【摘要】 目的 :了解人 REV3基因对化学致癌物 N-甲基 - N’-硝基 - N-亚硝基胍 (MNNG)诱发转录上调的机制。方法 :根据 BL AST序列比对、启动子和转录因子结合部位预测软件 ,对人 REV3基因促进子区进行生物信息学分析 ;用巢式PCR技术克隆了人 REV3基因启动子区 ;用瞬时转染二重荧光素酶报道基因系统检测人 REV3启动子对化学致癌物MNNG的反应。结果 :生物信息学分析显示 ,人 REV3基因启动子区位于 6号染色体 PAC克隆 RP3- 415 N12 ,假设的启动子区有启动子序列、富含 Cp G岛和包括 AP- 1/ c- Jun/ c- Fos、AP- 2、STAT、CREBP和 NF- κB等的转录因子识别部位。将启动子区 2 5 82 bp的片段插入荧光素酶报道基因载体 p GL3- Basic中 ,构建了重组体质粒 p GL3- 2 5 82。瞬时转染检测显示 ,该假设的启动子区确有启动子功能 ,对 MNNG发生反应。结论 :成功地克隆了人 REV3基因启动子区 ,并证明其对 MNNG发生反应。提示当细胞处于基因毒应激状态下可在转录水平调节 REV3基因。更多还原

【Abstract】 Objective: To understand the up retulatory mechanism of human REV3 gene induced by the chemical carcinogen N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). Methods: Bioinformatic analysis of human REV3 gene promoter region was based on BLAST alignment, promoter prediction software and recognition of transcriptional factor binding sites. Cloning of human REV3 gene promoter region was performed by nested PCR. Response of human REV3 gene promoter to the chemical carcinogen MNNG was measured by transient transfection assay based on the dual luciferase reporter assay system. Results: Bioinformatic analysis showed that human REV3 gene promoter region was located on chromosome 6 PAC clone RP3-415N12, and that the hypothetical promoter region contained promoter sequences, rich CpG islands, and putative recognition sites for several transcriptional factors, including AP-1/c-Jun/c-Fos, AP-2, STAT, CREBP, and NF-κB. Reconstructed reporter plasmid pGL3-2582 was established by inserting 2582 nucleotides from the promoter region into the luciferase reporter vector pGL3-Basic. Transient transfection assay showed the hypothetical REV3 promoter region had promoter function, and it responded to MNNG treatment (P<0.01). Conclusion: Human mutator REV3 gene promoter region has been successfully cloned. The response of REV3 promoter region to MNNG suggests that REV3 gene can be regulated at transcriptional level under conditions of genotoxic stress.更多还原