Search for specific DNAs in creutzfeldt-jakob infectious brain fractions using “nick translation”

Abstract

Scrapie agent has been reported to contain an “essential DNA” component in the viroid size range (R. F. Marsh, T. B. Malone, J. S. Semancik, W. D. Lancaster, and R. P. Hanson, Nature (London) 275, 146–147, 1978). The human transmissible encephalopathy, Creutzfeldt-Jakob disease (CJ) was searched for unique CJ DNA components in highly infectious brain fractions containing negligible amounts of nuclear DNA. DNA was quantitatively extracted from these fractions and labeled to high specific activity by “nick translation” with 32P-nucleotides under conditions where the labeled DNA maintained its original length. Labeled DNAs were analyzed by gel electrophoresis with special emphasis on molecules in the viroid size range. Reconstruction experiments showed that ⩽0.1 pg DNA could be detected with these procedures. No reproducible CJ specific DNA bands were detectable when compared to normal brain fractions. Restriction enzyme analysis of these DNAs also showed no CJ specific bands integrated into larger molecular weight DNA. Use of Brij 58 to release more infective physical particles into the rough endoplasmic reticulum also did not show CJ specific DNAs. Comparison of the amounts of known infective units by animal assay, assuming a 400-base DNA sequence, indicated that CJ specific DNAs should be detectable in these fractions with the methods used. These results appear to rule out usual forms of double-stranded or partially double-stranded DNAs as components of transmissible encephalopathy agents. A systematic analysis of other specific nucleic acid components of these agents is feasible using the highly sensitive in vitro labeling approach employed here.