O6-methylguanine-DNA methyltransferase (MGMT)is a ubiquitous protein responsible for repair of O6-alkylguanine, a mutagenic, carcinogenic and toxic lesion. To characterize the elements responsible for the regulation of the MGMT gene, a 2.6 kb Sstl fragment isolated from a genomic clone, was shown to contain 5′ flanking sequences of the gene. The promoter activity of this fragment as well as various subfragments were tested in NIH 3T3 mouse fibroblasts by transient expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to these fragments. Maximal promoter activity was observed in a 1.2 kb 3′ terminal fragment, which contains the first untranslated exon. The transcription initiation site was identified in this fragment by primer extension and S1 mapping. Sequence analysis of this fragment showed the absence of TATA and CAAT boxes but an abundance of extremely GC-rich sequences, including ten GC hexanucleotide motifs 5′CCGCCC. Reduced CAT expression with the minimal promoter sequence suggests the presence of multiple regulatory elements.
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