NR AAEL
AU Akowitz,A.; Sklaviadis,T.K.; Manuelidis,E.E.; Manuelidis,L.
TI Nuclease-resistant polyadenylated RNAs of significant size are detected by PCR in highly purified Creutzfeldt-Jakob disease preparations
QU Microbial Pathogenesis 1990 Jul; 9(1): 33-45
PT journal article
AB 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.
MH Base Sequence; Centrifugation, Density Gradient; Creutzfeldt-Jakob Syndrome/*microbiology; Micrococcal Nuclease/metabolism; Poly A/*analysis; Polymerase Chain Reaction; RNA/*analysis; RNA, Messenger; Support, U.S. Gov't, P.H.S.
AD Yale University School of Medicine, New Haven, Connecticut 06510.
SP englisch
PO England