Search for a Putative Scrapie Genome in Purified Prion Fractions Reveals a Paucity of Nucleic Acids

Abstract

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.

Prion distribution in hamster lung and brain following intraperitoneal inoculation.

“…Although the theory remains controversial, evidence in support of the protein-only model is substantial (Bolton et al, , 1987(Bolton et al, , 1991McKinley et al, 1983 ;Prusiner et al, 1984 ;Hsiao et al, 1989Hsiao et al, , 1990Scott et al, 1989Scott et al, , 1993Meyer et al, 1991 ;Bu$ eler et al, 1992).…”

Further analysis of nucleic acids in purified scrapie prion preparations by improved return refocusing gel electrophoresis

“…RRGE was described earlier (Meyer et al, 1991) and the improvements in RRGE are outlined briefly in the legend of Fig. 1.…”

“…Amounts of nucleic acid were determined by comparing intensities of silver-stained bands to those of nucleic acid standards used at three different concentrations (Table 1). Preparation of purified prion fractions for RRGE was similar to that used for previous studies (Meyer et al, 1991). In order to improve the recovery of prion infectivity during the collection of prion rods from sucrose gradient fractions, ultracentrifugation steps (2 × 4h, 100000g) were substituted for the ethanol precipitation previously used.…”

“…A recent search for a putative scrapie genome in purified prion preparations revealed a paucity of nucleic acids (Meyer et al, 1991). Purified fractions were extensively treated with nucleases and Zn 2+ ions, dispersed into detergent-lipid-protein complexes (DLPC), treated again with nucleases followed by deproteinization, and extracted for nucleic acids.…”

Nucleic acid binding proteins in highly purified Creutzfeldt-Jakob disease preparations.

“…Infectivity has never been separated from nucleic acids, even when only size-selected molecules are analyzed with lowsensitivity detection methods (34). Clearly reproducible filtration and physical studies indicate a viruslike core structure of significant size (7,8,10).…”