Effect of concentration on the subsequent fate of plasmid DNA in human fibroblasts

Abstract

The physical fate of plasmid DNA after entry into human fibroblasts was studied using Southern hybridisation and electron microscopy. Exposure of the cells (5 X 10(5) per well) to pC194 DNA-CaPi, containing 50 micrograms plasmid DNA, resulted in the occasional formation of interlocked molecules. Exposure to a co-precipitate containing 100 micrograms pC194 plasmid DNA per well resulted in an increase of interlocked molecules by a factor of 10-20 relative to the number of monomers. In addition, new classes of molecules were observed. After prolonged incubation of the cells exposed to the higher DNA concentration, the plasmid DNA was partly contained in structures with a very low electrophoretic mobility. Upon restriction endonuclease digestion of the re-extracted DNA, a pattern of bands was observed, suggesting the involvement of illegitimate recombination between non-random plasmid DNA sequences in the formation of the new classes of molecules.