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首页> 外文期刊>Molecular and Cellular Biology >Analysis by cell-free transcription of the liver-specific pyruvate kinase gene promoter.
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Analysis by cell-free transcription of the liver-specific pyruvate kinase gene promoter.

机译：肝特异性丙酮酸激酶基因启动子的无细胞转录分析。

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摘要

A DNA fragment spanning nucleotides -183 to -4 with respect to the cap site of the rat L-type pyruvate kinase (L-PK) gene contains at least four binding sites for putative transcriptional factors: hepatocyte nuclear factor 1 (HNF1), liver factor A1 (LF-A1), nuclear factor 1 (NF1), and major late transcription factor (MLTF). This fragment was used to direct transcription of a reporter sequence (a G-free cassette) in cell extracts. This L-PK promoter was active in liver nuclear extracts, but not in extracts from nonhepatic tissues. A reduction of 50% of the activity was obtained with a deleted L-PK promoter containing only the HNF1-binding site. In contrast, deletion of the HNF1-binding site inactivated the promoter by more than 90%. These results were confirmed by titration experiments with synthetic oligonucleotides. Titration of HNF1 resulted in an 85% decrease of transcriptional activity, while titration of LF-A1 resulted in only a 40% decrease. The influence of NF1 and MLTF seemed to be marginal in this system. The proximal 5'-flanking sequence of the L-PK gene therefore appears to function in vitro as an efficient liver-specific promoter which requires the binding of the liver factor HNF1 and which is also stimulated by the binding of another liver-specific factor, LF-A1.
机译：相对于大鼠L型丙酮酸激酶（L-PK）基因帽位的核苷酸-183至-4的DNA片段至少包含四个假定的转录因子结合位点：肝细胞核因子1（HNF1），肝因子A1（LF-A1），核因子1（NF1）和主要晚期转录因子（MLTF）。该片段用于指导细胞提取物中报道序列（无G盒）的转录。该L-PK启动子在肝核提取物中有活性，但在非肝组织的提取物中没有活性。用仅含有HNF1结合位点的缺失的L-PK启动子，活性降低了50％。相反，HNF1结合位点的缺失使启动子失活了90％以上。这些结果通过合成寡核苷酸的滴定实验得到证实。滴定HNF1导致转录活性降低85％，而滴定LF-A1仅导致降低40％。在该系统中，NF1和MLTF的影响似乎很小。因此，L-PK基因的近端5'侧翼序列似乎在体外起着有效的肝特异性启动子的作用，该启动子需要结合肝因子HNF1，并且还受到另一种肝特异性因子的结合的刺激， LF-A1。

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