Boukouvala Sotiria, Price Naomi, Plant Kathryn E, Sim Edith
University of Oxford, Department of Pharmacology, Mansfield Road, Oxford OX1 3QT, UK.
Biochem J. 2003 Nov 1;375(Pt 3):593-602. doi: 10.1042/BJ20030812.
Arylamine N-acetyltransferases (NATs) are polymorphic enzymes, well-known for their role in the metabolism of drugs and carcinogens. Mice have three NAT isoenzymes, of which NAT2 is postulated to be involved in endogenous, as well as xenobiotic, metabolism. To understand expression of the murine Nat2 gene, we have analysed its structure and transcriptional regulation. We have accurately mapped the transcription initiation site 6.5 kb upstream of the coding region of the gene, adjacent to a recently described non-coding exon. Transcription was demonstrated to start from this region in embryonic and adult liver, spleen, submaxillary gland, kidney, brain, thymus, lung and placenta, but not in the heart. Database searches and analyses of cDNA by PCR suggested alternative splicing of the single 6.2 kb intron of Nat2, and determined the position of the polyadenylation signal at 0.44 kb downstream of the coding region of the gene. Examination of the 13 kb sequence flanking the coding and non-coding exons of Nat2 revealed a single promoter, located close to the transcription-initiation site, and indicated regions likely to harbour control elements. The Nat2 promoter consists of an atypical TATA box and a Sp1 [SV40 (simian virus 40) protein 1] box identical with that found in many housekeeping gene promoters. Activity of the Nat2 promoter was severely reduced by deletion or mutation of either of these two elements, whereas the region of the Sp1 box bound cellular protein and resisted DNase I digestion. Finally, the ability of the promoter region to bind cellular protein was reduced by competition with oligonucleotides bearing the Sp1 consensus sequence.
芳胺N - 乙酰基转移酶（NATs）是多态性酶，因其在药物和致癌物代谢中的作用而闻名。小鼠有三种NAT同工酶，其中NAT2被认为参与内源性以及外源性物质的代谢。为了解小鼠Nat2基因的表达情况，我们分析了其结构和转录调控。我们已精确确定该基因编码区上游6.5 kb处的转录起始位点，该位点紧邻最近描述的一个非编码外显子。已证明在胚胎和成年肝脏、脾脏、颌下腺、肾脏、大脑、胸腺、肺和胎盘中，转录从该区域起始，但在心脏中未起始。数据库搜索和通过PCR对cDNA的分析表明Nat2的单个6.2 kb内含子存在可变剪接，并确定了多聚腺苷酸化信号在该基因编码区下游0.44 kb处的位置。对Nat2编码和非编码外显子两侧13 kb序列的检查揭示了一个位于转录起始位点附近的单一启动子，并指出了可能含有调控元件的区域。Nat2启动子由一个非典型的TATA盒和一个与许多管家基因启动子中发现的相同的Sp1 [猴病毒40（SV40）蛋白1]盒组成。这两个元件中的任何一个发生缺失或突变都会使Nat2启动子的活性严重降低，而Sp1盒区域能结合细胞蛋白并抵抗DNA酶I消化。最后，通过与带有Sp1共有序列的寡核苷酸竞争，启动子区域结合细胞蛋白的能力降低。
