Expression of the E.coli 3-methyladenine DNA glycosylase I gene in mammalian cells reduces the toxic and mutagenic effects of methylating agents.
AffiliationBiotechnology Centre of Oslo, Blindern, Norway.
MetadataShow full item record
AbstractIn order to investigate the importance of 3-methyladenine in cellular sensitivity to chemical methylating agents we have constructed retroviral vectors for the integration and expression of the Escherichia coli tag gene in mammalian cells. The tag gene encodes 3-methyladenine DNA glycosylase-1 which specifically removes 3-alkyladenines from DNA. The constructs were introduced into Chinese hamster V79 cells by liposome mediated transfection or into murine haemopoietic stem cells by cocultivation with a lipofected, virus-packaging cell line. In both cases, stable transfectants were selected for resistance to the antibiotic, G418, conferred by expression of the neo gene carried by the vector. Measurements of 3-methyladenine DNA glycosylase activity in cell extracts showed an up to 10-fold increase in cell lines with stably integrated tag gene sequences. These cell lines were significantly more resistant to the cytotoxic effects of methylmethanesulfonate and N-methyl-N-nitrosourea than their parent cell lines, indicating that 3-methyladenine repair is a limiting factor in cellular resistance to these methylating agents. Furthermore, the mutation frequency induced by methylmethanesulfonate was reduced to 50% of normal by expression of 3-methyladenine I activity in the Chinese hamster cells, indicating that m3A is not only a cytotoxic but also a premutagenic lesion in mammalian cells. It is concluded that an alkylation repair gene function of a type only thought to be present in bacteria can yield a hyperresistant phenotype when transferred to mammalian cells.
CitationExpression of the E.coli 3-methyladenine DNA glycosylase I gene in mammalian cells reduces the toxic and mutagenic effects of methylating agents. 1992, 11 (12):4439-44 EMBO J.
- Increased removal of 3-alkyladenine reduces the frequencies of hprt mutations induced by methyl- and ethylmethanesulfonate in Chinese hamster fibroblast cells.
- Authors: Klungland A, Bjørås M, Hoff E, Seeberg E
- Issue date: 1994 May 11
- Spectrum of mutations induced by methyl and ethyl methanesulfonate at the hprt locus of normal and tag expressing Chinese hamster fibroblasts.
- Authors: Klungland A, Laake K, Hoff E, Seeberg E
- Issue date: 1995 Jun
- Increased resistance of the Chinese hamster mutant irs1 cells to monofunctional alkylating agents by transfection of the E. coli or mammalian N3-methyladenine-DNA-glycosylase genes.
- Authors: Habraken Y, Laval F
- Issue date: 1993 Mar
- Effect of overexpression of E. coli 3-methyladenine-DNA glycosylase I (Tag) on survival and mutation induction in Salmonella typhimurium.
- Authors: Tomicic M, Franekic J
- Issue date: 1996 Oct 28
- 3T3 NIH murine fibroblasts and B78 murine melanoma cells expressing the Escherichia coli N3-methyladenine-DNA glycosylase I do not become resistant to alkylating agents.
- Authors: Imperatori L, Damia G, Taverna P, Garattini E, Citti L, Boldrini L, D'Incalci M
- Issue date: 1994 Mar