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: 3314931
• 负责人: ANITA H PAYNE
• 金额: $ 18.7万
• 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/3314931
• 关键词: Leydig cells; RNase protection assay; adrenal glands; androgens; complementary DNA; cytochrome P450; endoplasmic reticulum; gel electrophoresis; gene expression; genetic library; genetic manipulation; genetic mapping; genetic regulation; genetic strain; hydroxysteroid dehydrogenases; laboratory mouse; messenger RNA; molecular cloning; northern blottings; nucleic acid sequence; 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.

本项目的长期目标是确定 类固醇生成的遗传控制。 本申请的目的是 研究编码δ 5 - 3 β-羟基类固醇酶的基因 脱氢酶δ 5 → δ 4异构酶（3 β HSD）。 这种酶能将 δ 5 - 3 β-羟基类固醇双烯醇酮转化为孕酮， 脱氢表雄酮转化为雄烯二酮。 这种酶的活性 是肾上腺和性腺类固醇激素产生所必需的。 初步数据表明，小鼠肝脏也表达这种酶 并具有高的δ 5 - 3 β-羟基类固醇转化能力 到δ 4 -3-酮类类固醇。 一种针对纯化的人类 胎盘3 β HSD蛋白识别小鼠肾上腺中的单一蛋白 分子量与人类相同的腺体和睾丸 胎盘蛋白 相同的抗体识别单个蛋白质 在小鼠肝脏中有不同的分子量。 这些数据表明 肝酶可能不同于肾上腺和睾丸酶。 更多的证据表明，老鼠的基因组中含有不止一个 3 β HSD基因。 目前尚不清楚是否有一个以上的基因表达。 本提案的具体目标是：1.确定是否 存在两个或多个编码不同3 β HSD酶的基因; 2. 分离编码3 β HSD酶的小鼠基因并表征 结构; 3。检测小鼠3 β HSD mRNA的调节 睾丸肾上腺和肝脏 全长小鼠Leydig细胞3 β HSD cDNA已被分离和表征。 为了确定肾上腺 腺体和睾丸表达的3 β HSD基因不同于 在肝脏中，将使用放射性同位素进行RNA酶保护测定。 核糖核酸探针和来自不同组织的RNA。 保护不同 通过对来自肝脏的RNA比来自肾上腺和睾丸的RNA的大小确定片段的大小， 为肝脏中不同基因的表达提供了证据。 的 Leydig细胞3 β HSD cDNA将用作探针以分离3 β HSD 来自小鼠肝脏cDNA文库的cDNA。 来自小鼠肝脏的阳性克隆 文库将在COS-1细胞中表达。 这些实验将决定 cDNA的差异及其功能意义， 差异 确定3 β HSD结构基因的总数 和假基因，将从EMBL 3文库中分离基因组克隆， 表征了 3 β-HSD mRNA在胚胎发生过程中的表达， 青春期发育将在肝脏、肾脏、肾上腺 和生殖腺的基因组， 聚合酶链反应微量法 为了深入了解体内 糖皮质激素和睾酮对肝脏3 β HSD mRNA的调节 并确定循环睾酮是否调节肾上腺皮质激素 3 β HSD mRNA表达，去势和肾上腺切除的影响， 类固醇替代治疗将对肝脏表达进行研究。 3 β HDS mRNA和肾上腺及睾丸mRNA。 调控 将在小鼠原代培养物中研究3 β HSD mRNA的表达 睾丸间质细胞。 一个在肝脏中表达的3 β-HSD基因的鉴定 其不同于在肾上腺中表达的3 β HSD基因， 睾丸可能会改变我们对类固醇激素生物合成的看法 并可能为临床表现提供解释 在3 β HSD酶缺乏症患者中观察到。