Dissection of the mouse N-ras gene upstream regulatory sequences and identification of the promoter and a negative regulatory element.
Dissection of the mouse N-ras gene upstream regulatory sequences and identification of the promoter and a negative regulatory element.
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解剖小鼠 N-ras 基因上游调控序列并鉴定启动子和负调控元件。
DOI：
10.1128/mcb.11.3.1334-1343.1991
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发表时间：
1991
影响因子：
5.3
通讯作者：
Pellicer,A
中科院分区：
文献类型：
--
作者：
Paciucci,R;Pellicer,A
研究小鼠N-ras基因的5'侧翼区以确定控制该基因转录活性的元件。该启动子不含典型的TATA或CCAAT盒，并且根据引物延伸和RNA酶保护分析，转录起始于几个位点。这些测定也证实了插入在N-转录单位和先前描述的上游unrgene之间的短核苷酸距离。用DNA酶I消化细胞核进行染色质研究，发现存在4个超敏感位点：a、B、c和d。5'侧翼区的缺失诱变揭示了负责促进和抑制转录的序列。这些序列位于转录起始位点上游230 bp内。超敏位点B与具有启动子活性的76 bp片段共定位。在位置-180处的负调控元件与超敏位点a共定位，在稳定和瞬时测定中在N-ras启动子上是活性的，并且下调异源单纯疱疹病毒胸苷激酶启动子。足迹分析和体内转染竞争实验表明，atrans-acting factors对转录的负作用是由atrans-acting factors引起的。膜作用负调控元件和启动子区域之间的相互作用可能在N-ras基因的组织和发育阶段特异性表达模式中发挥作用。
The 5' flanking region of the mouse N-rasgene was investigated to determine the elements governing transcriptional activity of the gene. The promoter did not contain typical TATA or CCAAT boxes, and according to primer extension and RNase protection analyses, transcription started at several sites. These assays also confirmed the short nucleotide distance interposed between the N-rastranscription unit and the previously described upstreamunrgene. Chromatin studies performed by digestion of nuclei with DNase I revealed the presence of four hypersensitive sites: a, b, c, and d. Deletion mutagenesis of the 5' flanking region revealed sequences responsible for both promotion and inhibition of transcription. These sequences resided within 230 bp upstream of the transcription initiation site. Hypersensitive site b colocalized with the 76-bp segment with promoter activity. The negative regulatory element at position —180 colocalized with hypersensitive site a, was active on the N-raspromoter in stable as well as transient assays, and down-regulated the heterologous herpes simplex virus thymidine kinase promoter. Footprint analysis and in vivo transfectioncompetition experiments indicated that atrans-acting factor is responsible for the negative effect on transcription. The interaction between thecis-acting negative regulatory element and the promoter region may play a role in the tissue- and developmental-stage-specific patterns of expression of the N-rasgene.