NR ADDP
AU Dear,D.V.; Fitzmaurice,T.J.; Goymer,P.J.A.; Richards,S.J.
TI Rapid expression of polymorphic ovine prion proteins and studies on their protease sensitivity
QU Brain Research Bulletin 1999 Jan 1; 48(1): 89-92
PT journal article
AB We have used coupled and uncoupled in vitro transcription/translation to express rapidly aglycosyl ovine prion proteins from ovine genomic DNA genotyped for scrapie susceptible and nonsusceptible polymorphisms. Unlike previous in vitro studies of prion proteins, this method does not require cloning or laborious extractions [2]. To our knowledge, this is the first report of ovine PrP expression at low (ng) levels under the control of an Escherichia coli promoter and ribosome binding site both coded for in the polymerase chain reaction primer. The rapidity of this approach could form the basis of a high throughput screening assay for PrP interactions, as proteins were expressed in a matter of hours from genomic DNA as the starting material. There was no difference observed in proteinase K sensitivity between prion translation products containing either scrapie susceptible or nonsusceptible polymorphisms.
IN Die Autoren sind in der Lage, binnen Stunden genomische DNA in nicht glykosyliertes Protein umzusetzen. Dazu verwenden sie PCR Primer, in denen sich bereits ein Coil Promoter für E.cloi sowie eine Ribosomenbindungsstelle befinden. Sie fanden bei den damit produzierten Prionproteinen aus für Scrapie besonders empfindlichen, bzw. besonders unempfänglichen Schafen keine Unterschiede hinsichtlich der Empfindlichkeit gegenüber Proteinase K.
ZR 15
MH Animal; Drug Resistance; Endopeptidases/*pharmacology; Polymerase Chain Reaction; Polymorphism (Genetics)/*physiology; Prions/drug effects/*genetics/*metabolism; Sheep/*metabolism; Support, Non-U.S. Gov't; Time Factors; Transcription, Genetic/physiology; Translation, Genetic/physiology
AD Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, UK. dvd20@cam.ac.UK
SP englisch
PO USA