A. DNA SEQUENCES & CHROMOSOME STRUCTURE
o Discovery of tissue-specific arrangements of centromeres in interphase nuclei (6)
o Uncovering 3D movements of centromeres during differentiation & stress in neurons (7,8)
o Showing different species-specific DNA sequences maintain the same conserved tissue specific arrangements of centromeres in interphase neuron and glial nuclei during mammalian evolution (9)
o Glioblastomas (brain tumors) lose tissue-specific specific centromere arrangements
• Discovery of human Long interspersed Repeats (LINES) and their organization
o Identification, isolation and sequencing of LINES, incuding ORFs, and murine homologies (10-12)
o LINES collect in bands on chromosome arms, as shown by non-isotopic in-situ hybridization (12)
o LINES cluster with tissue specific genes in giemsa-dark chromosome bands whereas short ALU elements (SINES) concentrate with early replicating housekeeping genes in light bands by high resolution in-situ hybridization and pulse-field DNA analysis (13,14)
o Endogenous retroviruses have species-specific addresses on chromosome arms (15)
o Whole chromosomes are maintained as cohesive compact structures in interphase nuclei rather than in diffusely extended spaghetti-like strands (16)
o Application of in-situ detection of interphase chromosomes for tumors and genetic diseases (17,18)
o Genetic instability is a key feature of glioblastomas (18a)
o Unified model for chromosome DNA compaction in heterochromatin and local unfolding of chromatids for organized transcription and replication of long (megabase) genomic DNA (19-21)
o Transgenic insertion of tandem ORF DNA arrays into chromosome arms leads to gene silencing with heterochromatic ultrastructure, and its positioning with centromere domains in nuclei (22)
Summary: Identified DNA repeats are not “junk”, but are critical recognition elements that define specific chromosome regions and structures, and are part of the organized function of gene sets
B. TRANSMISSIBLE ENCEPHALOPATHIES (TSEs)
• Experimental transmissions of human Creutzfeldt-Jakob Disease (CJD) and TSEs
o Transmissions of human CJD to small laboratory animals (guinea pigs, hamsters, mice) (23-25). Previously it was believed that CJD and human agents could only infect primates. Experimental rodents reproduced typical spongiform lesions of humans with sporadic CJD (sCJD), and this agent was clearly different from sheep scrapie agents by incubation time and pathology.
o Infection transmitted by white blood cells with realization of silent inadvertent TSE spread (26,27)
o Positive CJD transmission from cornea inoculated into the eye (28)
o No maternal transmission from infected parent guinea pigs for 12 years, i.e., agent not germline (29)
o sCJD agent and Asiatic CJD agents markedly different, implicating environment-specific source (30)
o sCJD agent maintains its identity despite passage through different species, i.e. not host-encoded (30)
o Cultures derived from infected animals, or cells exposed to infectious material, can become transformed, and can also cause huge tumors in nude mice (31-33).
• Additional human agent-models and viral biology
o First demonstration of amyloid fibrils (SAF) with prion protein (PrP) in CJD (34-36)
o PrP is a glycoprotein of 34kd, and deglycosylation does not alter infectivity (36,37)
o The infectious agent replicates exponentially, whereas only after a long silent phase does it induce abnormal PrP, clinical signs and neurodegeneration (38). Thus abnormal PrP (PrP-res) appears as a pathological response to infection, rather than the infectious agent
o CJD agent, as other foreign viral agents, can induce innate immune responses early after infection. In contrast, PrP-res, a host-encoded molecule, does not induce these responses (39,40).
o Many experiments show that the PrP-res (the “infectious” prion) does not correlate with infectivity; CJD-infected microglia with minute amounts of PrP and no PrP-res have very high infectivity (41)
o Silent infection with the sCJD agent can prevent super-infection by the more virulent Asiatic (FU-CJD) agent in mice, and no intermediate mixed prion or “chimeric” agents are formed (42,43)
o sCJD cells in vitro, with no detectable PrP-res, also resist superinfection by other TSE agents (44)
o Altering prion protein bands by growth in different cell types does not alter agent properties (45)
o Warned MRC that mad-cow (BSE) could transmit to humans despite their different PrP sequences (46). Sterilization methods devised to prevent inadvertent spread of vCJD by medical instruments (47)
o Transmission of UK vCJD (BSE) and of New Guinea kuru with transgenic mouse studies showing the opposite result than predicted by species-specific PrP conversion (66,67). This further underscores an epidemic and geographic-specific environmental source of agents (that can be prevented) rather than spontaneous conversion of a host encoded protein to an infectious (prion) form
• Characterization of TSE infectious particles
o Gentle disaggregation of PrP (converting PrP-res back to sensitive PrP) does not alter infectivity
o After PrP disaggregation, infectious particles band with a defined virus-like size and density. These particles also separate from the majority of host PrP (68-70).
o Field flow fractionation and HPLC define infectious particles migrate with spheres that are ~25nm in diameter and with molecules that are~ 3e6 to1e7 daltons (71)
o Infectious particles, as many other viruses, resist nuclease digestion. Similarly, if nucleic acid-protein complexes are disrupted (by boiling in SDS or with GdnSCN) TSE infectivity is lost (69, 69a)
o With viral disruption, long nucleic acids are released, including proteins that bind nucleic acid. The RNAs and DNAs are in excess of that needed for a TSE genome, and include endogenous retroviral RNA and its protective gag protein (70, 70a, 72)
o More purified infectious material has 25nm dense particles ultrastructurally that do not bind PrP antibodies. Particles of 25nm also seen in viruslike arrays within some infected cells. These arrays are distinct and separated from intracellular PrP amyloid and normal membrane PrP (73)
o Development of a reliable, rapid tissue culture assay for many different CJD and scrapie strains. This has accelerated essential evaluations of infectivity, not just PrP-res. These assays have also facilitated analysis of many gradients and conditions not feasible in animal studies (74,75)
o Discovery of new environmental circular DNAs of 1.8-2.4kb (Sphinx DNA elements) that concentrate in highly infectious preparations (2,500x normal). These DNAs have been found in infectious fractions from cultures and brain and with a variety of CJD and scrapie agents (76). Parts of these sequences are linked to much larger plasmids that infect gram-negative antibiotic resistant Acinetobacteria strains and are common in the environment worldwide. They thus may have a significant role in covert infection, neurodegenration and tumor formation.
Summary: Infectious TSE particles have a virus like biology and structure that is inconsistent with prion protein models of infection. All highly infectious preparations contain significant amounts of nucleic acid when examined with modern molecular techniques.
A nucleic acid genome that can mutate is the most parsimonious way to explain all the distinct TSE agent strains, and a viral structure (nucleic acid protected by protein) also explains the resistance of TSE particles to digestion in the GI tract, a known route for BSE infection.
Do circular Sphinx DNA elements act as cofactors in TSE infection, or induce TSE disease?
1) Manuelidis, L., Repeating restriction fragments of human DNA. Nucleic Acids Res. 3:3063-3076, 1976.
2) Manuelidis, L., Complex and simple sequences in human repeated DNAs. Chromosoma 66:1-21, 1978.
3) Manuelidis, L., Chromosomal localization of complex and simple repeated human DNAs. Chromosoma 66:23-32, 1978.
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18a) Manuelidis, L., Genomic stability and instability in different neuroepithelial tumors: A role for chromosome structure? J. Neuro-Oncol, 18:225-239, 1994.
19) Sedat, J. and Manuelidis, L., A direct approach to the structure of eukaryotic chromosomes. Cold Spring Harbor Symp. Quant. Biol. 42:331-350, 1978.
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21) Manuelidis, L., A view of interphase chromosomes. Science 250:1533-1540, 1990.
22) Manuelidis, L., Heterochromatic features of an 11 Mb transgene in brain cells. Proc. Natl. Acad. Sci. USA 88:1049-1053, 1991.
23) Manuelidis, E.E., Kim, J, Angelo, J.N., Gorgacz, E.J. and Manuelidis, L., Serial propagation of Creutzfeldt-Jakob Disease to guinea pigs. Proc. Natl. Acad. Sci. USA 73: 223-7, 1976.
24) Manuelidis, E.E., Gorgacz, E.J. and Manuelidis, L., Interspecies transmission of Creutzfeldt-Jakob disease to Syrian hamsters with reference to clinical syndromes and strains of agent. Proc. Nalt. Acad. Sci. USA 75:3432-3436, 1978.
25) Manuelidis, E.E., Gorgacz, E.J. and Manuelidis, L., Transmission of Creutzfeldt-Jakob disease to mice with scrapie-like syndromes. Nature 271:778-779, 1978.
26) Manuelidis, E.E., Gorgacz, E.J. and Manuelidis, L., Viremia in experimental Creutzfeldt-Jakob disease. Science 200:1069-1071, 1978.
27) Manuelidis, E.E., Kim, J.H., Mericangas, J.R. and Manuelidis, L., Transmission to animals of Creutzfeldt-Jakob disease from human blood. Lancet ii:896-897,1985.
28) Manuelidis, E.E., Angelo, J.N., Gorgacz, E.J., Kim, J.H. and Manuelidis, L., Experimental Creutzfeldt-Jakob disease transmitted via the eye with infected cornea. N. Eng. J. Med. 296:1334-1336, 1977.
29) Manuelidis, E.E. and Manuelidis, L., Experiments on maternal transmission of Creutzfeldt-Jakob disease in guinea pigs. Proc. Soc. Exp. Biol. Med. 160:233-236, 1979.
30) Manuelidis, L., Murdoch, G. and Manuelidis, E.E., Potential involvement of retroviral elements in human dementias. Ciba Found. Symp. 135: 117-134, 1988.
31) Manuelidis, E.E., Kim, J.H. and Manuelidis, L., Novel biological properties of Creutzfeldt-Jakob infected brains in vitro. Banbury Rep. (Cold Spring Harbor) 15: 413-424, 1983.
32) Oleszak, E.L., Manuelidis, L. and Manuelidis, E.E., In vitro transformation elicited by Creutzfeldt-Jakob infected brain material. J. Neuropath. Exp. Neurol. 45:489-502, 1986.
33) Manuelidis, E.E., Fritch, W.W., Kim, J.H. and Manuelidis, L., Immortality of cell cultures derived from brains of mice and hamsters infected with Creutzfeldt-Jakob disease agent. Proc. Natl. Acad. Sci. USA 84:871-875, 1987.
34) Merz, P.A., Sommerville, R., Wisniewski, H.M., Manuelidis, L. and Manuelidis, E.E., Scrapie-associated fibrils in Creutzfeldt-Jakob disease. Nature 306:474-476, 1983. (see also Diringer on p 476-8 for PrP fibrils)
35) Manuelidis, L., Valley, S. and Manuelidis, E.E., Specific proteins in Creutzfeldt-Jakob disease and scrapie share antigenic and carbohydrate determinants. Proc. Nat. Acad. Sci. USA 82:4263-4267, 1985.
36) Sklaviadis, T., Manuelidis, L. and Manuelidis, E.E., Characterization of major peptides in Creutzfeldt-Jakob disease and scrapie. Proc. Natl. Acad. Sci. USA 83:6146-6150, 1986.
37) Manuelidis, L., Sklaviadis, T. and Manuelidis, E.E., Evidence suggesting that PrP is not the infectious agent in Creutzfeldt-Jakob disease. EMBO J. 6:341-347, 1987.
38) Manuelidis, L., Fritch W., Infectivity and host responses in Creutzfeldt-Jakob disease. Virology, 216:46-59, 1996.
39) Baker, CA. and Manuelidis, L. Unique inflammatory RNA profiles of microglia in Creutzfeldt-Jakob disease. Proc. Natl. Acad. Sci. USA 100: 675-679, 2003.
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41) Baker, C.A., Martin, D, Manuelidis, L. Microglia from Creutzfeldt-Jakob Disease-infected brains are infectious and show specific mRNA activation profiles. J. Virol. 76: 10905-13, 2002.
42) Manuelidis, L., Vaccination with an attenuated CJD strain prevents expression of a virulent agent. Proc. Nat. Acad. (USA) 95:2520-2525, 1998.
43) Manuelidis, and Lu, Z-Y Attenuated Creutzfeldt-Jakob Disease agents can hide more virulent infections. Neurosci. Lett. 293: 163-166, 2000.
44) Nishida, N., Katamine, S. Manuelidis, L. Reciprocal interference between specific CJD and scrapie agents in neural cell cultures. Science 310:493-496 2005.
45) Arjona A, Simarro L, Islinger F, Nishida N, Manuelidis L. Two Creutzfeldt-Jakob disease agents reproduce prion protein-independent identities in cell cultures. Proc Natl Acad Sci U S A. 101:8768-73, 2004.
46) Manuelidis, L. Penny wise, Pound foolish – a retrospective. Science 290: 2257, 2000 (letter)
47) Manuelidis, L., Decontamination of Creutzfeldt-Jakob Disease and other transmissible agents. J NeuroVirol., 3: 62-65, 1997.
66) Manuelidis, L, Liu, Y, Mullins, B.: Strain-specific viral properties of variant Creutzfeldt–Jakob Disease (vCJD) are encoded by the agent and not by host prion protein. J Cell Biochem 106:220–231, 2009.
67) Manuelidis, L, Chakrabarty, T, Miyazawa, K, Nduom, N-A, and Emmerling, K. The kuru infectious agent is a unique geographic isolate distinct from Creutzfeldt–Jakob disease and scrapie agents. Proc. Natl. Acad. Sci. USA 106: 13529-13534, 2009.
68) Sklaviadis T., Manuelidis L., and Manuelidis, E.E., Physical properties of the Creutzfeldt-Jakob disease agent. J. Virol. 63:1212-1222, 1989.
69) Sklaviadis, T., Akowitz, A., Manuelidis, E.E., and Manuelidis, L., Nuclease treatment results in high specific purification of Creutzfeldt-Jakob disease infectivity with a density characteristic of nucleic acid-protein complexes. Arch. Virol. 112:215-229, 1990.
69a) Manuelidis, L., Sklaviadis, T., Akowitz, A., Fritch, W., Viral particles are required for infection in neurodegenerative Creutzfeldt-Jakob disease. Proc. Natl. Acad. Sci. USA, 92:5124-5128, 1995.
70) Manuelidis, L. Transmissible Encephalopathies: Speculations and realities. Viral Immunology 16: 123-129, 2003
72) Akowitz, A., Manuelidis, E.E., and Manuelidis, L., Protected endogenous retroviral sequences copurify with infectivity in experimental Creutzfeldt-Jakob Disease. Arch. Virol. 130:301-316, 1993.
73) Manuelidis, L., Yu, Z.-X., Barquero, N., and Mullins, B. Cells infected with scrapie and Creutzfeldt-Jakob disease agents produce intracellular 25-nm virus-like particles. Proc Natl Acad Sci USA 104, 1965-1970, 2007. (also see review: J Cell Biochem. 100, 897-915, 2006 for isolated particles)
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75) Sun, R, Liu, Y., Zhang, H., and Manuelidis, L. Quantitative recovery of scrapie agent with minimal protein from highly infectious cultures. Viral Immunol. 21: 293-302, 2008.
76) Manuelidis, L. Nuclease resistant circular DNAs copurify with infectivity in scrapie and CJD. J Neurovirol eprint Dec 2010; print 17: 131-145, 2011 DOI: 10.1007/s13365-010-0007-0