Expression of the E.coli 3-methyladenine DNA glycosylase I gene in mammalian cells reduces the toxic and mutagenic effects of methylating agents.

2.50
Hdl Handle:
http://hdl.handle.net/10541/108857
Title:
Expression of the E.coli 3-methyladenine DNA glycosylase I gene in mammalian cells reduces the toxic and mutagenic effects of methylating agents.
Authors:
Klungland, A; Fairbairn, Leslie J; Watson, Amanda J; Margison, Geoffrey P; Seeberg, E
Abstract:
In 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.
Affiliation:
Biotechnology Centre of Oslo, Blindern, Norway.
Citation:
Expression 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.
Journal:
EMBO Journal
Issue Date:
Dec-1992
URI:
http://hdl.handle.net/10541/108857
PubMed ID:
1425578
Type:
Article
Language:
en
ISSN:
0261-4189
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorKlungland, Aen
dc.contributor.authorFairbairn, Leslie Jen
dc.contributor.authorWatson, Amanda Jen
dc.contributor.authorMargison, Geoffrey Pen
dc.contributor.authorSeeberg, Een
dc.date.accessioned2010-08-02T16:16:26Z-
dc.date.available2010-08-02T16:16:26Z-
dc.date.issued1992-12-
dc.identifier.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.en
dc.identifier.issn0261-4189-
dc.identifier.pmid1425578-
dc.identifier.urihttp://hdl.handle.net/10541/108857-
dc.description.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.en
dc.language.isoenen
dc.subject.meshAlkylating Agents-
dc.subject.meshAnimals-
dc.subject.meshBase Sequence-
dc.subject.meshCell Line-
dc.subject.meshCell Survival-
dc.subject.meshCricetinae-
dc.subject.meshCricetulus-
dc.subject.meshDNA Glycosylases-
dc.subject.meshDNA Repair-
dc.subject.meshDNA, Single-Stranded-
dc.subject.meshDrug Resistance-
dc.subject.meshEscherichia coli-
dc.subject.meshGenes, Bacterial-
dc.subject.meshMethyl Methanesulfonate-
dc.subject.meshMethylation-
dc.subject.meshMethylnitrosourea-
dc.subject.meshMolecular Sequence Data-
dc.subject.meshMutagens-
dc.subject.meshN-Glycosyl Hydrolases-
dc.subject.meshPlasmids-
dc.subject.meshTransfection-
dc.titleExpression of the E.coli 3-methyladenine DNA glycosylase I gene in mammalian cells reduces the toxic and mutagenic effects of methylating agents.en
dc.typeArticleen
dc.contributor.departmentBiotechnology Centre of Oslo, Blindern, Norway.en
dc.identifier.journalEMBO Journalen
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