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HORMONE-INDUCED GENE EXPRESSION: OOCYTE RECONSTITUTION

激素诱导的基因表达：卵母细胞重建

基本信息

批准号：
3469383
负责人：
CHERYL S WATSON
金额：
$ 8.15万
依托单位：
UNIVERSITY OF TEXAS MED BR GALVESTON
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-07-01 至 1993-06-30
项目状态：
已结题

项目摘要

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.

雌二醇激活编码卵母细胞-卵黄蛋白的基因雌、雄鼠肝细胞卵黄蛋白原前体卵生脊椎动物，而卵母细胞本身是对荷尔蒙没有反应以下实验计划建议演示如何人工引入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蟾卵母细胞是已知提供因子的方便的非靶细胞，细胞机械必须支持实验操纵转录和翻译。微量注射a 非洲爪蟾亲和层析纯化粗品肝雌激素结合蛋白进入卵母细胞，将35 S-甲硫氨酸掺入新合成卵黄蛋白原这些初步结果表明，可以用来研究一系列的问题，在类固醇激素诱导的机制中仍然没有解决，特异性基因表达进一步分馏和复溶结合蛋白制剂的定义可以确定哪些蛋白质对转录的启动和维持至关重要。可利用这一系统处理的其他问题包括：什么是受体“激活”，以及如何激活与受体的细胞内定位相关，受体的物种和激素特异性是严格的，哪些基因组元件与雌激素结合蛋白质或其他重要的调节蛋白。最终它可以使用卵母细胞作为“体内试管” 为基因工程提供了所有非特异性因子和原材料表达，而所有特定的因子，无论是调节蛋白（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于了解类固醇激素如何激活基因表达在正常和异常的生长和发育中，必须在开发治疗方法之前，预防癌症、出生缺陷和其他疾病生殖组织