Organization and expression of the mouse gene for DNA repair methyltransferase.
06-Methylguanine-DNA methyltransferase (MGMT) is present in various organisms, from bacteria to human cells, and plays an important role in preventing mutations caused by alkylating substances. To understand better the regulatory mechanism involved in the expression of the gene and to construct a mouse model to investigate roles of the enzyme in carcinogenesis, the genomic sequence for mouse methyltransferase was isolated and characterized. The gene consists of 5 exons and spans over 180 kb, whereas mRNA for the enzyme was less than 1 kb. The promoter region for the gene is GC-rich, contains many Sp1 recognition sequences and lacks typical TATA and CCAAT boxes. Primer extension and S1 mapping revealed the existence of multiple transcription initiation sites, among which a major site was defined as +1. The putative promoter region was placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and the construct was introduced into mouse NIH-3T3 cells. Deletion analyses revealed that a sequence from -262 to + 56 carries the basic promoter activity. In addition, an adjacent region, spanning from +56 to +95, carries an E2F-like element that greatly stimulates the frequency of transcription. Alteration of TTTTGGGGC to TTAACGGGC considerably reduced the activity.
Department of Biochemistry, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
T
T
Iwakuma
A
A
Shiraishi
M
M
Fukuhara
H
H
Kawate
M
M
Sekiguchi
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D005091:Exons; D015870:Gene Expression; D006801:Humans; D008780:Methyltransferases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism; D016385:TATA Box; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D015500:Chloramphenicol O-Acetyltransferase