PHYSIOLOGY OF PITUITARY CELL GLUCOCORTICOID BINDING

这个项目的目标是使用基因方法来识别 糖皮质激素反应机制的组成部分。战略将是 将一种基因结构引入AtT-20细胞，导致细胞 在新霉素类似物G418和 糖皮质激素。初步研究使用了ATT-20/D1.IDG8细胞，该细胞 在糖皮质激素阴性状态下含有新霉素耐药基因(Neor) 监管。这些细胞在G418或地塞米松的存在下生长 单独使用，但在地塞米松+G418存在下生长受阻。这个 用一种化学诱变剂和糖皮质激素抗药性处理细胞 含有地塞米松+的软琼脂上大克隆的鉴定 G418。对14个无性系进行了研究。在所有测试的克隆中，均为阴性 地塞米松对neor基因表达的调节作用被取消。在十四年中的第十年 阿片黑素皮质素原(POMC)是一种内源调控基因 保留表明突变是NEOR启动子的局部突变， 顺式突变。在其他四个克隆中，POMC和a的调控 报告基因催乳素-猫丢失，表明该突变是 一种常见的全球性因素，一种反式突变。四个缺乏受体的人之一 信使核糖核酸表明它是受体缺失、反式突变。另一个 其中3个含有正常大小和丰度的受体mRNA，表明 它们可能是正常受体，反式突变。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 吉恩。如果不能通过正常的表达来纠正调节的缺陷 受体，其正常受体、反式突变状态将得到确认。 这些研究表明，信息性突变的基因选择可以 通过这种方法来实现。项目的第一阶段将是 稳定表达新霉素的细胞系的建立 POMC启动子控制下的抗性基因(POMNEO基因)。在……里面 第二阶段，将产生突变克隆，分离并分类到 受体缺陷、其他反式缺陷和顺式缺陷。第三阶段将 确定顺式和反式突变体的特征。顺式基因的POMNEO启动子 将对突变体进行测序，以确定改变的核苷酸序列。自.以来 反式突变可能导致其他DNA结合蛋白的变化 而不是受体，或者蛋白质-蛋白质相互作用中的改变 对于受体功能，将对反式突变细胞进行测试，以确定 蛋白质与POMNEO启动子结合的模式已经改变。 该项目的成功完成将：确定细胞过程 对受体功能很重要；以及识别和表征顺式元件 识别其他反式因子的糖皮质激素反应机制 而不是受体。

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.