Promoter Region of the Rat m4 Muscarinic Acetylcholine Receptor Gene Contains a Cell Type-specific Silencer Element (*)
Promoter Region of the Rat m4 Muscarinic Acetylcholine Receptor Gene Contains a Cell Type-specific Silencer Element (*)
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大鼠 m4 毒蕈碱乙酰胆碱受体基因的启动子区域包含细胞类型特异性沉默元件 (*)
DOI：
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发表时间：
1996
影响因子：
4.8
通讯作者：
D. Saffen
中科院分区：
文献类型：
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作者：
Michihiro Mieda;T. Haga;D. Saffen
我们描述了大鼠m4毒蕈碱乙酰胆碱受体基因的特征及其调控区域的鉴定。两个5 ' -非编码外显子位于编码外显子上游约5千碱基处，至少两个m4 mRNA的可选剪接变体在神经元细胞系PC12D中表达。有两个转录起始位点。启动子区域富含gc，不含TATA-box，但有两个潜在的CAAT box和几个转录因子Sp1和AP-2的推定结合位点。我们使用荧光素酶作为报告基因，在瞬时表达试验中对m4启动子活性进行了功能性评估。在表达m4的神经元细胞系（PC12D和NG108-15）和非神经元细胞系（L6和3Y1B）中，5 '侧区近435碱基对（bp）序列均产生荧光素酶活性。含有额外638 bp序列的较长片段仅在表达m4的神经细胞系中产生荧光素酶活性。这些数据表明，近435 bp的序列包含一个组成启动子，而更远的上游638 bp的序列包含一个细胞类型特异性沉默元件。在这个638 bp的片段中发现了一个一致的神经限制性沉默元件序列。
We describe here the characterization of the rat m4 muscarinic acetylcholine receptor gene and the identification of its regulatory region. Two 5′-noncoding exons are located approximately 5 kilobases upstream from the coding exon, and at least two alternatively spliced variants of m4 mRNA are expressed in the neuronal cell line PC12D. There are two transcription initiation sites. The promoter region is GC-rich, contains no TATA-box, but has two potential CAAT boxes and several putative binding sites for transcription factors Sp1 and AP-2. We assessed the m4 promoter activity functionally in transient expression assays using luciferase as a reporter. The proximal 435-base pair (bp) sequence of the 5′-flanking region produced luciferase activity in both m4-expressing neuronal cell lines (PC12D and NG108-15) and non-neuronal cell lines (L6 and 3Y1B). A longer fragment containing an additional 638-bp sequence produced luciferase activity only in m4-expressing neuronal cell lines. These data suggest that the proximal 435-bp sequence contains a constitutive promoter and that a 638-bp sequence farther upstream contains a cell type-specific silencer element. A consensus sequence for the neural-restrictive silencer element is found within this 638-bp segment.