Isolation and partial characterisation of a Chinese hamster O6-alkylguanine-DNA alkyltransferase cDNA.
AuthorsRafferty, Joseph A
Elder, Rhoderick H
Watson, Amanda J
Potter, P M
Margison, Geoffrey P
AffiliationCRC Department of Chemical Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK.
MetadataShow full item record
AbstractThe cDNA encoding Chinese hamster O6-alkylguanine-DNA-alkyltransferase (ATase) has been isolated from a library prepared from RNA isolated from V79 lung fibroblasts which had an upregulated level of this repair activity following stepwise selection with a chloroethylating agent (1, 2). Expression of the cDNA in E. coli produced functionally active ATase at levels of 2.5% of total cellular protein as determined by in vitro assay. The recombinant hamster protein has a molecular weight of 28 kDa as estimated by SDS-PAGE and fluorography and this was identical to that in the upregulated cells. The characteristic PCHRV pentapeptide of the alkyl acceptor site has been identified and there is a 68 amino acid residue region which is 90% conserved across all the mammalian proteins so far analysed: in contrast, the N- and C-terminal domains diverge by as much as 50% between species. Polyclonal antibodies to the human and rat ATases hybridised to the hamster protein on western analysis suggesting at least one common epitope shared across species. However, in antibody inhibition experiments neither of the antisera cross reacted with the hamster ATase in a way which interfered with functional activity whereas the anti-human antibodies inhibited the human ATase and the anti-rat antibodies inhibited the rat and mouse ATases. There may therefore be significant tertiary structural differences between the hamster protein and the other mammalian ATases.
CitationIsolation and partial characterisation of a Chinese hamster O6-alkylguanine-DNA alkyltransferase cDNA. 1992, 20 (8):1891-5 Nucleic Acids Res.
JournalNucleic Acids Research
- Isolation and partial characterization of murine O6-alkylguanine-DNA-alkyltransferase: comparative sequence and structural properties.
- Authors: Santibanez-Koref M, Elder RH, Fan CY, Cawkwell L, McKie JH, Douglas KT, Margison GP, Rafferty JA
- Issue date: 1992
- Expression in mammalian cells of the Escherichia coli O6 alkylguanine-DNA-alkyltransferase gene ogt reduces the toxicity of alkylnitrosoureas.
- Authors: Harris LC, Margison GP
- Issue date: 1993 Jun
- Upregulation of O6-alkylguanine-DNA-alkyltransferase expression and the presence of double minute chromosomes in alkylating agent selected Chinese hamster cells.
- Authors: Morten JE, Bayley L, Watson AJ, Ward TH, Potter PM, Rafferty JA, Margison GP
- Issue date: 1992 Mar
- Mammalian cells expressing Escherichia coli O6-alkylguanine-DNA alkyltransferases are hypersensitive to dibromoalkanes.
- Authors: Abril N, Margison GP
- Issue date: 1999 Jun
- Characterisation and nucleotide sequence of ogt, the O6-alkylguanine-DNA-alkyltransferase gene of E. coli.
- Authors: Potter PM, Wilkinson MC, Fitton J, Carr FJ, Brennand J, Cooper DP, Margison GP
- Issue date: 1987 Nov 25