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A promoter function of the CCCGGG Sma I recognition sequence and its specific role in determining p53 status and
1MRC Toxicology Unit, University of Leicester, Lancaster Road, Leicester LE1 9HN, United Kingdom. aly1@le.ac.uk
Abstract:
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.
Insights
Researchers identified the CCCGGG sequence as a transcriptionally active DNA-binding site for the tumor suppressor p53 protein. This finding enables the pGL3-Basic vector to indicate p53 activity and identify p53-regulated genes.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- The tumor suppressor p53 protein is crucial for cellular responses to DNA damage.
- A common in vivo DNA-binding site for p53 has remained unidentified.
- The pGL3-Basic vector is designed for studying regulatory sequences without endogenous promoters.
Purpose of the Study:
- To identify a transcriptionally active DNA-binding site for p53.
- To evaluate the pGL3-Basic vector's potential for assessing p53 activity.
- To explore the CCCGGG sequence as a potential p53 consensus site.
Main Methods:
- Electrophoretic mobility shift assay (EMSA) to assess p53 binding to DNA in vitro.
- Utilizing the pGL3-Basic vector with its CCCGGG sequence to monitor p53-dependent transcription.
- Analyzing the presence of the CCCGGG sequence in known p53-regulated genes.
Main Results:
- The CCCGGG sequence (Sma I site) in the pGL3-Basic vector promoted p53-dependent transcription of the luciferase gene.
- Human p53 demonstrated in vitro binding to the CCCGGG sequence via EMSA.
- The CCCGGG sequence was identified in promoter regions of several p53-regulated genes.
Conclusions:
- The CCCGGG sequence is the first identified transcriptionally active DNA-binding site for p53.
- The pGL3-Basic vector can serve as an indicator of p53 transcriptional activity.
- The CCCGGG sequence is proposed as a consensus site for p53 recognition in vivo, aiding in the identification of p53-controlled genes.