DNA-mediated transfer of complex I genes into three different respiration-deficient Chinese hamster mutant cell lines with defects in complex I of electron transport chain
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We have used genomic DNA from human or mouse cells as a calcium phosphate precipitate to transfect three different respiration-deficient Chinese hamster mutant cell lines with defects in complex I of the electron transport chain. Transformants were selected in DMEM containing galactose, a medium in which respiration-deficient cells do not grow. Evidence for the DNA-mediated transformation of these respiration-deficient cells with a putative complex I gene includes: (1) the clones are respiration-positive and respire at rates comparable to those of wild-type human, hamster, or mouse cells; (2) the clones have rotenone-sensitive NADH oxidase activities, indicating a functional complex I of the electron transport chain; and (3) the clones appear to be true transformants, as demonstrated by hybridization and Southern blot analyses. These experiments provide the basis for the isolation and subsequent characterization of several of the genes involved with complex I of the mammalian electron transport chain.
KeywordsCalcium Blot Analysis Galactose Southern Blot Oxidase Activity
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