Visualization of viral candidate cDNAs in infectious brain fractions from Creutzfeldt-Jakob disease by representational difference analysis.
Creutzfeldt-Jakob Disease (CJD), a neurodegenerative and dementing disease of later life, is caused by a viruslike entity that is incompletely characterized. As in scrapie, all more purified infectious brain preparations contain nucleic acids. However, it has not been possible to visualize unique bands that may derive from a viral genome. We here used a subtractive strategy known as representational difference analysis (RDA) to uncover such sequences. To reduce the complexity of starting target nucleic acids, sucrose gradients were first used to select nuclease resistant particles with a defined 120S size. In CJD this single 120S gradient peak is highly enriched for infectivity, and contains reduced amounts of PrP (Proc. Natl. Acad. Sci. 92, 5124-8, 1995). Parallel 120S fractions from uninfected brain were made to generate subtractor sequences. 120S particles were lysed in GdnSCN, and ng amounts of released RNA were purified for random-primed cDNA synthesis. To capture representative fragments of 100-500 bp, cDNAs were cleaved with Mbo I for adaptor ligation and amplification. In the first experiment with moderate RDA selection, it was possible to visualize clones from CJD cDNA that did not hybridize to control cDNA. In the second experiment, more exhaustive subtractions yielded a discrete set of CJD derived gel bands. Competitive hybridization showed a subset of these bands were not present in either the control 120S cDNA or in the hamster genome. This represents the first demonstration of apparently CJD-specific nucleic acid bands in more purified infectious preparations. Although exhaustive cloning, sequencing and correlative titration studies need to be done, it is encouraging that most of the viral candidates selected thus far have no significant homology with any previously described sequence in the database.
Dron
M
M
Yale Medical School, New Haven, Connecticut 06510, USA.
Manuelidis
L
L
D000818:Animals; D001923:Brain Chemistry / Q000235:genetics; D003001:Cloning, Molecular; D007562:Creutzfeldt-Jakob Syndrome / Q000235:genetics* / Q000821:virology*; D006224:Cricetinae; D018076:DNA, Complementary / Q000032:analysis / Q000302:isolation & purification; D004279:DNA, Viral / Q000032:analysis* / Q000302:isolation & purification; D004195:Disease Models, Animal; D005784:Gene Amplification; D005820:Genetic Testing; D009693:Nucleic Acid Hybridization; D010761:Phosphorus Radioisotopes; D016133:Polymerase Chain Reaction
D018076:DNA, Complementary; D004279:DNA, Viral; D010761:Phosphorus Radioisotopes