Scrapie can be transmitted by novel infectious pathogens termed prions. No evidence for a scrapie-specific nucleic acid has been detected to date. To investigate amounts, types and sizes of nucleic acid molecules associated with prions in purified preparations, aliquots were deproteinized, and the nucleic acids analysed by PAGE and silver staining. Digestion with nucleases and exposure to Zn z+ prior to analysis substantially diminished the content of nucleic acids, but did not alter the prion titre indicating that those nucleic acids which were removed are not essential for infectivity. Since a single species of scrapie-specific nucleic acid could not be identified, we explored the unprecedented possibility of scrapie-specific nucleic acids of variable length which are biologically active. If such molecules of variable length exist then they might be hidden within the background smear on silverstained gels after PAGE. A new procedure designated return refocusing gel electrophoresis (RRGE) was developed to identify heterogeneous nucleic acids in purified prion fractions. The content of variable length nucleic acids was reduced by a factor of 10 by exhaustive Bal 31 exonuclease digestion after dispersion of purified prions into detergent-lipid-protein complexes. For example, a typical sample after Bal 31 digestion contained approximately 4 ng of nucleic acid of variable length and 10 s'7 IDso units of scrapie prion infectivity. Consideration of different models for a hypothetical scrapie-specific nucleic acid suggests that such a molecule would have to be: (i) quite small (< 100 nucleotides), (ii) possess a particle-to-infectivity ratio near unity or (iii) heterogeneous in size. Although our results do not eliminate the possibility that prions possess a scrapie-specific nucleic acid of variable length, they narrow considerably the spectrum of features specifying such a candidate molecule.
A fluorescence detection system was developed for the analytical ultracentrifuge Spinco model E. Fluorescence is excited by a laser beam which is focussed into the cell and illuminates an area with a dimension of 60 ~tm in radial direction. For scanning the laser beam is moved in radial direction. After passing the cell, the laser beam is quenched by a carbon light trap and a set of optical filters. Fluorescence emission intensity is monitored by a photomultiplier located behind the light trap and the set of filters. The sensitivity of the detection system was tested by applying it to the sedimentation analysis of proteins and nucleic acids. Bovine serum albumin (BSA) was covalently labelled with the fluorescence-dye fluorescein-isothiocyanate (FITC), and its sedimentation coefficient could be determined even if BSA was analyzed in a concentration as low as 10-10 M. Nucleic acids were labelled non-covalently by the intercalating dye ethidium bromide. Only 8 ng RNA were needed for the determination of the sedimentation coefficient. The particular advantages of the fluorescence detection system were exploited for the establishment of a new method for quantitative virus detection. To tobacco mosaic virus (TMV) a monoclonal anti-TMV antibody from mouse was bound, and to this a second, anti-mouse antibody that carried the fluorescence-label HTC was attached. Either by UV-irradiation or by incubation with glutaraldehyde, the first antibody was covalently crosslinked to TMV, and the second antibody to the first. In CsC1 density centrifugation with fluorescence detection as little as 3.2 ng virus/80 IA or 6 x 10 s virus particles/ml were recorded in a well expressed band at the corresponding buoyant density. Tenfold lower concentration would result still in a significant band. The sensitivity compares well with those of the most advanced techniques from immunology. Due to the specific labelling of viruses by antibodies it will be possible to carry out quantitative physical characterization of virus containing samples without purifying the virus. Future applications of the fluorescence detection system and of the virus detection technique are discussed.
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