cDNA cloning and chromosomal assignment of the human O6-methylguanine-DNA methyltransferase. cDNA expression in Escherichia coli and gene expression in human cells.
The O6-methylguanine-DNA methyltransferases are the most common form of cellular defense against the biological effects of O6-methylguanine in DNA. By screening a cDNA library with oligonucleotide probes derived from the active site amino acid sequence of the bovine methyltransferase, we have isolated a cDNA clone for the human enzyme. The cDNA contains a single open reading frame encoding a protein of Mr 21,700 which exhibits considerable homology to three bacterial methyltransferases. When provided with an Escherichia coli lac promoter, the encoded polypeptide can be expressed in E. coli to produce an active methyltransferase which is indistinguishable in size from the protein from human cells. The enzyme expressed in this way is functional in vivo and protects an E. coli methyltransferase deletion mutant against the mutational and cytotoxic properties of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. The methyltransferase gene spans at least 15 kilobases and is located on human chromosome 10. Alkylating agent-resistant Mex+ cells which express the methyltransferase protein contain a methyltransferase mRNA of about 1 kilobase. However, this mRNA is absent from three alkylation sensitive Mex- human cell lines indicating that the regulation of methyltransferase gene expression in these cell lines may be transcriptional.
Imperial Cancer Research Fund, South Mimms, Hertfordshire, United Kingdom.
B
B
Rydberg
N
N
Spurr
P
P
Karran
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002874:Chromosome Mapping; D002879:Chromosomes, Human, Pair 10; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D015342:DNA Probes; D015246:Deoxyribonuclease EcoRI; D015247:Deoxyribonuclease HindIII; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008780:Methyltransferases / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009693:Nucleic Acid Hybridization; D019853:O(6)-Methylguanine-DNA Methyltransferase; D012689:Sequence Homology, Nucleic Acid
D015342:DNA Probes; D004247:DNA; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D015246:Deoxyribonuclease EcoRI; D015247:Deoxyribonuclease HindIII