GENETIC DETERMINANTS OF STEROID HORMONE BIOSYNTHESIS

类固醇激素生物合成的遗传决定因素

• 批准号: 3314924
• 负责人: ANITA H PAYNE
• 金额: $ 18.92万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-09-01 至 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3314924
• 关键词: Leydig cells; adrenal glands; androgens; complementary DNA; cytochrome P450; endoplasmic reticulum; gel electrophoresis; gene expression; genetic library; genetic manipulation; genetic mapping; genetic regulation; genetic strain; laboratory mouse; messenger RNA; molecular cloning; northern blottings; regulatory gene; scintillation counter; steroid 17alpha monooxygenase; steroid hormone biosynthesis; structural genes; testis; testosterone

The long term objective of this project is to determine the influence of genetic control on steroidogenesis. The aims of this application are to study the gene(s) that encode the enzyme, delta5-3beta-hydroxysteroid dehydrogenase delta5->delta4 isomerase (3betaHSD). This enzyme converts the delta5-3beta-hydroxysteroid pregnenolone to progesterone and dehydrodroepiandrosterone to androstenedione. The activity of this enzyme is essential for adrenal and gonadal steroid hormone production. Preliminary data indicate that the mouse liver also expresses this enzyme and has a high capacity for the conversion of delta5-3beta-hydroxysteroids to delta4-3-ketosteroids. An antibody generated against a purified human placental 3betaHSD protein recognizes a single protein in mouse adrenal glands and testes that is identical in molecular weight to the human placental protein. The same antibody recognizes a single protein of different molecular weight in the mouse liver. These data suggest that the liver enzyme may be distinct from the adrenal and testicular enzyme. Additional evidence indicates that the mouse genome contains more than one 3betaHSD gene. It is not known whether more than one gene is expressed. The specific aims of this proposal are designed to: 1. determine whether there are two or more genes which encode distinct 3betaHSD enzymes; 2. isolate the mouse genes that encode the 3betaHSD enzyme(s) and characterize their structure; 3. examine the regulation of 3betaHSD mRNA in mouse testes, adrenal glands and liver. A full length mouse Leydig cell 3betaHSD cDNA has been isolated and characterized. To determine whether adrenal glands and testes express a 3betaHSD gene distinct from the one expressed in liver, RNAse protection assays will be performed using radioactive riboprobes and RNA from the different tissues. Protection of different size fragments by RNA from liver than from adrenal glands and testes will provide evidence for the expression of a different gene in liver. The Leydig cell 3betaHSD cDNA will be used as a probe to isolate a 3betaHSD cDNA from a mouse liver cDNA library. Positive clones from the mouse liver library will be expressed in COS-1 cells. These experiments will determine the differences in the cDNA's and the functional significance of these differences. To determine the total number of 3betaHSD structural genes and pseudogenes, genomic clones will be isolated from an EMBL3 library and characterized. The expression of 3betaHSD mRNA during embryogenesis and pubertal development will be determined in livers, kidneys, adrenal glands and gonads of both male and female mice using a reverse transcriptase- polymerase chain reaction micromethod. To gain insight into the in vivo regulation of hepatic 3betaHSD mRNA by glucocorticoids and by testosterone and to determine whether circulating testosterone modulates adrenal expression of 3betaHSD mRNA, the effect of castration and adrenalectomy and steroid replacement therapy will be studied on the expression of hepatic 3betaHDS mRNA and on adrenal and testicular mRNA, respectively. Regulation of 3betaHSD mRNA expression will be studied in primary cultures of mouse Leydig cells. The identification of a 3betaHSD gene expressed in liver which is distinct from the 3betaHSD gene expressed in adrenal glands and testes could change our thinking about the biosynthesis of steroid hormones in general and may provide an explanation for the clinical manifestations observed in patients with 3betaHSD enzyme deficiency.

该项目的长期目标是确定 类固醇合成的遗传控制。此应用程序的目标是 研究编码β-5-3β-羟基类固醇酶的基因(S) 脱氢酶delta5-gt；delta4异构酶(3betaHSD)。这种酶可以转化为 孕烯醇酮对孕酮和孕酮的作用 脱氢表雄酮为雄烯二酮。这种酶的活性 对肾上腺和性腺类固醇激素的产生是必不可少的。 初步数据表明，小鼠肝脏也表达这种酶。 并具有较高的转化5-3β-羟基类固醇的能力 至4-3-酮类固醇。一种针对纯净人产生的抗体 胎盘3betaHSD蛋白识别小鼠肾上腺单一蛋白 与人类分子量相同的腺体和睾丸 胎盘蛋白。相同的抗体识别单一的蛋白质 小鼠肝脏中的不同相对分子质量。这些数据表明， 肝酶可能不同于肾上腺和睾丸的酶。 更多的证据表明，小鼠的基因组包含不止一个 3β-HSD基因。目前尚不清楚是否有多个基因表达。 这项提案的具体目的是：1.确定是否 有两个或多个基因编码不同的3betaHSD酶； 编码3betaHSD酶的小鼠基因(S)的分离和鉴定 3.检测3betaHSD基因在小鼠体内的表达规律。 睾丸、肾上腺和肝脏。全长小鼠间质细胞3betaHSD 该基因已被分离、鉴定和鉴定。以确定肾上腺是否 腺体和睾丸表达3betaHSD基因，与表达的基因不同 在肝脏，核糖核酸酶保护分析将使用放射性 不同组织中的核糖体探针和RNA。保护不同的 大小片段由来自肝脏的RNA而不是来自肾上腺和睾丸的 为另一种基因在肝脏中的表达提供了证据。这个 本研究将以间质细胞3betaHSD基因为探针，分离3betaHSD 从小鼠肝脏的c DNA文库中提取c DNA。小鼠肝脏中的阳性克隆 文库将在COS-1细胞中表达。这些实验将决定 它们之间的差异及其功能意义 不同之处。测定3betaHSD结构基因总数 和假基因，基因组克隆将从EMBL3文库中分离出来 特色化的。3betaHSD基因在胚胎发育和发育过程中的表达 青春期发育将由肝脏、肾脏、肾上腺决定。 以及雄性和雌性小鼠的性腺使用逆转录酶- 聚合酶链式反应微量方法。为了深入了解体内的 糖皮质激素和睾酮对肝脏3betaHSD基因表达的调节 并确定循环中的睾丸素是否调节肾上腺 3betaHSD基因表达与去势和肾上腺切除的关系 类固醇替代疗法将研究对肝脏表达的影响 3β-HDS m RNA和肾上腺、睾丸m RNA表达。监管 3betaHSD基因在小鼠原代培养中的表达 间质细胞。3betaHSD基因在肝脏中表达的鉴定 它不同于3betaHSD基因在肾上腺和 睾丸可能会改变我们对类固醇激素生物合成的看法 并可为临床表现提供解释。 对3betaHSD酶缺乏症患者进行观察。