IMPORT OF PLASMID DNA INTO THE NUCLEUS IS SEQUENCE-SPECIFIC

Nuclear import of plasmid DNA in nondividing cells is a process essent ial to the success of numerous viral life cycles, gene therapy protoco ls, and gene expression experiments, Here, intact protein-free SV40 DN A was cytoplasmically injected into cells and its subcellular localiza tion was followed by in situ hybridization. SV40 DNA localized to the nucleus consistent with a mechanism of transport through the nuclear p ore complex (NPC): import was inhibited by the addition of the NPC-inh ibitory agents wheat germ agglutinin and an anti-nucleoporin antibody as well as by energy depletion. DNA transport appeared to be a multist ep process with the DNA accumulating at the nuclear periphery before i ts import. Most importantly, nuclear import was sequence specific: a r egion of SV40 DNA containing the origin of replication and the early a nd late promoters supported import, whereas bacterial sequences alone and other SV40-derived sequences did not. The majority of the imported DNA colocalized with the SC-35 splicing complex antigen, suggesting t hat the intranuclear DNA localizes to areas of transcription or messag e processing. This link to transcription was strengthened by the findi ng that inhibition of transcription blocked DNA import but not protein nuclear import. Taken together, these results support a model in whic h plasmid DNA nuclear import occurs by a mechanism similar to that use d by nuclear localization signal-containing proteins but is also depen dent on transcription. (C) 1997 Academic Press.