CHARACTERIZATION OF THE PROMOTER REGION OF THE HUMAN O6-METHYLGUANINE-DNA METHYLTRANSFERASE GENE
CHARACTERIZATION OF THE PROMOTER REGION OF THE HUMAN O6-METHYLGUANINE-DNA METHYLTRANSFERASE GENE
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DOI：
10.1093/nar/19.22.6163
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发表时间：
1991-11-25
影响因子：
14.9
通讯作者：
BRENT, TP
中科院分区：
文献类型：
--
作者：
HARRIS, LC;POTTER, PM;BRENT, TP
O 6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）是一种普遍存在的蛋白质，负责修复O 6-烷基鸟嘌呤，这是一种致突变、致癌和毒性损伤。为了表征负责调节MGMT基因的元件，从基因组克隆分离的2.6kb SstI片段显示含有该基因的5'侧翼序列。在NIH 3 T3小鼠成纤维细胞中通过瞬时表达与这些片段连接的细菌氯霉素乙酰转移酶（CAT）基因来测试该片段以及各种亚片段的启动子活性。在含有第一个非翻译外显子的1.2kb 3'端片段中观察到最大启动子活性。通过引物延伸和S1作图，确定了该片段的转录起始位点。序列分析表明，该片段不含TATA和CAAT盒，但含有大量富含GC的序列，包括10个GC六核苷酸基序5 'CCGCCC。最小启动子序列的CAT表达减少表明存在多种调控元件。
O6-methylguanine-DNA methyltransferase (MGMT)is a ubiquitous protein responsible for repair of O6-alkylguanine, a mutagenic, carcinogenic and toxic lesion. To characterize the elements responsible for the regulation of the MGMT gene, a 2.6 kb Sstl fragment isolated from a genomic clone, was shown to contain 5' flanking sequences of the gene. The promoter activity of this fragment as well as various subfragments were tested in NIH 3T3 mouse fibroblasts by transient expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to these fragments. Maximal promoter activity was observed in a 1.2 kb 3' terminal fragment, which contains the first untranslated exon. The transcription initiation site was identified in this fragment by primer extension and S1 mapping. Sequence analysis of this fragment showed the absence of TATA and CAAT boxes but an abundance of extremely GC-rich sequences, including ten GC hexanucleotide motifs 5'CCGCCC. Reduced CAT expression with the minimal promoter sequence suggests the presence of multiple regulatory elements.