Institute for Neurodegenerative Diseases1 and Departments of Neurology2 and Biochemistry and Biophysics3, Box 0518, University of California, San Francisco, CA 94143-0518, USA
Author for correspondence: Stanley Prusiner. Fax +1 415 476 8386.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
When different strains of prions are propagated within the same species, they are known to retain their original properties (Bruce & Dickinson, 1987 ; Dickinson & Meikle, 1969
; Pattison & Millson, 1961
). These strains are described by characteristic neuropathological patterns of plaque deposition, vacuolation, neuronal loss and astrogliosis, as well as different incubation times and, in part, different biochemical characteristics (Bessen & Marsh, 1994
; Safar et al., 1998
; Scott et al., 1997
).
In the absence of an identifiable nucleic acid in highly purified prion preparations (Kellings et al., 1994 ), differences between strains can only be explained by a replicative mechanism that occurs post-translationally. Strong evidence suggests that strain characteristics are enciphered in the conformation of PrPSc itself (Bessen & Marsh, 1994
; Safar et al., 1998
; Scott et al., 1997
; Telling et al., 1996
). The molecular mechanism of replication of strain-specific features through modulation of PrPSc conformation is not yet understood. Distinct prion isolates induce accumulations of PrPSc in the brain that are region-specific (Bruce et al., 1989
). This fact has led to the hypothesis that distinct cell types in the central nervous system dictate the strain-specific traits enciphered in the specific structure of each PrPSc (Hecker et al., 1992
).
Another hypothesis concerning strain replication is based on the observation that strains of prions seem to maintain a distinctive pattern of PrPSc glycosylation site occupancy (Collinge et al., 1996 ; Parchi et al., 1997
). From these findings, it has been hypothesized that strain characteristics are encrypted in their glycosylation patterns as assessed by the banding patterns of proteinase K-digested PrPSc on SDSPAGE gels (Collinge et al., 1996
). However, glycoform patterns of PrPSc have been found to differ when comparing infected tonsil and brain tissues from the same CreutzfeldtJakob diseased patient (Hill et al., 1999
), spleen and brain samples from infected mice (Rubenstein et al., 1991
) and different brain regions within the same mouse brain (Somerville, 1999
). Also, the passaging of different mouse prion strains in N2a cells overexpressing mouse PrP was shown to alter the glycosylation patterns of PrPSc (Nishida et al., 2000
), arguing that glycosylation is unlikely to specify strain-specific properties of prions.
In addition to the studies noted above that question the proposed role of glycoforms in prion strains, investigations of fatal insomnia argue persuasively that strain-specified properties are not enciphered in the asparagine-linked oligosaccharides (Mastrianni et al., 1999 ; Parchi et al., 1999
). The familial form of fatal insomnia (FFI) is a genetic prion disease linked to a mutation of aspartate to asparagine at residue 178 (D178N) and a methionine polymorphism at residue 129 in the human prion protein (Goldfarb et al., 1992
; Lugaresi et al., 1986
). The disease phenotype of FFI is sleep loss and thalamic degeneration. Recently, patients have been described with phenotypes indistinguishable from FFI, but without any mutation in the PrP gene. These patients carry the diagnosis of sporadic fatal insomnia (sFI) (Mastrianni et al., 1999
; Parchi et al., 1999
). Moreover, patients with sporadic and familial forms of fatal insomnia exhibit a prion disease phenotype that is indistinguishable even though the levels of di- and monoglycosylated PrPSc molecules are markedly different (Mastrianni et al., 1999
). Additionally, extracts from the brains of patients with either the sporadic or familial forms of fatal insomnia transmit disease to transgenic mice with similar incubation times and neuropathological lesion profiles (Mastrianni et al., 1999
). The foregoing studies argue that although some strains may be correlated with certain glycosylation patterns, these patterns are inconsistent and cannot encipher strain-specific properties.
In this study, we investigated the influence of glycosylation on the conversion of PrPC by mouse prion strains. While it has been shown that inhibition of glycosylation by tunicamycin in scrapie-infected cells results in protease-resistant unglycosylated PrPSc (Lehmann & Harris, 1997 ; Taraboulos et al., 1990
), attempts to express a metabolically stable, mutated unglycosylated PrPC have been unsuccessful (DeArmond et al., 1997
; Lehmann & Harris, 1997
; Rogers et al., 1990
). The present study introduces a novel mutation, replacing the asparagines (mouse PrP codons 180 and 196) which carry the carbohydrate side-chains with glutamines (N180Q,N196Q), rather than mutating threonines in the glycosylation consensus sequence (T182A,T198A) as previously described (DeArmond et al., 1997
; Lehmann & Harris, 1997
; Taraboulos et al., 1990
).
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Vectors.
Expression of recombinant epitope-tagged PrP using the CMV promoter-based pSPOX vector in N2a cells has been described (Kaneko et al., 1997 ; Scott et al., 1992
). Introduction of the epitope for monoclonal antibody (MAb) 3F4 (Kascsak et al., 1987
) into mouse PrP (designated MHM2PrP) allows detection of newly formed PrPSc against the background of cell- and inoculum-resident PrPSc, which is not recognized by MAb 3F4 (Scott et al., 1992
). Although mutations at residues constituting the MAb 3F4 epitope have been implicated in altering the species barrier between prions (Priola et al., 1994
), the MHM2PrP construct has not been found to exert a significant impact on the susceptibility to mouse prions when compared to mouse PrPa/a (Scott et al., 1993
). Generation of pSPOX vectors containing the 3F4 epitope and the Q218K mutation have been described (Kaneko et al., 1997
; Scott et al., 1992
). Mutations at codons 180 and 196 were introduced using mismatched primers in PCR of particular PrP templates. The resulting fragments were substituted into unique cloning sites of pSPOX. Specifically, primers 5' GGCAGATCTACCATGGCGAACCTTGGC 3' (sense) and 5' GTGGTGGTGGTGACCGTGTGCTGCTTGATGGTGATCTGGACGCAGTC 3' (antisense) were used to create a fragment in which codon 180 was mutated to glutamine. The fragment was then cloned into unique sites BglII and BstEII. Primers 5' CAGCACACCGTCACCACCACCACCAAGGGGGAGCAGTTCACCGAG 3' (sense) and 5' CACTATAGAACTCGAGCAGCCTCCCT 3' (antisense) were used to create a fragment in which codon 196 was mutated to glutamine. The fragment was then cloned into unique sites BstEII and XhoI. The correct nucleotide sequence of constructs was verified by sequencing the pSPOX inserts with an ABI prism 377 sequencer.
Transient transfection of ScN2a cells.
A confluent 100 mm dish of ScN2a cells was washed, detached with 0·05% trypsin and resuspended in a total volume of 5 ml. These detached ScN2a cells were added in 0·5 ml aliquots to a 60 mm dish filled with 2 ml of fresh MEM medium. pSPOX vector (15 µg) carrying the respective PrP constructs was resuspended in DOTAP (Roche) according to the manufacturer and applied to each 60 mm dish of freshly split ScN2a cells; the next day, another 2 ml of fresh MEM medium was added. Four days after transfection, cells were harvested after being washed three times in PBS with 0·5 ml lysis buffer (150 mM NaCl, 10 mM Tris pH 8·0, 0·5% NP-40, 0·5% deoxycholate) per 60 mm dish. Lysate (0·4 ml) was digested with 20 µg/ml proteinase K (Roche) for 30 min at 37 °C. The digested lysate was then ultracentrifuged at 100000 g for 45 min in a TLA 55 rotor (Beckman table-top ultracentrifuge). The supernatant was discarded and the pellet was resuspended in SDSPAGE sample buffer. Finally, all samples were processed for Western blotting using standard procedures. The primary antibodies used were MAb 3F4 (Kascsak et al., 1987 ) or Ro73. The secondary antibodies were HRP-labelled goat anti-mouse IgG and goat anti-rabbit IgG, respectively. The ECL/Hyperfilm detection system was obtained from Amersham. All experiments were repeated independently at least three times.
Immunofluorescence labelling.
ScN2a cells were transfected as described above in dishes containing coverslips. Four days after transfection, coverslips were processed for surface staining and intracellular staining. For surface staining, coverslips were washed three times with cold PBS and then incubated for 1 h at 4 °C with MAb 3F4 diluted 1:50 with 1% BSA. Subsequent steps were performed at room temperature. Coverslips were washed again with PBS and fixed with 4% paraformaldehyde for 30 min. After blocking with 5% milk, 1% BSA in PBS, coverslips were washed in PBS and goat anti-mouse IgGFITC-labelled secondary antibody (Roche) was added at a dilution of 1:100 for 30 min. Intracellular staining was carried out at room temperature as follows: coverslips were washed, incubated in 4% paraformaldehyde for 30 min and blocked with 5% milk, 1% BSA, 0·5% saponin for 30 min. Coverslips were then incubated in MAb 3F4 diluted 1:50 with 1% BSA, 0·5% saponin for 1 h. After washing in PBS, goat anti-mouse IgGFITC-labelled secondary antibody (Roche) was added at a dilution of 1:100 for 30 min. All coverslips were finally washed three times in PBS, mounted on a glass slide with 5 µl mounting medium (Vetashield) and examined under a Leitz microscope.
Inoculation of transiently transfected N2a cells.
Homogenates (10%) of whole mouse brains in PBS were prepared by passing them five times through successively smaller syringe needles from 16 to 26 gauge. The origins of the strains RML (Chandler, 1961 ), Me7 (Dickinson & Meikle, 1969
) and 301V (Farquhar et al., 1996
) have been described. Homogenates were kept at -80 °C.
Confluent N2a cells on a 10 cm dish were split into five 10 cm dishes; 37 µg of pSPOX encoding different MHM2PrP constructs was suspended in 300 µl sterile 20 mM HEPES pH 7·5, and an equal volume of 20 mM HEPES pH 7·5 DOTAP (Roche) was added, incubated for approximately 12 min and added to the freshly split neuroblastoma cells. After 24 h, 150 µl of a 10% brain homogenate in PBS was added. Fresh MEM medium was added 24 h and 72 h after transfection. Five days after transfection (4 days after inoculation) cells were washed three times in PBS, lysed in 1 ml lysis buffer and processed for Western blotting as described above. All experiments were repeated independently at least three times.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Neuroblastoma cells transiently expressing unglycosylated MHM2PrP(N180Q,N196Q) can be infected with mouse prions from brain homogenates
Based on the results in ScN2a cells (Figs 1 and 2
), a protocol was developed to infect transiently transfected N2a cells expressing MHM2PrP(N180Q,N1996Q) with 10% scrapie-infected mouse brain homogenates. Four days after the inoculation of N2a cells expressing MHM2PrP(N180Q,N196Q) with 10% brain homogenate, newly formed MHM2PrPSc-(N180Q,N196Q) could be detected by its epitope tag using MAb 3F4. As shown in Fig. 3
, 100 µl of 10% RML-infected mouse brain homogenate was sufficient to stimulate conversion of PrP(N180Q,N196Q) expressed in N2a cells into PrPSc after 4 days. Because the immunoreactive band at 16 kDa corresponding to protease-resistant MHM2PrP(N180Q,N196Q) is weak compared to undigested MHM2PrP(N180Q,N196Q), the immunoblots were overexposed (Figs 3
and 4
). The overexposure increased both the PrPSc and background bands due to cross-reactivity with the secondary antibody, as can be seen in the undigested lysates on the left side of the immunoblot and the cross-reactive band of proteinase K in the digested lysates.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An homogenate of scrapie-infected brain was sufficient to induce conversion of MHM2PrP(N180Q,N196Q) that was transiently expressed in neuroblastoma cells. In the inoculation protocol, no purification or concentration steps for prions in the inoculum were necessary to achieve MHM2PrPSc(N180Q,N196Q) formation after 4 days of incubation. The low efficiency of glycosylated PrP conversion compared to that of unglycosylated PrP is consistent with data showing that detection of mouse PrPSc in non-transfected N2a cells after inoculation with prions takes several weeks (Bosque & Prusiner, 2000 ; Butler et al., 1988
). Apparently, glycosylation of asparagines 180 and 196 delays conversion of wild-type PrPC to PrPSc, a conclusion that is consistent with earlier findings (Taraboulos et al., 1990
). Inhibition of PrP(N180Q,N196Q,Q218K) conversion by RML-infected brain homogenates demonstrates that the acquisition of protease resistance is an active process rather than protection of expressed PrP from protease digestion.
That transiently expressed MHM2PrP(N180Q,N196Q) is converted into PrPSc by different strains of mouse prions contrasts with infection of non-transfected cell lines (Bosque & Prusiner, 2000 ; Butler et al., 1988
; Race et al., 1987
; Schätzl et al., 1997
). We did not see any strain specificity with respect to the susceptibility of MHM2PrP(N180Q,N196Q) to conversion into PrPSc; however, we do not know if strain-specific characteristics were conserved during MHM2PrPSc(N180Q,N196Q) formation. Notably, we did find similar levels of PrPSc formation during inoculation with RML, Me7 or 301V. Although Me7 and RML have similar incubation times in CD-1 mice of approximately 150 days, 301V prions have an incubation time of approximately 230 days. These findings suggest that multiple prion strains more readily convert unglycosylated than glycosylated PrPC in ScN2a cells into PrPSc and that strain-specified properties of prions do not alter the conversion of MHM2PrP(N180Q,N196Q) into PrPSc. It is noteworthy that other investigators have found that N2a cells overexpressing wild-type mouse PrP are more susceptible to prion infection than untransfected cells (Nishida et al., 2000
). They also reported that the Chandler, 139A and 22L prion strains infected these N2a cells overexpressing mouse PrP. The RML strain used in our studies was derived from the Chandler strain, as was 139A.
Even though inocula from very different strains convert transiently expressed MHM2PrP(N180Q,N196Q) to protease-resistant MHM2PrPSc(N180Q,N196Q) equally well, the question remains whether characteristics such as the neuronal cell loss, astrocytic gliosis, PrP amyloid plaque deposition, and vacuolation patterns in brain as well as incubation times are preserved within the conformation of protease-resistant, unglycosylated PrPSc(N180Q,N196Q). Experiments with prion strains propagated in mice expressing PrP(N180Q,N196Q) transgenes may provide reliable answers to such questions. It is notable that characterization of prion strains passaged in cultured cells requires at least two passages in mice since the titres of prions in cultured cells are usually low (Butler et al., 1988 ) and this results in a prolonged incubation time. Only on second passage in mice can a reliable incubation time for a particular strain be established.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bosque, P. J. & Prusiner, S. B. (2000). Cultured cell sublines highly susceptible to prion infection. Journal of Virology 74, 4377-4386.
Bruce, M. E. & Dickinson, A. G. (1987). Biological evidence that scrapie agent has an independent genome. Journal of General Virology 68, 79-89.[Abstract]
Bruce, M. E., McBride, P. A. & Farquhar, C. F. (1989). Precise targeting of the pathology of the sialoglycoprotein, PrP, and vacuolar degeneration in mouse scrapie. Neuroscience Letters 102, 1-6.[Medline]
Butler, D. A., Scott, M. R. D., Bockman, J. M., Borchelt, D. R., Taraboulos, A., Hsiao, K. K., Kingsbury, D. T. & Prusiner, S. B. (1988). Scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins. Journal of Virology 62, 1558-1564.[Medline]
Chandler, R. L. (1961). Encephalopathy in mice produced by inoculation with scrapie brain material. Lancet i, 1378-1379.
Collinge, J., Sidle, K. C. L., Meads, J., Ironside, J. & Hill, A. F. (1996). Molecular analysis of prion strain variation and the aetiology of new variant CJD. Nature 383, 685-690.[Medline]
DeArmond, S. J., Sánchez, H., Yehiely, F., Qiu, Y., Ninchak-Casey, A., Daggett, V., Camerino, A. P., Cayetano, J., Rogers, M., Groth, D., Torchia, M., Tremblay, P., Scott, M. R., Cohen, F. E. & Prusiner, S. B. (1997). Selective neuronal targeting in prion disease. Neuron 19, 1337-1348.[Medline]
Dickinson, A. G. & Meikle, V. M. (1969). A comparison of some biological characteristics of the mouse-passaged scrapie agents, 22A and ME7. Genetic Research 13, 213-225.
Endo, T., Groth, D., Prusiner, S. B. & Kobata, A. (1989). Diversity of oligosaccharide structures linked to asparagines of the scrapie prion protein. Biochemistry 28, 8380-8388.[Medline]
Farquhar, C. F., Dornan, J., Moore, R. C., Somerville, R. A., Tunstall, A. M. & Hope, J. (1996). Protease-resistant PrP deposition in brain and non-central nervous system tissues of a murine model of bovine spongiform encephalopathy. Journal of General Virology 77, 1941-1946.[Abstract]
Goldfarb, L. G., Petersen, R. B., Tabaton, M., Brown, P., LeBlanc, A. C., Montagna, P., Cortelli, P., Julien, J., Vital, C. & Pendelbury, W. W. (1992). Fatal familial insomnia and familial CreutzfeldtJakob disease: disease phenotype determined by a DNA polymorphism. Science 258, 806-808.[Medline]
Hecker, R., Taraboulos, A., Scott, M., Pan, K.-M., Torchia, M., Jendroska, K., DeArmond, S. J. & Prusiner, S. B. (1992). Replication of distinct scrapie prion isolates is region specific in brains of transgenic mice and hamsters. Genes & Development 6, 1213-1228.[Abstract]
Hill, A. F., Butterworth, R. J., Joiner, S., Jackson, G., Rossor, M. N., Thomas, D. J., Frosh, A., Tolley, N., Bell, J. E., Spencer, M., King, A., Al-Sarraj, S., Ironside, J. W., Lantos, P. L. & Collinge, J. (1999). Investigation of variant CreutzfeldtJakob disease and other human prion diseases with tonsil biopsy samples. Lancet 353, 183-189.[Medline]
Kaneko, K., Zulianello, L., Scott, M., Cooper, C. M., Wallace, A. C., James, T. L., Cohen, F. E. & Prusiner, S. B. (1997). Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation. Proceedings of the National Academy of Sciences, USA 94, 10069-10074.
Kascsak, R. J., Rubenstein, R., Merz, P. A., Tonna-DeMasi, M., Fersko, R., Carp, R. I., Wisniewski, H. M. & Diringer, H. (1987). Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins. Journal of Virology 61, 3688-3693.[Medline]
Kellings, K., Prusiner, S. B. & Riesner, D. (1994). Nucleic acids in prion preparations: unspecific background or essential component? Philosophical Transactions of the Royal Society of London B Biological Sciences 343, 425-430.
Lehmann, S. & Harris, D. A. (1997). Blockade of glycosylation promotes acquisition of scrapie-like properties by the prion protein in cultured cells. Journal of Biological Chemistry 272, 21479-21487.
Locht, C., Chesebro, B., Race, R. & Keith, J. M. (1986). Molecular cloning and complete sequence of prion protein cDNA from mouse brain infected with the scrapie agent. Proceedings of the National Academy of Sciences, USA 83, 6372-6376.[Abstract]
Lugaresi, E., Medori, R., Montagna, P., Baruzzi, A., Cortelli, P., Lugaresi, A., Tinuper, P., Zucconi, M. & Gambetti, P. (1986). Fatal familial insomnia and dysautonomia with selective degeneration of thalamic nuclei. New England Journal of Medicine 315, 997-1003.[Medline]
Mastrianni, J. A., Nixon, R., Layzer, R., Telling, G. C., Han, D., DeArmond, S. J. & Prusiner, S. B. (1999). Prion protein conformation in a patient with sporadic fatal insomnia. New England Journal of Medicine 340, 1630-1638.
Nishida, N., Harris, D. A., Vilette, D., Laude, H., Frobert, Y., Grassi, J., Casanova, D., Milhavet, O. & Lehmann, S. (2000). Successful transmission of three mouse-adapted scrapie strains to murine neuroblastoma cell lines overexpressing wild-type mouse prion protein. Journal of Virology 74, 320-325.
Parchi, P., Capellari, S., Chen, S. G., Petersen, R. B., Gambetti, P., Kopp, P., Brown, P., Kitamoto, T., Tateishi, J., Giese, A. & Kretzschmar, H. (1997). Typing prion isoforms. Nature 386, 232-233.[Medline]
Parchi, P., Capellari, S., Chin, S., Schwarz, H. B., Schecter, N. P., Butts, J. D., Hudkins, P., Burns, D. K., Powers, J. M. & Gambetti, P. (1999). A subtype of sporadic prion disease mimicking fatal familial insomnia. Neurology 52, 1757-1763.
Pattison, I. H. & Millson, G. C. (1961). Scrapie produced experimentally in goats with special reference to the clinical syndrome. Journal of Comparative Pathology 71, 101-108.
Priola, S. A., Caughey, B., Race, R. E. & Chesebro, B. (1994). Heterologous PrP molecules interfere with accumulation of protease-resistant PrP in scrapie-infected murine neuroblastoma cells. Journal of Virology 68, 4873-4878.[Abstract]
Prusiner, S. B. (1998). Prions. Proceedings of the National Academy of Sciences, USA 95, 13363-13383.
Race, R. E., Fadness, L. H. & Chesebro, B. (1987). Characterization of scrapie infection in mouse neuroblastoma cells. Journal of General Virology 68, 1391-1399.[Abstract]
Rogers, M., Taraboulos, A., Scott, M., Groth, D. & Prusiner, S. B. (1990). Intracellular accumulation of the cellular prion protein after mutagenesis of its Asn-linked glycosylation sites. Glycobiology 1, 101-109.[Abstract]
Rubenstein, R., Merz, P. A., Kascsak, R. J., Scalici, C. L., Papini, M. C., Carp, R. I. & Kimberlin, R. H. (1991). Scrapie-infected spleens: analysis of infectivity, scrapie-associated fibrils, and protease-resistant proteins. Journal of Infectious Diseases 164, 29-35.[Medline]
Rudd, P. M., Endo, T., Colominas, C., Groth, D., Wheeler, S. F., Harvey, D. J., Wormald, M. R., Serban, H., Prusiner, S. B., Kobata, A. & Dwek, R. A. (1999). Glycosylation differences between the normal and pathogenic prion protein isoforms. Proceedings of the National Academy of Sciences, USA 96, 13044-13049.
Safar, J., Wille, H., Itri, V., Groth, D., Serban, H., Torchia, M., Cohen, F. E. & Prusiner, S. B. (1998). Eight prion strains have PrPSc molecules with different conformations. Nature Medicine 4, 1157-1165.[Medline]
Schätzl, H. M., Laszlo, L., Holtzman, D. M., Tatzelt, J., DeArmond, S. J., Weiner, R. I., Mobley, W. C. & Prusiner, S. B. (1997). A hypothalamic neuronal cell line persistently infected with scrapie prions exhibits apoptosis. Journal of Virology 71, 8821-8831.[Abstract]
Scott, M. R., Köhler, R., Foster, D. & Prusiner, S. B. (1992). Chimeric prion protein expression in cultured cells and transgenic mice. Protein Science 1, 986-997.
Scott, M., Groth, D., Foster, D., Torchia, M., Yang, S.-L., DeArmond, S. J. & Prusiner, S. B. (1993). Propagation of prions with artificial properties in transgenic mice expressing chimeric PrP genes. Cell 73, 979-988.[Medline]
Scott, M. R., Groth, D., Tatzelt, J., Torchia, M., Tremblay, P., DeArmond, S. J. & Prusiner, S. B. (1997). Propagation of prion strains through specific conformers of the prion protein. Journal of Virology 71, 9032-9044.[Abstract]
Somerville, R. A. (1999). Host and transmissible spongiform encephalopathy agent strain control glycosylation of PrP. Journal of General Virology 80, 1865-1872.[Abstract]
Taraboulos, A., Rogers, M., Borchelt, D. R., McKinley, M. P., Scott, M., Serban, D. & Prusiner, S. B. (1990). Acquisition of protease resistance by prion proteins in scrapie-infected cells does not require asparagine-linked glycosylation. Proceedings of the National Academy of Sciences, USA 87, 8262-8266.[Abstract]
Telling, G. C., Scott, M., Mastrianni, J., Gabizon, R., Torchia, M., Cohen, F. E., DeArmond, S. J. & Prusiner, S. B. (1995). Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein. Cell 83, 79-90.[Medline]
Telling, G. C., Parchi, P., DeArmond, S. J., Cortelli, P., Montagna, P., Gabizon, R., Mastrianni, J., Lugaresi, E., Gambetti, P. & Prusiner, S. B. (1996). Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity. Science 274, 2079-2082.
Zulianello, L., Kaneko, K., Scott, M., Erpel, S., Han, D., Cohen, F. E. & Prusiner, S. B. (2000). Dominant negative inhibition of prion formation diminished by deletion mutagenesis of the prion protein. Journal of Virology 74, 4351-4360.
Received 3 March 2000;
accepted 27 June 2000.