1887

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 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 silver-stained 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 ID 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.

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1991-01-01
2022-11-30
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References

  1. Aiken J. M., Williamson J. L., Marsh R. F. 1989; Evidence of mitrochondrial involvement in scrapie infection. Journal of Virology 63:1686–1694
    [Google Scholar]
  2. Alper T., Haig D. A., Clarke M. C. 1966; The exceptionally small size of the scrapie agent. Biochemical and Biophysical Research Communications 22:278–284
    [Google Scholar]
  3. Alper T., Camp W. A., Haig D. A., Clarke M. C. 1967; Does the agent of scrapie replicate without nucleic acid?. Nature, London 214:764–766
    [Google Scholar]
  4. Basler K., Oesch B., Scott M., Westaway D., Walchli M., Groth D. F., McKinley M. P., Prusiner S. B., Weissmann C. 1986; Scrapie and cellular PrP isoforms are encoded by the same chromosomal gene. Cell 46:417–428
    [Google Scholar]
  5. Bellinger-Kawahara C., Cleaver J. E., Diener T. O., Prusiner S. B. 1987a; Purified scrapie prions resist inactivation by UV irradiation. Journal of Virology 61:159–166
    [Google Scholar]
  6. Bellinger-Kawahara C., Diener T. O., McKinley M. P., Groth D. F., Smith D. R., Prusiner S. B. 1987b; Purified scrapie prions resist inactivation by procedures that hydrolyze, modify, or shear nucleic acids. Virology 160:271–274
    [Google Scholar]
  7. Bellinger-Kawahara C. G., Kempner E., Groth D., Gabizon R., Prusiner S. B. 1988; Scrapie prion liposomes and rods exhibit target sizes of 55, 000 Da. Virology 164:537–541
    [Google Scholar]
  8. Bolton D. C., McKinley M. P., Prusiner S. B. 1982; Identification of a protein that purifies with the scrapie prion. Science 218:1309–1311
    [Google Scholar]
  9. Bolton D. C., McKinley M. P., Prusiner S. B. 1984; Molecular characteristics of the major scrapie prion protein. Biochemistry 23:5898–5905
    [Google Scholar]
  10. Braig H. R., Diringer H. 1985; Scrapie: concept of a virus-induced amyloidosis of the brain. EMBO Journal 4:2309–2312
    [Google Scholar]
  11. Bruce M. E., Dickinson A. G. 1987; Biological evidence that scrapie agent has an independent genome. Journal of General Virology 68:79–89
    [Google Scholar]
  12. Carlson G. A., Westaway D., DeArmond S. J., Peterson-Torchia M., Prusiner S. B. 1989; Primary structure of prion protein may modify scrapie isolate properties. Proceedings of the National Academy of Sciences, U.S.A. 86:7475–7479
    [Google Scholar]
  13. Chesebro B., Race R., Wehrly K., Nishio J., Bloom M., Lechner D., Bergstrom S., Robbins K., Mayer L., Keith J. M., Garon C., Haase A. 1985; Identification of scrapie prion protein-specific mRNA in scrapie-infected and uninfected brain. Nature, London 315:321–333
    [Google Scholar]
  14. Cho H. J. 1983; Inactivation of the scrapie agent by pronase. Canadian Journal of Comparative Medicine 47:494–496
    [Google Scholar]
  15. Czub M., Braig H. R., Diringer H. 1986; Pathogenesis of scrapie: study of the temporal development of clinical symptoms, of infectivity titres and scrapie-associated fibrils in brains of hamsters infected intraperitoneally. Journal of General Virology 67:2005–2009
    [Google Scholar]
  16. Czub M., Braig H. R., Diringer H. 1988; Replication of the scrapie agent in hamsters infected intracerebrally confirms the pathogenesis of an amyloid-inducing virosis. Journal of General Virology 69:1753–1756
    [Google Scholar]
  17. Davis B. D., Dulbecco R., Eisen H. N., Ginsberg H. S. 1980 Microbiology: Including Immunology and Molecular Genetics 3rd edn., p 876 Philadelphia: Harper & Row;
    [Google Scholar]
  18. Dees C., Wade W. F., German T. L., Marsh R. F. 1985; Inactivation of the scrapie agent by ultraviolet irradiation in the presence of chlorpromazine. Journal of General Virology 66:845–849
    [Google Scholar]
  19. Diener T. O., McKinley M. P., Prusiner S. B. 1982; Viroids and prions. Proceedings of the National Academy of Sciences, U.S.A. 79:5220–5224
    [Google Scholar]
  20. Diedrich J., Wietgrefe S., Zupancic M., Staskus K., Retzel E., Haase A. T., Race R. 1987; The molecular pathogenesis of astrogliosis in scrapie and Alzheimer’s disease. Microbial Pathogenesis 2:435–442
    [Google Scholar]
  21. Dubois M., Gilles K. A., Hamilton J. K., Rebers P. A., Smith F. 1956; Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28:350–356
    [Google Scholar]
  22. Duguid J. R., Rohwer R. G., Seed B. 1988; Isolation of cDNAs of scrapie-modulated RNAs by substractive hybridisation of a cDNA library. Proceedings of the National Academy of Sciences, U.S.A. 85:5738–5742
    [Google Scholar]
  23. Duguid J. R., Bohmont C. W., Liu N., Tourtellotte W. W. 1989; Changes in brain gene expression shared by scrapie and Alzheimer disease. Proceedings of the National Academy of Sciences, U.S.A. 86:7260–7264
    [Google Scholar]
  24. Follett E. A. C., Desselberger U. 1983; Cocirculation of different rotavirus strains in a local outbreak of infantile gastronen-teritis: monitoring by rapid and sensitive nucleic acid analysis. Journal of Medical Virology 11:39–52
    [Google Scholar]
  25. Gabizon R., McKinley M. P., Prusiner S. B. 1987; Purified prion proteins and scrapie infectivity copartition into liposomes. Proceedings of the National Academy of Sciences, U.S.A. 84:4017–4021
    [Google Scholar]
  26. Gabizon R., McKinley M. P., Groth D. F., Kenaga L., Prusiner S. B. 1988a; Properties of scrapie prion protein liposomes. Journal of Biological Chemistry 263:4950–4955
    [Google Scholar]
  27. Gabizon R., Groth D., McKinley M. P., Prusiner S. B. 1988b; Immunoaffinity purification and neutralization of scrapie prion infectivity. Proceedings of the National Academy of Sciences, U.S.A. 85:6617–6621
    [Google Scholar]
  28. German T. L., McMillan B. C., Castle B. E., Dees C., Wade W. F., Marsh R. F. 1985; Comparison of RN A from healthy and scrapie-infected hamster brain. Journal of General Virology 66:839–844
    [Google Scholar]
  29. Hsiao K., Baker H. F., Crow T. J., Poulter M., Owen F., Terwilliger J. D., Westaway D., Ott J., Prusiner S. B. 1989; Linkage of a prion protein missense variant to Gerstmann-Sträussler syndrome. Nature, London 338:342–345
    [Google Scholar]
  30. Hundley F., Biryahwaho B., Gow M., Desselberger U. 1985; Genome rearrangements of bovine rotavirus after serial passage at high multiplicity of infection. Virology 143:88–103
    [Google Scholar]
  31. Kimberlin R. H., Cole S., Walker C. A. 1987; Temporary and permanent modifications to a single strain of mouse scrapie on transmission to rats and hamsters. Journal of General Virology 68:1875–1881
    [Google Scholar]
  32. Lax A. J., Millson G. C., Manning E. J. 1983; Involvement of protein in scrapie agent infectivity. Research in Veterinary Science 34:155–158
    [Google Scholar]
  33. McKinley M. P., Masiarz F. R., Isaacs S. T., Hearst J. E., Prusiner S. B. 1983; Resistance of the scrapie agent to inactivation by psoralens. Photochemistry and Photobiology 37:539–545
    [Google Scholar]
  34. Murdoch G. H., Sklaviadis T., Manuelidis E. E., Manuelidis L. 1990; Potential retroviral RNAs in Creutzfeldt-Jakob disease. Journal of Virology 64:1477–1486
    [Google Scholar]
  35. Narang H. K., Asher D. M., Gajdusek D. C. 1988; Evidence that DNA is present in abnormal tubulofilamentous structures found in scrapie. Proceedings of the National Academy of Sciences, U.S.A. 85:3575–3579
    [Google Scholar]
  36. Oesch B., Groth D. F., Prusiner S. B., Weissmann C. 1988; Search for a scrapie-specific nucleic acid: a progress report. In Novel Infectious Agents and the Central Nervous System, Ciba Foundation Symposium 135 pp. 209–223 Edited by Bock G., Marsh J. New York & Chichester: John Wiley;
    [Google Scholar]
  37. Oesch B., Westaway D., Wälchli M., McKinley M. P., Kent S. B. H., Aebersold R., Barry R. A., Tempst P., Teplow D. B., Hood L. E., Prusiner S. B., Weissmann C. 1985; A cellular gene encodes scrapie PrP 27–30 protein. Cell 40:735–746
    [Google Scholar]
  38. Palukaitis P., Zaitlin M. 1987; The nature and biological significance of linear potato spindle tuber viroid molecules. Virology 157:199–210
    [Google Scholar]
  39. Prusiner S. B. 1982; Novel proteinaceous infectious particles cause scrapie. Science 216:136–144
    [Google Scholar]
  40. Prusiner S. B. 1989; Scrapie prions. Annual Review of Microbiology 43:345–374
    [Google Scholar]
  41. Prusiner S. B., McKinley M. P., Groth D. R., Bowman K. A., Mock N. I., Cochran S. P., Masiarz F. R. 1981; Scrapie agent contains a hydrophobic protein. Proceedings of the National Academy of Sciences, U.S.A 78:6675–6679
    [Google Scholar]
  42. Prusiner S. B., Bolton D. C., Groth D. F., Bowman K. A., Cochran S. P., McKinley M. P. 1982a; Further purification and characterization of scrapie prions. Biochemistry 21:6942–6950
    [Google Scholar]
  43. Prusiner S. B., Cochran S. P., Groth D. F., Downey D. E., Bowman K. A., Martinez H. M. 1982b; Measurement of the scrapie agent using an incubation time interval assay. Annals of Neurology 11:353–358
    [Google Scholar]
  44. Prusiner S. B., McKinley M. P., Bowman K. A., Bolton D. C., Bendheim P. E., Groth D. R., Glenner G. G. 1983; Scrapie prions aggregate to form amyloid-like birefringent rods. Cell 35:349–358
    [Google Scholar]
  45. Sammons D. W., Adams D. L., Nishizawa E. E. 1981; Ultrasensitive silver-based color staining of polypeptides in polyacrylamide gels. Electrophoresis 2:135–141
    [Google Scholar]
  46. Sӓnger H. L., Ramm K., Domdey H., Gross H. J., Henco K., Riesner D. 1979; Conversion of circular viroid molecules to linear strands. FEBS Letters 99:117–122
    [Google Scholar]
  47. Schumacher J., Meyer N., Weidemann H. L., Riesner D. 1986; Diagnostic procedure for detection of viroids and viruses with circular RNAs by ‘return’-gel electrophoresis. Journal of Phytopathology 115:332–343
    [Google Scholar]
  48. Scott M., Foster D., Mirenda C., Serban D., Coufal F., Walchli M., Torchia M., Goth D., Carlson G., DeArmond S. J., Westaway D., Prusiner S. B. 1989; Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques. Cell 59:847–857
    [Google Scholar]
  49. Sklaviadis T. K., Manuelidis L., Manuelidis E. E. 1989; Physical properties of the Creutzfeldt-Jakob disease agent. Journal of Virology 63:1212–1222
    [Google Scholar]
  50. Tabler M., Sӓnger H. L. 1984; Cloned single- and double-stranded DNA copies of potato spindle tuber viroid (PSTV) RNA and co-inoculated subgenomic DNA fragments are infectious. EMBO Journal 3:3055–3062
    [Google Scholar]
  51. Wletgrefe S., Zupancic M., Haase A., Chesebro B., Race R., Frey W. I., Rustan T., Friedman R. L. 1985; Cloning of a gene whose expression is increased in scrapie and in senile plaques in human brain. Science 230:1177–1181
    [Google Scholar]
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