O6-methylguanine-DNA methyltransferase (MGMT) is responsible for repair of the promutagenic and probably procarcinogenic base, O6-alkylguanine, produced in DNA by many alkylating carcinogens as well as antitumor drugs, and is tightly regulated in both bacterial and mammalian cells. Although the E. coli MGMT gene has been cloned and its regulation partially elucidated, little is yet known regarding regulation of the corresponding mammalian genes. The level of MGMT varies widely in different mammalian tissues and cell lines and also in many tumors, in which the susceptibility to certain alkylating drugs is inversely relate to the enzyme level. In order to investigate the molecular mechanism of MGMT regulation in human cells, the human MGMT gene will be cloned from a transformed CHO cell line carrying this gene, and will subsequently be used as a probe for cloning the cDNA. A number of other current approaches to the cloning of human MGMT cDNA will also be pursued, including enrichment by differential hybridization of cDNA's from MGMT+ and MGMT- human cell lines, and the preparation of antibodies to MGMT as probes for use with expression vectors and of synthetic DNA probes based on the N-terminal amino acid sequence. The genomic and cDNA clones will be used for mutual identification. The genomic clone will be physically characterized for organization of the gene including identification of its regulatory sequence and the cDNA will be sequenced for deriving the amino acid sequence of the protein. The cloned human gene and cDNA will also be used (1) for chromosomal mapping of the structural gene and possible regulatory sequences in human/rodent cell hybrids containing segregated human chromosomes, (2) to test whether gene amplification is the basis for drug resistance in cell lines, (3) to determine whether regulation of transcription is responsible for tissue-specific variation in MGMT level, (4) to investigate the mechanism for loss of MGMT activity in many transformed cell lines, including potential methylation of the regulatory sequence, (5) to determine whether MGMT expression is inducible by alkylating agents as well as other general inducers, and (6) to clone the mouse MGMT gene, based on its (assumed) sequence homology, for regulation studies. Preliminary experiments will also be initiated on the cloning of human N- methylpurine-DNA glycosylases by in vivo complementation of E. coli deficient in N-methylpurine repair with a human cDNA library in an E. coli expression vector.
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