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Structure of the gene encoding the human cyclin-dependent kinase inhibitor p18 and mutational analysis in breast
1Laboratory of Molecular Endocrinology, CHUL Research Center, University Hospital of Quebec, Canada.
Abstract:
The cyclin-dependent kinase (CDK) inhibitor p18 blocks progression of the cell cycle by associating with the cyclin D-dependent kinases CDK6 and CDK4. To better understand the regulation of p18 gene expression, we isolated full-length cDNA clones from a human BT-20 breast cancer cell cDNA library. These clones were then used to isolate the human gene from a human genomic DNA library. The human p18 gene spans at least 7.5 kb and is composed of three exons, two of which encode the p18 protein. The genomic clone we isolated contained 5 kb of putative promotor sequence which directed expression of the luciferase reporter gene in transient transfection experiments. The longest cDNA that we isolated from BT-20 cells contained 2103 nucleotides which corresponds to the size of the major RNA transcript detected by Northern analysis in these cells. Transcription start sites mapping to the 5' end of the putative full-length cDNA were identified by ribonuclease protection assays. A novel polymorphism was identified in the 3' untranslated region of BT-20 cell cDNA clones that contained the previously described codon 72 mutation. The codon 72 mutation was also detected in 3 of 35 breast tumors analyzed using a mismatch PCR/RFLP strategy.
Insights
Researchers characterized the human p18 gene, a cyclin-dependent kinase inhibitor, identifying its structure and regulatory elements. They also found a common mutation in breast cancer cells and tumors, aiding in understanding cell cycle control.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- The cyclin-dependent kinase (CDK) inhibitor p18 regulates cell cycle progression by binding to CDK6 and CDK4.
- Understanding p18 gene expression is crucial for insights into cell cycle control and potential therapeutic strategies.
Purpose of the Study:
- To isolate and characterize the human p18 gene, including its regulatory promoter regions.
- To identify the full-length cDNA and major RNA transcript of the p18 gene.
- To investigate genetic variations, such as polymorphisms and mutations, within the p18 gene in breast cancer.
Main Methods:
- Isolation of full-length cDNA clones from a human breast cancer cell line (BT-20).
- Isolation of the human p18 gene from a genomic DNA library.
- Luciferase reporter gene assays to assess promoter activity.
- Northern analysis to determine RNA transcript size.
- Ribonuclease protection assays for transcription start site mapping.
- Mismatch PCR/RFLP for mutation detection.
Main Results:
- The human p18 gene spans at least 7.5 kb and comprises three exons, with two encoding the p18 protein.
- A 5 kb putative promoter region was identified and shown to be functional in directing gene expression.
- The major p18 RNA transcript is 2103 nucleotides long.
- A novel polymorphism in the 3' untranslated region was found, alongside the previously described codon 72 mutation.
- The codon 72 mutation was present in 3 out of 35 analyzed breast tumors.
Conclusions:
- The study provides a comprehensive characterization of the human p18 gene structure and its regulatory promoter sequence.
- Identification of transcription start sites and transcript size enhances understanding of p18 gene expression.
- The presence of the codon 72 mutation in breast tumors suggests its potential role in breast cancer development or progression.