对各种类固醇的cdna探针 羟化酶mRNAs。在接下来的授权期内，我们建议 在转录水平上建立反式作用因子 而顺式调控序列涉及这种复杂的模式 类固醇羟基酶基因表达的调控。利用基因 编码人和牛P-450 17α人和牛P-450scc‘ 人P-450C21和牛肾上腺素我们将鉴定肾上腺 核蛋白对这些基因的转录反应 CAMP(所谓的船只)及其绑定的顺式序列。 凝胶延迟、DNA足迹、CAT活性的组合 体外转录试验将被用于鉴定这些 其次是因子的纯化和表征。在……里面 此外，我们还将确定满足以下要求的类似因素 胎儿类固醇羟基酶基因在胎儿体内的印迹 牛的肾上腺皮质使用同样的技术。我们也会 确定细胞特异性调控的分子基础 牛网状束膜P-450 17α与带状核的比较 肾小球糖蛋白以及缺乏表达的原因 P-450 17α在大鼠肾上腺皮质中的表达。同样，特定于细胞的 肾上腺激素基因表达的调控因素 大脑皮层、肝脏和肾脏将被识别。最后利用 CAMP依赖蛋白激酶的突变调节亚单位我们将 确定该酶是否在类固醇羟基酶中起作用 基因表达。总括而言，在拟议的五年内，我们会 识别、描述和比较哪些反式作用因素 激素调控肾上腺类固醇羟基酶基因表达 介导性、组织特异性和发育水平。结果是 这些研究将允许评估复杂性和 这些过程的关联性。此外，他们还将展示 通往一组新基因的道路，这些基因编码反式作用因子 它调节类固醇羟基酶基因的表达。

Normal reproduction and the maintenance of life in all mammalian species requires complex patterns of steroidogenic activities. In this renewal application it is proposed to elucidate in biochemical terms the multifactorial regulation of steroid hydroxylase gene expression in the adrenal cortex. During the past granting period we have investigated three levels of regulation of expression of these genes (maintenance of optimal steroidogenic capacity regulated in part by ACTH via cAMP, tissue-specific, and developmental) using cDNA probes specific for the various steroid hydroxylase mRNAs. In the coming granting period we propose to establish at the transcriptional level, the trans-acting factors and cis-regulatory sequences involved this complex pattern of regulation of steroid hydroxylase gene expression. Utilizing genes encoding human and bovine P-450 17 alpha human and bovine P-450scc' human P-450C21 and bovine adrenodoxin we will identify adrenal nuclear proteins which transcription of these genes in response to cAMP (so-called SHIPs) and the cis-sequences to which they bind. A combination of gel retardation, DNA footprinting, CAT activities and in vitro transcription assays will be used to identify these factors followed by their purification and characterization. In addition we will identify similar factors which are required for the fetal imprinting of steroid hydroxylase genes in the fetal bovine adrenal cortex using the same techniques. Also we will determine the molecular basis of the cell specific regulation of P-450 17 alpha in bovine fasiculata-reticularis compared to zona glomerulose as well as the reason for the absence of expression of P-450 17 alpha in rat adrenal cortex. Likewise the cell specific factors which regulate adrenodoxin gene expression in adrenal cortex, liver and kidney will be identified. Finally utilizing mutant regulatory subunits of cAMP-dependent protein kinase we will establish whether this enzyme plays a role in steroid hydroxylase gene expression. In summary, during the proposed 5 years we will identify, characterize and compare trans-acting factors which regulate adrenal steroid hydroxylase gene expression at the hormone mediated, tissue-specific and developmental levels. The results of these studies will permit assessment of both the complexity and the relatedness of these processes. Furthermore they will show the way to a new set of genes, those encoding trans-acting factors which regulate steroid hydroxylase gene expression.