Use of polylysine for adsorption of nuclei acids and enzymes to electron microscope specimen films

Abstract

Enzymes and nucleic acids, both free and as bound in binary complexes, adsorb to electron microscope specimen films in well-distributed fashion if a dilute solution of polylysine is previously applied to the films. Electron micrographs are exhibited that demonstrate the usefulness of the technique in visualizing double- and single-stranded DNA, Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) in negative stain, and polymerase complexed to poly(dA-dT) and to an 1100 base-pair restriction fragment of bacteriophage T7 DNA containing the early promoters. The base-pair spacing of DNA prepared for electron microscopy by the polylysine method was found to be 0.326 nm. Four promoter sites on the T7 fragment were located at 215, 440, 560, and 670 base-pair distances from the left terminus. When poly(dA-dT) was incubated with a 20-to-1 weight ratio of polymerase the bound enzyme particles were found to be about two-thirds as closely packed as is sterically permissible.