Nuclease-resistant polyadenylated RNAs of significant size are detected by PCR in highly purified Creutzfeldt-Jakob disease preparations.
The molecular nature of the 'unconventional viruses' that cause slow, progressive brain deterioration is still poorly understood. As part of a reinvestigation of potential agent-specific nucleic acids, we developed a protocol for enriching agent-specific sequences. This protocol uses extensive micrococcal nuclease digestion followed by rate zonal sucrose sedimentation. Most of the infectivity in the gradient (84%) had a characteristic mean size of approximately 120S, and was resolved from 70% of a host glycoprotein (PrP) that can cosediment with infectivity. In infectious size fractions, nucleic acids were reduced approximately one million-fold with respect to starting brain homogenate, and specific purification of infectivity was approximately 100,000-fold with respect to nucleic acid. Using a novel polymerase chain reaction strategy, we were able to amplify RNA species in these fractions. Remarkably, host polyadenylated sequences of 1 to over 4 kb were detected in the nuclease-protected infectious fractions. These strategies set the stage for the identification of similar nucleic acids that may be specific for the CJD agent.
Akowitz
A
A
Yale University School of Medicine, New Haven, Connecticut 06510.
Sklaviadis
T
T
Manuelidis
E E
EE
Manuelidis
L
L
D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D007562:Creutzfeldt-Jakob Syndrome / Q000382:microbiology*; D008836:Micrococcal Nuclease / Q000378:metabolism; D011061:Poly A / Q000032:analysis*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis*; D012333:RNA, Messenger
D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D008836:Micrococcal Nuclease