Related Experiment Videos
Organization and expression of the human gene for O6-methylguanine-DNA methyltransferase
Y Nakatsu1, K Hattori, H Hayakawa
1Department of Biochemistry, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Abstract:
O6-Methylguanine-DNA methyltransferase plays an important role in cellular defence against mutagens and carcinogens with alkylating activity. Certain tumor-derived cell lines, termed Mer-, are defective in the enzyme activity and have an increased sensitivity to alkylating agents. We cloned the genomic sequence coding for the human O6-methylguanine-DNA methyltransferase and elucidated the structure. The gene consisted of 5 exons and spanned more than 170 kb, while mRNA for the enzyme was 950 nucleotides long. No or only little mRNA for the enzyme was formed in Mer- cells, though there was no gross difference in the coding and promoter regions of the gene between Mer+ and Mer- cells. The putative promoter region, derived from Mer+ cells, was placed upstream of the chloramphenicol acetyltransferase reporter gene and the constructs were introduced into Mer+ and Mer- cells. In Mer- cells, a lowered level of transient expression of the gene was observed as compared with Mer+ cells, but this difference alone does not account for the in vivo difference of expression of the gene in the two types of cells; there might be difference in cis-acting elements. The DNA sequence in the 5' upstream region of the gene was extremely GC-rich and there were no consensus sequences, such as the TATA and CAAT boxes. There were lower levels of methylation in the putative promoter of various Mer- cells, as compared with findings in Mer+ cells. Methylation in this region may be involved in regulating expression of the gene.
Insights
O6-Methylguanine-DNA methyltransferase protects cells from alkylating agents. Defective enzyme activity in Mer- cells correlates with lower gene expression, potentially due to promoter methylation.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- O6-Methylguanine-DNA methyltransferase (MGMT) is crucial for cellular defense against DNA alkylating agents.
- Tumor cell lines with defective MGMT activity (Mer-) exhibit increased sensitivity to these agents.
Purpose of the Study:
- To clone and structurally elucidate the human MGMT gene.
- To investigate the molecular basis for the reduced MGMT expression in Mer- cells.
Main Methods:
- Genomic DNA cloning and sequencing of the human MGMT gene.
- Analysis of gene structure, including exons and mRNA length.
- Reporter gene assays (chloramphenicol acetyltransferase) to assess promoter activity.
- DNA methylation analysis of the promoter region.
Main Results:
- The human MGMT gene comprises 5 exons, spanning over 170 kb with a 950-nucleotide mRNA.
- Mer- cells show significantly reduced MGMT mRNA levels compared to Mer+ cells.
- Reporter gene assays revealed lower transient expression in Mer- cells, suggesting regulatory differences beyond coding/promoter regions.
- The 5' upstream region is GC-rich, lacking TATA/CAAT boxes.
- Mer- cells exhibit lower methylation levels in the putative promoter region compared to Mer+ cells.
Conclusions:
- Reduced MGMT expression in Mer- cells is linked to epigenetic modifications, specifically lower promoter methylation.
- Methylation of the MGMT promoter region likely plays a role in regulating gene expression.
- Potential differences in cis-acting elements may also contribute to expression variability.