A promoter function of the CCCGGG Sma I recognition sequence and its specific role in determining p53 status and identifying DNA damaging agents.
The tumour suppressor p53 protein regulates many genes involved in cellular responses to DNA damage. To date, a common transcriptionally active DNA-binding site for p53 in vivo has not been identified. The pGL3-Basic vector contains a modified fire-fly luciferase cDNA designated luc+ and is designed for studying putative regulatory sequences as it lacks any known eukaryotic promoter sequences. We report here that the CCCGGG sequence, a Sma I site, in the cloning region of the pGL3-Basic vector can promote p53-dependent transcription of the luc+ gene. We have demonstrated, by electrophoretic mobility shift assay (EMSA), that human p53 is able to bind to the CCCGGG sequence in vitro. These data provide the first demonstration that the CCCGGG sequence is a transcriptionally active DNA-binding site for p53. Thus, the pGL3-Basic vector could be used as an indicator of p53 transcriptional activity, to determine the p53 status of cell lines and to identify DNA damaging agents that initiate the activation of p53. The CCCGGG sequence has been found to be present in a number of promoter regions of p53-regulated genes. This and the present study suggest that the CCCGGG sequence may be a consensus sequence recognized by p53 in vivo and may be used to identify genes whose expression may be controlled by p53.
Yang
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AL
MRC Toxicology Unit, University of Leicester, Lancaster Road, Leicester LE1 9HN, United Kingdom. aly1@le.ac.uk
Festing
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MF
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D004247:DNA / Q000187:drug effects* / Q000235:genetics; D004249:DNA Damage; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D000903:Antibiotics, Antineoplastic; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D004317:Doxorubicin; D004247:DNA; D008156:Luciferases; D004262:DNA Restriction Enzymes