Organization and expression of the human gene for O6-methylguanine-DNA methyltransferase.
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.
Nakatsu
Y
Y
Department of Biochemistry, Faculty of Medicine, Kyushu University, Fukuoka, Japan.
Hattori
K
K
Hayakawa
H
H
Shimizu
K
K
Sekiguchi
M
M
D001483:Base Sequence; D003001:Cloning, Molecular; D004260:DNA Repair; D005091:Exons; D005796:Genes; D006801:Humans; D066298:In Vitro Techniques; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D016415:Sequence Alignment; D014162:Transfection; D014407:Tumor Cells, Cultured
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase