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: 3314929
• 负责人: ANITA H PAYNE
• 金额: $ 16.87万
• 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/3314929
• 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; laboratory mouse; 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.

该项目的长期目标是确定 类固醇合成的遗传控制。在这篇文章中提出的研究 应用程序旨在检查结构之间的关系 和影响酶表达的调控基因(S) 参与睾丸类固醇激素的生物合成。具体地说，它是 提出了确定染色体位置的基因决定 下列类固醇激素的数量和结构变异 酶：3β-羟基类固醇脱氢酶异构酶(3β-HSD)， 胆固醇侧链裂解(P-450scc)和17α-羟基酶/C17-20 裂解酶(P-450-17α)。完整的睾丸制剂，精制的莱迪格 来自多个品系近交系小鼠的细胞或肾上腺将被 调查了每一个人在数量和质量上的差异 酵素。重组自交系(RI)，来源于祖先系 它们显示了最大的菌株间差异，将被用来绘制 结构调控基因(S)的染色体定位 酵素。3种β-HSD的数量差异将通过以下方式确定 测量酶的活性；这种酶的结构变体将是 通过测量热失活率(热稳定性)来检测。 将确定P-450scc和P-450-17α的数量差异 在纯化的间质细胞或肾上腺的裂解物中使用特异性 针对这些酶产生的兔抗血清加上125I标记的第二 抗体检测及免疫印迹分析。P-450scc的结构变体 和P-450-17α将通过分析 不同自交系基因组DNA的限制性内切酶片段长度 通过与P-450scc和P-450scc特异的cDNAs探针杂交 P-450-17α。P-450scc的特定抗药的可用性和 P-450-17α和每种酶的特异性cDNA克隆 为定量和定量检测提供灵敏的试剂 分别是质的变种。从建议的 研究将提供对正常生理的更好的理解。 间质细胞的功能及其对病理状态的解释 由于睾丸和睾丸类固醇生物合成酶的遗传缺陷 肾上腺。