PHYSIOLOGY OF PITUITARY CELL GLUCOCORTICOID BINDING

垂体细胞糖皮质激素结合的生理学

• 批准号: 3182911
• 负责人: ROBERT W HARRISON
• 金额: $ 20.68万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-08-01 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3182911
• 关键词: DNA binding protein; DNA footprinting; clone cells; dexamethasone; electroporation; fusion gene; gel electrophoresis; gene expression; gene mutation; genetic library; genetic promoter element; genetic regulatory element; genetic transcription; glucocorticoids; hormone regulation /control mechanism; molecular cloning; natural gene amplification; neomycin; northern blottings; nuclear runoff assay; nucleic acid sequence; phosphotransferases; polymerase chain reaction; posttranscriptional RNA processing; proopiomelanocortin; southern blotting; steroid hormone receptor; transcription factor; transfection

This project's objective is to use a genetic approach to identify components of the glucocorticoid response mechanism. The strategy will be to introduce a gene construct into AtT-20 cells that results in cells that are growth-arrested in the presence of the neomycin analog, G418, and glucocorticoids. Preliminary studies have used AtT-20/D1.IDG8 cells which contain the neomycin resistance gene (neor) under negative glucocorticoid regulation. These cells grow in the presence of G418 or dexamethasone alone but are growth-arrested in the presence of dexamethasone + G418. The cells were treated with a chemical mutagen and glucocorticoid-resistant clones identified as large colonies on soft agar containing dexamethasone + G418. Fourteen clones were studied. In all clones tested, negative regulation of neor mRNA by dexamethasone was abolished. In ten of fourteen clones, regulation of an endogenous gene, pro-opiomelanocortin (POMC) was retained indicating that the mutation was local to the neor promoter, a cis-mutation. In the other four clones, regulation of POMC and of a reporter gene, prolactin-CAT, was lost indicating that the mutation was of a common, global factor, a trans-mutation. One of four lacked receptor mRNA indicating that it was a receptor-minus, trans-mutation. The other three contained receptor mRNA of normal size and abundance indicating that they might be normal-receptor, trans-mutations. They will be analyzed further by co-introduction of a receptor expression vector and a reporter gene. If the deficient regulation is not corrected by expression of normal receptor, their normal-receptor, trans-mutation status will be confirmed. These studies indicate that genetic selection of informative mutations can be accomplished by this approach. Phase one of the project will be establishment of a cell line stably-transfected with the neomycin resistance gene under control of the POMC promoter (the "POMNEO" gene). In phase two, mutant clones will be produced, isolated and sorted into receptor defects, other trans defects and cis defects. Phase three will characterize the cis- and trans-mutants. The POMNEO promoter of cis- mutants will be sequenced to identify altered nucleotide sequences. Since a trans-mutation could result in a change of a DNA binding protein other than the receptor, or alteration in a protein-protein interaction necessary for receptor function, trans-mutant cells will be tested to determine if the pattern of protein binding to the POMNEO promotor has been altered. Successful completion of this project will: identify cell processes important to receptor function; and, identify and characterize cis elements of the glucocorticoid response mechanism that recognize trans-factors other than the receptor.

该项目的目标是使用遗传学方法来识别 糖皮质激素反应机制的组成部分。 该战略将 将基因构建体导入AtT-20细胞， 在新霉素类似物G418的存在下生长停滞， 糖皮质激素 初步研究使用AtT-20/D1.IDG8细胞， 在糖皮质激素阴性下含有新霉素抗性基因（neor） 调控 这些细胞在G418或地塞米松存在下生长 但是在地塞米松+ G418存在下生长停滞。 的 用化学诱变剂和糖皮质激素抗性处理细胞 在含有地塞米松+的软琼脂上鉴定为大菌落的克隆 G418筛选 研究了14个克隆。 在所有测试的克隆中，阴性 地塞米松对neor mRNA的调节作用消失。 在十四个中的十个中 克隆，调节内源性基因，前阿黑皮素（POMC）， 保留表明突变是neor启动子的局部， 顺式突变 在其他四个克隆中，POMC和一个 报告基因，催乳素-CAT，丢失，表明突变是 一个共同的，全球性的因素，一种转变。 四分之一缺乏受体 这表明它是一种受体缺失的转化突变。 另 三个含有正常大小和丰度的受体mRNA，表明 它们可能是正常的受体，也可能是转基因的。 他们将被分析 进一步通过共引入受体表达载体和报告基因 基因 如果调节不足不能通过表达正常的 受体，其正常受体，转化状态将被确认。 这些研究表明，信息突变的遗传选择可以 通过这种方式来实现。 该项目的第一阶段将是 新霉素稳定转染细胞系的建立 P0 MC启动子控制下的抗性基因（“P0 MNE 0”基因）。 在 第二阶段，将产生突变克隆，分离并分选成 受体缺陷、其它反式缺陷和顺式缺陷。 第三阶段将 表征顺式和反式突变体。 POMNEO顺式启动子 将对突变体进行测序以鉴定改变的核苷酸序列。 以来 一个转化突变可能会导致DNA结合蛋白的变化， 或者改变蛋白质间的相互作用 对于受体功能，将测试反式突变细胞以确定是否 蛋白质与POMNEO启动子结合的模式已经改变。 成功完成本项目将：确定细胞过程 对受体功能很重要;并且，识别和表征顺式元件 糖皮质激素的反应机制，识别反式因子， 而不是受体。