Response of REV3 promoter to N-methyl-N'-nitro-N-nitrosoguanidine.
Previously, we have shown that low concentration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) led to the upregulation of REV3 gene at transcriptional level in cultured human amnion FL cells. In this study, using bioinformatic analysis the putative binding sites for different transcription factors were found to exist in REV3 gene promoter region. A 2570-bp fragment of the 5' flanking region of REV3 gene was amplified by PCR from PAC clone RP3-415N12 and inserted into the pGL3-Basic reporter vector. Dual-luciferase reporter assay demonstrated that the reconstructed plasmid did respond to MNNG exposure in transfected FL cells. Several variants of the reporter plasmids with different deletions of the REV3 promoter region were also constructed and their promoter strength was analyzed. It was found that the MNNG response element might locate at the REV3 gene promoter region -404 to -102 between two Sma1 sites. The shortest responsive fragment containing the putative binding sites for transcription factors CREBP, AP-2, NF-kappaB, and SP1 was also identified.
Yu
Yingnian
Y
Department of Pathology and Pathophysiology, Center for Environmental Genomics, Zhejiang University School of Medicine, 353 Yan An Road, Hangzhou, Zhejiang 310031, China. ynyu@hzcnc.com
Yang
Jun
J
Zhu
Feng
F
Xu
Fang
F
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D019295:Computational Biology; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D017353:Gene Deletion; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008769:Methylnitronitrosoguanidine; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009153:Mutagens; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012984:Software; D013997:Time Factors; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D009153:Mutagens; D008769:Methylnitronitrosoguanidine; D008156:Luciferases; D004259:DNA-Directed DNA Polymerase; C118285:REV3L protein, human