Experimental scrapie in the mouse: electrophoretic and sedimentation properties of the partially purified agent.

Some biochemical and biophysical properties of the scrapie agent in a partially purified fraction P5 from murine spleen are described in this communication. The agent was stable in the nonionic detergents Triton X-100 and Nonidet P40 and stable in the nondenaturing, anionic detergents sodium cholate and sodium N-lauroyl sarcosinate. In contrast, sodium dodecyl sulfate (SDS) inactivated the agent at high concentrations (1% or >) when the detergent-to-protein ration approached 1.5 g SDS/g protein. The agent was resistant to inactivation by nucleases and proteases, even in the presence of 0.1% SDS. A broad peak of infectivity was exhibited in modified colloidal silica (Percoll) density gradients. Maximal titers were found at a Percoll density of 1.10 g/cm3 in the presence and absence of 0.05% SDS. Gel electrophoresis of the agent in the presence of 0.1% SDS resulted in inactivation of > 95% of the agent loaded onto the gel. Free-flow electrophoresis showed that > 99% of the agent in fraction P5 migrated toward the anode, but not as a discrete species. Sedimentation analysis of the agent in fraction P5 in the presence of 1% lysolecithin showed that the agent has a sedimentation coefficient of < 300S but > 30S. Heating P5 preparations caused the agent to associate with cellular elements and form aggregates with sedimentation coefficients > 10,000S. Removal by differential centrifugation of the large forms of the agent produced upon heating permitted characterization of a discrete subpopulation of scrapie agent particles. Rate-zonal sucrose gradient studies showed that > 95% of the infectivity in this subpopulation sedimented as uniform particles with a sedimentation coefficient of 240S.

Partial purification and evidence for multiple molecular forms of the scrapie agent.

A procedure for the partial purification of the scrapie agent from mouse spleen was developed based on its sedimentation profile. Differential centrifugation and detergent treatment with sodium deoxycholate yielded a fraction designated "P5" which was enriched for scrapie infectivity approximately 20-fold with respect to cellular protein. The P5 fraction was devoid of cellular membranes but heavily contaminated with ribosomes as judged by electron microscopy. On centrifugation of the fraction P5 to near equilibrium in a sucrose gradient scrapie infectivity was distributed over a range of densities from 1.08 to 1.30 g/cm3. Parallel rate-zonal analysis showed that the infectivity was distributed over a range of particle sizes with s20.w values from approximately 40 S to greater than 500 S. Incubation of P5 at 37 or 80 degrees C, under conditions that disrupt ribosomes, dramatically altered the rate-zonal gradient profile of the agent. Under these conditions, the agent sedimented as particles with s20.w greater than 500 S. The apparent heterogeneity of the scrapie agent with respect to both size and density and its ability to shift from one form to another suggest that the agent may contain hydrophobic domains on its surface.

Mitogen stimulation of splenocytes from mice infected with scrapie agent.

Spleen weights and mitogen responsiveness of splenocyte cultures from scrapie agent-infected and control-inoculated mice were compared over two-month periods following inoculation. Splenocytes from Swiss, C57B1, and BALB/c mice were stimulated with the T (thymus-derived) cell mitogens phytohemagglutinin or concanavalin A, the B (bone marrow-derived) cell mitogen bacterial lipopolysaccharide, or pokeweed mitogen, a stimulator of both T and B cells. Although significant splenomegaly was associated with scrapie infection, we failed to observe any significant differences in the activation of experimental and control cells. Studies with BALB/c mice suggested the possibility, however, that with both phytohemagglutinin and lipopolysaccharide, specific decreases in lymphocyte activation might occur with more optimal culture conditions. The data are consistent with the idea that the scrapie agent stimulates only subtle immunological changes within the host as it destroys the cells of the central nervous system.

Suppression of polyclonal B cell activation in scrapie-infected C3H/HeJ mice.

A consistent modification in B lymphocyte activation has been observed 1 month after infection of C3H/HeJ mice with scrapie. The mitogenic response to lipopolysaccharide of splenocyte cultures from experimental mice was reduced 30 to 60% as compared to controls. This reduction in mitogen responsiveness was transient but coincided with the onset of detectable splenomegaly and with the reported recovery of maximum yield of infectious scrapie agent in the spleen. The DNA synthetic response to lipopolysaccharide stimulation of splenocytes from scrapie-infected C3H/HeJ mice was depressed relative to controls only between 20 and 40 days after intracerebral inoculation. At all other times, experimental and control responses were identical. Scrapie-associated decreases in mitogenesis were found whether the spleen cell cultures contained splenocytes from individual mice, splenocytes pooled from several mice, or gradient-purified mononuclear cells. The responses of C3H/HeJ splenocyte cultures to phytohemagglutinin or concanavalin A stimulation were unaffected by scrapie infection.

Sequences spanning the EcoRI substrate site.

Substrate recognition by the EcoRI restriction endonuclease was investigated by analysis of the nucleotide sequences at the sites of enzymatic cleavage in various DNA molecules. 5'-end labeling and homochromatographic fingerprinting led to the determination of a 17-base-pair sequence spanning the EcoRI site of simian virus 40 DNA and a 15-base-pair sequence overlapping the EcoRI site of Col El plasmid DNA. Three other DNAs were similarly tested, although extended sequences were not determined in these cases. The EcoRI site was shown to be symmetric double-stranded equivalent of -N-G-A-A-T-T-C-N-.

Specificity of substrate recognition by the EcoRI restriction endonuclease.

The substrate specificity of the EcoRI restriction endonuclease can be varied in vitro by changing the pH and the ionic environment of the reaction. Phosphodiester bond cleavage occurs at a DNA hexanucleotide sequence d(N-G-A-A-T-T-C-N)/d(N-C-T-T-A-A-G-N) when the ionic strength is high, 100 mM Tris-HCl, 50 mM NaCl, 5 mM MgCl2, and the pH is approximately 7.3. Lowering the ionic strength to 25 mM Tris-HCl, 2 mM MgCl2, and adjusting the pH to 8.5 reduces the recognition specificity of the EcoRI endonuclease to the tetranucleotide sequence, d(N-A-A-T-T-N)/d(N-T-T-A-A-N). The enzymatic activity responsible for this substrate recognition is referred to as EcoRI. Cleavage of pVH51 plasmid DNA under EcoRI conditions results in a number of partial digest fragments, some of which disappear slowly over a prolonged digestion period. This suggests that different recognition sites are cleaved at different rates. Comparison of DNA fragment patterns of modified and unmodified pVH51 DNA indicates that the canonical EcoRI sequence is the most rapidly cleaved site under EcoRI conditions. DNA modified in vivo by the EcoRI methylase is not cleaved by the EcoRI endonuclease under standard conditions, but is cleaved under EcoRI conditions at sites other than the standard EcoRI substrate.