MOLECULAR-CLONING OF SINGLE-STRANDED-DNA PURIFIED FROM SCRAPIE-INFECTED HAMSTER BRAIN

Homogenized normal and scrapie-infected hamster brains were subjected to subcellular fractionation. A single band of ssDNA corresponding to about 1.2 kb was purified by alkaline gel-electrophoresis from the nucleic acid content of enriched preparations of mitochondria/tubulofilamentous particles. The ssDNA was synthesized into double-stranded DNA using Taq polymerase with four dNTP for extension. The cDNA synthesized was inserted in M13mp10, cloned and sequenced. An unusual palindromic six-base TACGTA repeat sequence was obtained and confirmed by an independent automated pathway and by cutting with a specific restriction enzyme. Comparison of the nucleotide sequence of the inserted DNA with the GenBank nucleotide database revealed no significant homology with those sequences. A probe prepared from the Nar 50 clone hybridized with the scrapie DNA band of about 1.2 kb noted above; however, no hybridization was observed with normal DNA, thus confirming the presence of ssDNA in scrapie. The presence of palindromic sequences in the scrapie genome could explain why many previous searches have revealed no evidence for a scrapie-specific nucleic acid.