Clustered point mutation analysis of the rat prolactin promoter.
Clustered point mutation analysis of the rat prolactin promoter.
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DOI：
10.1210/mend-4-10-1564
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发表时间：
1990-10
影响因子：
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通讯作者：
Ronald A. Iverson;Kathleen H. Day;Michael C. D'Emden;R. N. Day;R. A. Maurer
中科院分区：
文献类型：
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作者：
Ronald A. Iverson;Kathleen H. Day;Michael C. D'Emden;R. N. Day;R. A. Maurer
为了确定对大鼠PRL基因的基础转录和激素刺激转录重要的DNA区域，在紧邻的5‘侧翼区域准备了一系列聚集点突变。将代表PRL基因野生型和19种不同接头扫描器突变的DNA片段分别连接到一个荧光素酶标记基因，并通过电穿孔将这些DNA构建物转入GH3垂体瘤细胞。分别用0.5 mM氯苯硫代cAMP、100 nM TRH和100 nM佛波醇醋酸酯处理对照细胞24 h后测定荧光素酶活性。单个聚集点突变覆盖了从TATA盒上游(位置-30)到转录开始处上游193个碱基的区域。检测到五个区域的突变导致标记基因的基础和cAMP、TRH或佛波酯刺激的表达显著降低。其中三个区域(位置-41到-58，-113到-124，和-149到-156)对应于先前发现的垂体特异的同源框蛋白Pit-1/GHF-1的结合位点。第四和第五个区域不对应于Pit-1/GHF-1结合位点，可能代表一个未知因子的结合位点。在这些区域内，已经检测到与共识cAMP反应元件和AP-2结合位点具有一定相似性的序列。这些数据证实了Pit-1/GHF-1作为PRL基因转录的关键因子的重要性。此外，研究结果还表明，PRL基因的高效表达可能需要额外的转录因子。
To identify DNA regions important for basal and hormone-stimulated transcription of the rat PRL gene, a series of clustered point mutations were prepared within the immediate 5' flanking region. DNA fragments representing the wild-type and 19 different linker-scanner mutations of the PRL gene were each linked to a luciferase marker gene, and the DNA constructs were transferred into GH3 pituitary tumor cells by electroporation. Luciferase activity was determined 24 h after transfection in extracts from control cells or cells treated with 0.5 mM chlorophenylthio-cAMP, 100 nM TRH, or 100 nM phorbol myristate acetate. The individual clustered point mutations covered a region from just up-stream of the TATA box (position -30) to a position 193 basepairs up-stream from the start of transcription. Five regions in which mutations produced substantial decreases in both basal and cAMP-, TRH-, or phorbol ester-stimulated expression of the marker gene were detected. Three of these regions (positions -41 to -58, -113 to -124, and -149 to -156) correspond to previously identified binding sites for the pituitary-specific, homeobox protein, Pit-1/GHF-1. The fourth and fifth regions do not correspond to Pit-1/GHF-1-binding sites and presumably represent sites for an unidentified factor. Within these regions, sequences with some similarity to a consensus cAMP response element and an AP-2-binding site have been detected. These data confirm the importance of Pit-1/GHF-1 as a key factor in PRL gene transcription. In addition, the results suggest that additional transcription factors are probably required for efficient expression of the PRL gene.(ABSTRACT TRUNCATED AT 250 WORDS)