HORMONE-INDUCED GENE EXPRESSION: OOCYTE RECONSTITUTION

激素诱导的基因表达：卵母细胞重建

• 批准号: 3469381
• 负责人: CHERYL S WATSON
• 金额: $ 8.54万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3469381
• 关键词: Anura; androgens; chromatography; egg /ovum; estrogens; fresh water environment; gene expression; genetic library; genetic transcription; genetic translation; genome; glucocorticoids; hormone binding protein; hormone receptor; laboratory rabbit; ligands; messenger RNA; molecular cloning; molecular genetics; nucleic acid sequence; radioassay

Estradiol activates the genes coding for the oocyte-yolk protein precursor, vitellogenin, in hepatocytes of male and female oviparous vertebrates, while the oocytes themselves are unresponsive to the hormone. The following experimental plan proposes to demonstrate how an artificially introduced functional estrogen binding protein can activate hormone specified, but dormant genes in a non-target cell. The Xenopus oocyte is a convenient non-target cell which is know to supply the factors and cellular machinery necessary to support experimentally manipulated transcription and translation. Microinjection of a crude affinity chromatography purified preparation of Xenopus liver estrogen binding proteins into oocytes resulted in incorporation of 35S-methionine into newly synthesized vitellogenin. These preliminary results suggest that this system can be used to investigate an entire range of questions which remain unsolved in the mechanism of steroid hormone induction of specific gene expression. Further fractionation and reconstitution of the binding protein preparation might define which proteins(s) are essential for initiation and maintenance of transcription. Other questions which could be approached using this system include: what constitutes receptor 'activation' and how is this correlated to the intracellular localization of the receptor, how stringent are the species and hormone specificities for receptors, and which genomic elements interact with estrogen binding proteins or other important regulatory proteins. Eventually it may be possible to use the oocyte as and 'in vivo test tube' providing all nonspecific factors and raw materials for gene expression while all specific factors, both regulatory proteins (including other steroid hormone receptors) and their nucleic acid targets, would be injected into the systems. These kinds of very basic findings are important to the understanding of how steroid hormones activate gene expression in both normal and abnormal growth and development and thus, must precede development of methods for treatment and prevention of cancer, birth defects and other diseases of reproductive tissues.

雌二醇激活编码卵黄蛋白的基因 雄性和雌性肝细胞中的前体卵黄蛋白原 卵生脊椎动物，而卵母细胞本身 对荷尔蒙没有反应。以下是实验计划 建议演示人工引入的功能 雌激素结合蛋白可以激活特定的激素，但 非靶细胞中的休眠基因。非洲爪哇卵母细胞是一种 方便的非靶细胞，已知提供因子和 实验支持所需的细胞机械 操纵转录和翻译。微量注射A 非洲爪哇粗亲和层析纯化制剂的研究 肝脏雌激素结合蛋白进入卵母细胞导致 新合成的~(35)S-蛋氨酸的掺入 卵黄蛋白原。这些初步结果表明，该系统 可用于调查一系列问题，这些问题 类固醇激素诱导骨质疏松症的机制尚不清楚 特定的基因表达。进一步的分馏和重组 结合蛋白制剂可能决定哪些蛋白(S) 对于转录的启动和维持是必不可少的。 可以使用该系统解决的其他问题 包括：什么是受体‘激活’，这是如何实现的 与受体在细胞内的定位有关，如何 受体的种类和激素特异性是严格的， 以及哪些基因组元件与雌激素结合相互作用 蛋白质或其他重要的调节蛋白质。最终，它 有可能将卵母细胞用作体内试管 为基因提供所有非特异性因子和原料 在表达所有特定因子的同时，这两种调节蛋白 (包括其他类固醇激素受体)及其核酸 目标，将被注入系统。 这些非常基本的发现对 对类固醇激素如何激活基因表达的理解 在正常和异常的生长和发育中，因此， 必须在开发治疗和治疗的方法之前 预防癌症、出生缺陷和其他疾病 生殖组织。