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: 3314926
• 负责人: ANITA H PAYNE
• 金额: $ 15.19万
• 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 1991-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3314926
• 关键词: Leydig cells; adrenal glands; complementary DNA; cytochrome P450; endoplasmic reticulum; gel electrophoresis; gene expression; genetic manipulation; genetic mapping; genetic regulation; genetic strain; 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 studies proposed in this application are designed to examine the relationships among the structural and regulatory gene(s) that influence the expression of the enzymes involved in testicular steroid hormone biosynthesis. Specifically, it is proposed to determine the chromosomal location of genes determining quantitative and structural variation of the following steroidogenic enzymes: 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD), cholesterol side-chain cleavage (P-450scc) and 17 alpha-hydroxylase/C17-20 lyase (P-450-17 alpha). Whole testicular preparations, purified Leydig cells or adrenal glands from numerous strains of inbred mice will be surveyed for quantitative and qualitative differences of each of the enzymes. Recombitant inbred (RI) lines, derived from progenitor strains which show the greatest interstrain differences, will be used to map the chromosomal location of the structural and regulatory gene(s) for each enzyme. Quantitative differences in 3 beta-HSD will be determined by measuring enzyme activity; structural variants of this enzyme will be detected by measuring rate of heat inactivation (thermostability). Quantitative differences in P-450scc and P-450-17 alpha will be determined in lysates of purified Leydig cells or of adrenal glands using specific rabbit antisera generated against these enzymes plus 125I-labeled second antibody and analysis by immunoblotting. Structural variants of P-450scc and P-450-17 alpha will be detected by analyzing for variations in restriction fragment length of genomic DNA, from different inbred strains of mice, through hybridization with cDNA probes specific for P-450scc and P-450-17 alpha. The availability of specific antisers to the P-450scc and P-450-17 alpha and the cDNA clones specific for each of these enzymes provide sensitive reagents for the detection of quantitative and qualitative variants, respectively. Information gained from the proposed studies will provide a better understanding of the normal physiologic function of the Leydig cell as well as explanations for pathologic states due to genetic defects in steroid biosynthesis enzymes in testes and adrenal glands.

本项目的长期目标是确定 类固醇生成的遗传控制。 本报告中提出的研究 应用程序旨在检查结构之间的关系， 和影响酶表达的调节基因 参与睾丸类固醇激素的生物合成。 具体来说就是 建议确定基因的染色体位置， 以下类固醇生成的数量和结构变化 酶：3 β-羟基类固醇脱氢酶-异构酶（3 β-HSD）， 胆固醇侧链裂解（P-450 SCC）和17 α-羟化酶/C17-20 裂解酶（P-450-17 α）。 全睾丸制备物，纯化Leydig 来自许多品系的近交系小鼠的细胞或肾上腺将被 调查了每一个国家在数量和质量上的差异， 内切酶 重组近交系（RI），来源于祖先品系 显示出最大的菌株间差异，将被用来绘制 每个基因的结构和调控基因的染色体定位 酵素 3 β-HSD的定量差异将通过以下方法确定： 测量酶活性;该酶的结构变体将被 通过测量热失活率（热稳定性）进行检测。 将确定P-450 SCC和P-450-17 α的定量差异 在纯化的Leydig细胞或肾上腺的裂解物中，使用特异性 产生的抗这些酶的兔抗血清加上125 I标记的第二 抗体并通过免疫印迹进行分析。 P-450 SCC的结构变体 和P-450-17 α将通过分析 来自不同近交系的基因组DNA的限制性片段长度 通过与P-450 SCC特异性cDNA探针杂交， P-450-17阿尔法。 P-450 SCC的特定抗毒剂的可用性， P-450-17 α和对这些酶中的每一种特异的cDNA克隆 提供灵敏的定量检测试剂， 质的变化。 从拟议的 研究将提供一个更好的了解正常的生理 间质细胞的功能以及病理状态的解释 由于睾丸中类固醇生物合成酶的遗传缺陷， 肾上腺