REGULATION OF THE PREPRO GRP GENE

哺乳动物蛙皮素样肽、胃泌素释放肽(GRP)和 神经介素B(NMB)是一种重要的调节神经肽 包括平滑肌收缩在内的一系列生物反应， 刺激分泌，调节神经元活动，并控制 细胞增殖。该实验室之前的研究表明 即：1)在肿瘤细胞系中，前-GRP基因在 初级转录水平；以及2)可检测到的表达 仅限于中枢神经和外周神经中的一部分神经元 系统、神经内分泌细胞和衍生的肿瘤细胞系。我们计划 明确前GRP的重要分子机制 表达细胞的转录激活和抑制 不表达的细胞。此分析的第一步是 顺式作用的前-GRP启动子元件的鉴定 抄写。一系列融合基因被组装在一起来检测 前GRP启动子中序列元件的转录效应。 在这些结构中，萤火虫荧光素酶报告基因被置于 不同长度人前GRP的转录调控 启动子区域。在转染和随后的瞬时表达之后 在不同的宿主细胞系中，荧光素酶活性被确定为 含有不同前GRP启动子元件的构建。使用这个 在检测系统中，我们做了以下观察：1)启动子 介于-5000和-1600之间的序列(原点定义为 转录起始点)发挥大约两倍的作用 对转录的负面影响，而-1600到-400之间的序列 发挥微弱的积极作用。在这个正值区域，数百个 碱基长的5‘上游序列在大鼠之间高度保守 和人类前GRP前基因(-720到-450)；以及2)构建体表现 在正常表达或不表达前GRP的宿主细胞中也是如此 吉恩。我们得出结论，仅-5000和+100之间的启动子元件 不确定转录调控的细胞类型特异性。

The mammalian bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB), are important regulatory neuropeptides which mediate a range of biologic responses including smooth muscle contraction, stimulation of secretion, modulation of neuronal activity, and control of cell proliferation. Previous studies from this laboratory have shown that: 1) in tumor cell lines, the prepro-GRP gene is regulated at the level of primary transcription; and 2) detectable expression is restricted to a subset of neurons in the central and peripheral nervous system, neuroendocrine cells, and derivative tumor cell lines. We plan to define the molecular mechanisms important in prepro-GRP transcriptional activation in expressing cells, and suppression in nonexpressing cells. The first step in this analysis is the identification of cis-acting prepro-GRP promoter elements important for transcription. A series of fusion genes were assembled to assay the transcriptional effects of sequence elements in the prepro-GRP promoter. In these constructs, the firefly luciferase reporter gene is placed under transcriptional control of varying lengths of the human prepro-GRP promoter region. After transfection and subsequent transient expression in various host cell lines, luciferase activity is determined for each construct containing different prepro-GRP promoter elements. Using this assay system, we have made the following observations: 1) Promoter sequences between -5000 and -1600 (the origin is defined as the initiation site for transcription) exert an approximately two-fold negative effect on transcription, while sequences between -1600 and -400 exert a weak positive effect. In this positive domain, a several hundred base long 5' upstream sequence is very highly conserved between the rat and human prepro-GRP gene (-720 to -450); and 2) The constructs behave the same in host cells that do or do not normally express the prepro-GRP gene. We conclude that promoter elements between -5000 and + 100 alone do not determine cell-type specificity of transcription regulation.