NUCLEASE-RESISTANT POLYADENYLATED RNAS OF SIGNIFICANT SIZE ARE DETECTED BY PCR IN HIGHLY PURIFIED CREUTZFELDT-JAKOB DISEASE PREPARATIONS

被引:38
作者
AKOWITZ, A [1 ]
SKLAVIADIS, T [1 ]
MANUELIDIS, EE [1 ]
MANUELIDIS, L [1 ]
机构
[1] YALE UNIV,SCH MED,NEW HAVEN,CT 06510
关键词
Creutzfeldt-Jakob disease; nucleic acid; PCR; RNA; scrapie; slow virus;
D O I
10.1016/0882-4010(90)90038-R
中图分类号
R392 [医学免疫学]; Q939.91 [免疫学];
学科分类号
100102 ;
摘要
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 ~ 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 ~ 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. © 1990.
引用
收藏
页码:33 / 45
页数:13
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