NR AITO
AU Narang,H.K.
TI Molecular cloning of single-stranded DNA purified from scrapie-infected hamster brain
QU Research in Virology 1993 Sep-Oct; 144(5): 375-87
PT journal article
AB 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.
IN Nachdem er die normalen zellulären Nukleinsäuren durch Abzentrifugieren der Zellkerne und enzymatisches Verdauen von DNA und RNA entfernt hatte, reicherte Narang die tubulofilamentösen Partikel an, die er Nemaviren nennt. Durch eine Proteinasebehandlung und Phenolextraktion isolierte er daraus eine anscheinend scrapiespezifische DNA mit einer scheinbaren Länge von 1,2 kb, die erstaunlicherweise nach Ethidiumbromidfärbung selbst in einem alkalischen Gel kräftig leuchtet. Diese DNA klonierte er in M13 und sequenzierte sie an. Sie bestand aus einer endlosen Wiederholung von nur 6 palindromischen Basenpaaren. Da diese klonierte DNA aber mit der 1,2 kb-Bande hybridisiert, scheint es sich nicht einfach um einen Artefakt zu handeln.
MH Amino Acid Sequence; Animal; Base Sequence; Brain/*microbiology; Cloning, Molecular; DNA, Single-Stranded/*chemistry; DNA, Viral/*chemistry; Female; Hamsters; Mesocricetus; Molecular Sequence Data; Prions/*genetics; Scrapie/*microbiology; Support, Non-U.S. Gov't
AD Public Health Laboratory, General Hospital, Newcastle Upon Tyne, UK
SP englisch
PO Frankreich
OR Prion-Krankheiten 6