Interaction in vitro of non-epithelial intermediate filament proteins with supercoiled plasmid DNA.
Sucrose gradient analysis of reaction products obtained from non-epithelial intermediate filament (IF) subunit proteins and a mixture of supercoiled, relaxed and linearized plasmid pBR322 DNA at low ionic strength revealed that limited amounts of these polypeptides interacted exclusively with the supercoiled form of the plasmid DNA. These results were corroborated by electron-microscopic analysis of the reaction products, which showed that only circles of supercoiled pBR322 DNA were completely and smoothly covered with vimentin. IFs reconstituted from pure vimentin reacted with supercoiled pBR322 DNA only through their physical ends. The reaction of an aged preparation of vimentin with supercoiled pBR322 DNA produced large aggregates consisting of a central, axially oriented protein scaffold to which individual loops of DNA were attached at their bases in a halo-like arrangement. The electron-microscopic appearance of such complexes was very reminiscent of that of histone-depleted metaphase chromosomes. Together with the previous observations that non-epithelial IF proteins have high affinities for single-stranded DNA and core histones and that they are structurally and functionally closely related to the nuclear lamins, these results were used to advance a novel hypothesis on the biological role of IF proteins in eukaryotic cells.
Kühn
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Max-Planck-Institut für Zellbiologie, Ladenburg/Heidelberg, Federal Republic of Germany.
Vorgias
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Traub
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D000818:Animals; D002499:Centrifugation, Density Gradient; D004278:DNA, Superhelical / Q000187:drug effects / Q000648:ultrastructure*; D066298:In Vitro Techniques; D007381:Intermediate Filament Proteins / Q000494:pharmacology*; D008854:Microscopy, Electron; D010957:Plasmids / Q000187:drug effects*; D014746:Vimentin / Q000378:metabolism
D004278:DNA, Superhelical; D007381:Intermediate Filament Proteins; D014746:Vimentin