UNIQUE BIOASSAY SCREENING FOR STEROIDOGENIC ENZYME CLONE

我们实验室的长期目标是了解 类固醇激素的产生和先天性 类固醇生成酶紊乱。 为此，我们已经克隆了 肾上腺细胞色素P450三种酶的cDNA和基因组DNA 类固醇生产，胆固醇侧链裂解酶（P450 SCC）， 21-羟化酶（P450 c21）和17 α-羟化酶/17，20裂解酶（P450 c17）。 我们 已经用这些来研究胆固醇侧链裂解的调节 酶和17 α-羟化酶/17，20裂解酶与遗传病变 胆固醇侧链断裂和21-羟化酶缺乏。 然而，在这方面， 目前可用的技术都没有成功地分离， 表征或克隆关键酶的蛋白质、cDNA或基因 3 β-羟基类固醇脱氢酶（3 BetaHSD）和δ 4，δ 5异构酶 （Isom）. 虽然3 β HSD缺乏通常被认为是罕见的， 最近的数据表明3 β HSD和/或Isom中的轻度酶缺陷 是多毛症，男性化，多囊卵巢疾病的常见原因， 两性不育。 3BetaHSD/Isom的调节也是 由于它们在胎儿肾上腺中的活性较低， 出生;相比之下，肾上腺癌的特点往往是缺乏 3 BetaHSD/Isom活性。 目的：获得cDNA探针，研究其结构、表达调控和功能。 我们提出了一种全新的， 方法对它们的cDNA克隆进行生物学鉴定。 富集 mRNA来源将用于在已建立的酵母载体中克隆cDNA 已知允许哺乳动物细胞表达和膜插入 需要NAD的酶。 将筛选酵母重组体的表达 通过适当转化3 H标记的甾体化合物， 高压液相色谱法测定甾体类化合物的底物及含量 层析 这个拟议的系统将首先进行测试和优化 利用我们最近克隆的另一种类固醇生成酶cDNA（P450 c17）， 实验室

The long term objective of our laboratory is to understand the regulation of steroid hormone production and the molecular bases of congenital disorders in the steroidogenic enzymes. To this end we have already cloned cDNA and genomic DNA for three adrenal cytochrome P450 enzymes involved in steroid production, the cholesterol side-chain cleavage enzyme (P450scc), 21-hydroxylase (P450c21) and 17Alpha-hydroxylase/17,20 lyase (P450c17). We have used these to study regulation of the cholesterol side-chain cleavage enzyme and 17Alpha-hydroxylase/17,20 lyase and genetic lesions in cholesterol side-chain cleavage and 21-hydroxylase deficiency. However, none of the presently available technology has succeeded in isolating, characterizing or cloning proteins, cDNAs or genes for the key enzymes 3Beta-hydroxysteroid dehydrogenase (3BetaHSD) and delta4, delta5 isomerase (Isom). While deficiency of 3BetaHSD has generally been regarded as rare, recent data indicate that mild enzymatic defects in 3BetaHSD and/or Isom are a common cause of hirsutism, virilism, polycystic ovarian disease, and infertility in both sexes. The regulation of 3BetaHSD/Isom is also of great interest as their activity is low in the fetal adrenal and induced at birth; by contrast, adrenal carcinomas are often characterized by deficient 3BetaHSD/Isom activity. To obtain cDNA probes to study the structure, regulated expression and disease-causing mutations in these key enzymes we propose a radical new approach to biological identification of their cDNA clones. An enriched source of mRNA will be used to clone cDNA in an established yeast vector known to permit expression and membrane insertion of mammalian NAD-requiring enzymes. Yeast recombinants will be screened for expression of 3BetaHSD or Isom by appropriate conversion of 3H-labeled steroidal substrates and assay of the steroidal products by high pressure liquid chromatography. This proposed system will first be tested and optimized using another steroidogenic enzyme cDNA (P450c17) recently cloned in our lab.