Transcription stimulates homologous recombination in mammalian cells.
Transcription stimulates homologous recombination in Saccharomyces cerevisiae and has been implicated in the control of recombinational events during the development of mammalian immune systems. Here, we describe a plasmid-based system in which an inducible promoter from the mouse mammary tumor virus is located upstream of heteroallelic neomycin genes carried on two plasmids. Pairs of plasmids are introduced into Chinese hamster ovary cells by electroporation, and recombination is monitored by scoring colonies resistant to the aminoglycoside G418. When transcription is induced with dexamethasone, a synthetic glucocorticoid hormone, and double-strand breaks are introduced at mutation sites, recombination is stimulated sixfold over noninduced levels. Inducing transcription in circular substrates or in substrates cleaved at sites distant from the mutations has no detectable effect on recombination between neomycin genes. Results are presented that are consistent with the observed stimulation of recombination occurring before plasmids integrate into the cellular DNA. Our results are discussed in relation to molecular models for extrachromosomal recombination in mammalian cells.
Nickoloff
J A
JA
Life Sciences Division, Los Alamos National Laboratory, New Mexico 87545.
Reynolds
R J
RJ
D000483:Alleles; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D003907:Dexamethasone / Q000494:pharmacology; D008324:Mammary Tumor Virus, Mouse / Q000187:drug effects / Q000235:genetics; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D015183:Restriction Mapping; D013539:Simian virus 40 / Q000187:drug effects / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D003907:Dexamethasone