HUMAN NAD(P)H - QUINONE OXIDOREDUCTASE (NQO1) GENE STRUCTURE AND INDUCTION BY DIOXIN
HUMAN NAD(P)H - QUINONE OXIDOREDUCTASE (NQO1) GENE STRUCTURE AND INDUCTION BY DIOXIN
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DOI：
10.1021/bi00108a007
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发表时间：
1991-11-05
期刊：
影响因子：
2.9
通讯作者：
JAISWAL, AK
中科院分区：
文献类型：
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作者：
JAISWAL, AK
已测定了人NAD（P）H：醌氧化还原酶（NQO 1）基因5'侧翼区的1850个碱基对（bp）和3'侧翼区的67个碱基对。人NQO 1基因长约20 kb，有6个外显子，被5个内含子中断。通过引物延伸分析确定转录起始位点。第一个外显子长118 bp，编码两个氨基酸，包括起始甲硫氨酸和第二个外显子的第一个密码子的一个G。第六外显子是所有外显子中最大的一个，长度为1833 bp。第六外显子的序列分析显示存在四个潜在的多聚腺苷酸化信号序列（AATAAA）和一个单拷贝的人Alu重复序列。第二个内含子是所有内含子中最小的（116 bp）。用从2，3，7，8-四氯二苯并-对-二恶英（TCDD）处理和未处理的人肝母细胞瘤（Hep-G2）细胞中分离的细胞核进行的核连续实验表明，TCDD处理使内源性NQO 1基因的转录速率增加3倍。对NQO 1基因5 ′侧翼区的序列分析表明，在-37 ~-32 bp区域存在一个TATA盒，在-649位存在一个CCAAT盒，在-462位存在一个AP 1结合位点，在-156位存在一个AP 2位点，和一个拷贝的核苷酸序列GCGTG（在位置-740）类似于在异生素/二恶英反应元件中发现的存在于细胞色素P-4501 A1（CYP 1A 1）基因中的XRE和DRE，已知与Ah受体-诱导物复合物结合，并增加CYP 1A 1基因对多环芳烃（PAH）和TCDD的表达。序列分析还显示，在人NQO 1基因的残基-467和-447之间存在一个抗氧化反应元件（ARE）拷贝。ARE序列最近已被确定在谷胱甘肽S-转移酶基因和大鼠NAD（P）H：醌还原酶基因的ya亚基的调控区，并显示介导的基础表达和其诱导响应平面芳香族化合物和酚类抗氧化剂。值得注意的是，与大鼠醌还原酶基因中ARE的3'位置相比，人NQO 1基因中的XRE位于ARE的5'。在大鼠醌还原酶基因中表征的ARE的核苷酸序列在人NQO 1基因中高度保守。有趣的是，与AP 1蛋白结合的共有序列（TGACTCA）包含在人NQO 1基因的ARE序列（TCACAGTGACTCAGCAGAATC）中。NQO 1基因5'端侧翼区1850 bp（XRE和ARE各1个拷贝）与CAT基因连接后，转染人肝母细胞瘤（Hep-G2）和小鼠肝癌（Hepa-1）细胞，经TCDD处理后，CAT基因的表达增加。通过缺失突变和转染研究，我们已经确定了人NQO 1基因上游区域（位置-780和-365之间）的DNA片段，其在肝癌细胞中高水平表达所需的和TCDD诱导的。
The human NAD(P)H:quinone oxidoreductase (NQO1) gene, 1850 base pairs (bp) of the 5' flanking region, and 67 bp of the 3' flanking region have been sequenced. The human NQO1 gene is approximately 20 kb in length and has six exons interrupted by five introns. The start site of transcription was determined by primer extension analysis. The first exon is 118 bp in length and codes for two amino acids including the initiating methionine and one G for the first codon of the second exon. The sixth exon is the largest among the exons and is 1833 bp in length. The sequence analysis of the sixth exon revealed the presence of four potential polyadenylation signal sequences (AATAAA) and a single copy of human Alu repetitive sequence. The second intron is the smallest of all the introns (116 bp). Nuclear run-on experiments performed using nuclei isolated from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated and untreated human hepatoblastoma (Hep-G2) cells demonstrated that TCDD treatment increases the rate of transcription of endogenous NQO1 gene by 3-fold. The sequence analysis of the 5' flanking region of NQO1 gene showed the presence of a TATA box in the -37 to -32 bp region, one CCAAT box at nucleotide -649, an AP1 binding site at position -462, an AP2 site at nucleotide position -156, and one copy of the nucleotide sequence GCGTG (at position -740) similar to that found in the xenobiotic/dioxin response elements (XREs and DREs) present in the cytochrome P-4501A1 (CYP1A1) gene and known for binding to the Ah receptor-inducer complex and increasing the expression of CYP1A1 gene in response to polycyclic aromatic hydrocarbons (PAHs) and TCDD. The sequence analysis also showed the presence of one copy of an antioxidant response element (ARE) between residues -467 and -447 of the human NQO1 gene. The ARE sequence has recently been identified in the regulatory region of ya subunit of glutathione S-transferase gene and rat NAD(P)H:quinone reductase gene and shown to mediate the basal expression and its induction in response to planar aromatic compounds and phenolic antioxidants. It is noteworthy that XRE in human NQO1 gene is located 5' to the ARE compared to its 3' location in the rat quinone reductase gene. The nucleotide sequence of the ARE characterized in the rat quinone reductase gene is highly conserved in the human NQO1 gene. Interestingly, the consensus sequence for binding to AP1 protein (TGACTCA) is contained within the ARE sequence (TCACAGTGACTCAGCAGAATC) of the human NQO1 gene. The 1850 bp of 5' flanking region of the NQO1 gene containing one copy each of the XRE and the ARE, when attached to the CAT gene and transfected into human hepatoblastoma (Hep-G2) and mouse hepatoma (Hepa-1) cells, increased the expression of CAT gene upon treatment with TCDD. By deletion mutagenesis and transfection studies, we have identified a segment of DNA in the upstream region of the human NQO1 gene (between positions -780 and -365) required for a high level of expression in hepatoma cells and its induction by TCDD.