Nucleic acids in prion preparations: unspecific background or essential component?
As recently published (Kellings et al. J. gen Vir. 73, 1025-1029 (1992)), the analysis of purified scrapie prions by return refocusing gel electrophoresis revealed remaining nucleic acids in the size range up to 1100 nucleotides. The results defined the possible characteristics of a hypothetical scrapie-specific nucleic acid. If homogeneous in size, such a molecule would be less than 80 nucleotides in length at a particle-to-infectivity ratio (P:I) near unity; if heterogeneous, scrapie-specific nucleic acids would have to include molecules smaller than 240 nucleotides. To decrease the amount of nucleic acids, several modifications of the PrPSc purification scheme were introduced. Instead of sucrose gradient, ultrafiltration was applied as a purification step and nucleic acids were degraded by Benzonase after ultrafiltration, but significant reduction of the P:I ratio could not be achieved. To prevent trapping of nucleic acids in prion rods, nuclease (Benzonase) was added into the tissue homogenate and incubated at 37 degrees C, overnight. The Benzonase treatment revealed no loss of infectivity, but the whole procedure of nucleic acid analysis did not lead to a reduction of the P:I ratio. In another approach the number of nucleic acid degradations steps was reduced to essentially two steps: Zn2+ hydrolysis and Benzonase digestion. Higher Zn2+ concentrations and prolonged incubation times resulted in a more efficient nucleic acid degradation. The bioassays yielded complete recovery of infectivity. Large-scale preparations for determining the P:I ratio are still underway.
Kellings
K
K
Heinrich-Heine-Universität Düsseldorf, Institut für Physikalische Biologie, Germany.
Prusiner
S B
SB
Riesner
D
D
D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D006224:Cricetinae; D004586:Electrophoresis / Q000379:methods; D006868:Hydrolysis; D008970:Molecular Weight; D009696:Nucleic Acids / Q000737:chemistry / Q000302:isolation & purification*; D018620:PrPSc Proteins; D017096:Prion Diseases / Q000209:etiology; D011328:Prions / Q000737:chemistry / Q000302:isolation & purification*; D012608:Scrapie / Q000209:etiology; D014462:Ultrafiltration; D015032:Zinc
D009696:Nucleic Acids; D018620:PrPSc Proteins; D011328:Prions; D015032:Zinc