MOLECULAR MECHANISMS OF HFSH/B GENE REGULATION

HFSH/B 基因调控的分子机制

• 批准号: 2904971
• 负责人: KATHRYN G SCHUFF
• 金额: $ 12.18万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2904971
• 关键词: DNA binding protein; DNA footprinting; adrenocorticotropic hormone; androgen receptor; corticosteroid receptors; corticotropin releasing factor; cyclic AMP; endorphins; estrogen receptors; follicle stimulating hormone; gel mobility shift assay; gene expression; genetic enhancer element; genetic regulation; genetic transcription; genetically modified animals; gonadotropin releasing factor; hormone regulation /control mechanism; hypothalamic pituitary axis; laboratory mouse; luteinizing hormone; nuclear runoff assay; receptor binding; reproductive system; tissue /cell culture

DESCRIPTION (Taken from the applicant's Abstract) One of the most common and difficult to manage disorders of the reproductive system is altered gonadotropin regulation related to overactivity of the hypothalamic-pituitary-adrenal (HPA) axis. The first Specific Aim of this mentored project is directed towards understanding the normal regulation of the reproductive system, in particular the regulation of the hFSH-beta gene by gonadotropin specific factors, gonadotropin releasing hormone (GnRH) and gonadal steroids. This will be accomplished by l) comparison of the hFSH-beta gene to known cAMP-responsive, gonadotropin-specific, estrogen, and androgen response elements and GATA-binding sites known to regulate other gonadotropin genes, 2) electromobility shift and DNase protection assays to demonstrate DNA binding and mutation of any binding sites found to assess their importance for expression and 3) expression of 3' deleted gene constructs in transgenic mice to map candidate enhancers. In Specific Aim Two, the importance of beta-endorphin in mediating stress-induced anovulation will be evaluated in physiologic experiments using beta-endorphin knockout mice. The importance of glucocorticoids for this effect will be tested by demonstrating DNA binding of glucocorticoid receptor and in physiologic experiments of stress-induced anovulation in adrenalectomized beta-endorphin deficient mice. Work in this field has been hampered by the lack of a convenient expression system; therefore, continued efforts to develop an FSH-producing cell line from mouse gonadotropin cells immortalized with the SV40tsTAg linked to the hFSH-beta promoter and optimizing transient transfections is the third Specific Aim of this project. An improved understanding of the physiologic and molecular regulation of the reproductive system and the interaction of the HPA axis with that system will lead to improved treatment of the reproductive effects of disorders of the HPA axis. Through a mentored career development plan, the Principal Investigator will enhance knowledge of biochemistry and molecular biology through course work, enhance skills in application of basic science research to understanding endocrinology, gain expertise in research design and statistical analysis, and gain appreciation of principles of responsible scientific conduct. By the end of the five year mentorship period, this will result in achievance of independence in the design and implementation of research projects.

描述（摘自申请人摘要） 最常见和最难管理的生殖系统疾病之一 系统是改变促性腺激素调节有关的过度活跃的 下丘脑-垂体-肾上腺（HPA）轴。 第一个具体目标是 指导项目是为了了解正常的规则， 生殖系统，特别是hFSH-β基因的调节 促性腺激素释放激素（GnRH）， 性腺类固醇 这将通过l）比较 hFSH-β基因与已知cAMP反应性、促性腺激素特异性、雌激素， 雄激素反应元件和GATA结合位点已知调节 其他促性腺激素基因，2）电迁移率改变和DNA酶保护 证明DNA结合和发现的任何结合位点突变的试验， 评估它们对表达的重要性和3）3'缺失基因的表达 在转基因小鼠中构建以定位候选增强子。 具体目标 二、β-内啡肽在介导应激诱导的 无排卵将在生理学实验中进行评价， β-内啡肽敲除小鼠。 糖皮质激素对此的重要性 将通过证明糖皮质激素的DNA结合来测试效果 受体和生理实验中的应激诱导的无排卵， 肾上腺切除的β-内啡肽缺乏小鼠。 这一领域的工作一直是 由于缺乏方便的表达系统， 从小鼠促性腺激素细胞中开发FSH生产细胞系的努力 用与hFSH-β启动子连接的SV 40 tsTAg永生化， 优化瞬时转染是本发明的第三个具体目标， 项目 提高对生理和分子生物学的理解， 调节生殖系统和HPA轴的相互作用 将改善对生殖影响的治疗 下丘脑-垂体-肾上腺轴紊乱 通过指导职业发展计划， 主要研究者将提高生物化学知识， 分子生物学通过课程工作，提高技能的应用 基础科学研究，了解内分泌学，获得专业知识， 研究设计和统计分析，并获得赞赏 负责任的科学行为原则。 到五年结束时， 在指导期间，这将导致独立性的丧失， 研究项目的设计和实施。