Prions and nucleic acids: search for "residual" nucleic acids and screening for mutations in the PrP-gene.
Studies on prions involve nucleic acid chemistry under two headings: i) do infectious prion particles contain nucleic acids? ii) is it possible by a simple procedure to screen the prion protein (PrP)-gene for mutations? The return refocusing gel electrophoresis technique was developed to detect by sensitive silver staining homogeneous and heterogeneous nucleic acids extracted from highly purified scrapie prion preparations. With this method all kinds of nucleic acids from a length of 13 nucleotides up to several thousand could be recovered and detected with a yield over 90%. Despite extensive nuclease digestion some small polynucleotides remained. The results define clear restrictions for a hypothetical scrapie-specific nucleic acid. If homogeneous in size, such a molecule would be smaller than 80 nucleotides in length at a particle-to-infectivity ratio near unity; if heterogeneous, scrapie-specific nucleic acids would have to include molecules smaller than 240 nucleotides. To detect mutations in the PrP-gene, either known mutations from human prion diseases or artificial ones in transgenic animals, or to screen for not yet identified mutations in patients, a method is required which guarantees detection of mutations which might occur in every single position of the whole PrP-ORF. It will be shown that a combination of PCR and temperature-gradient gel electrophoresis fulfils these requirements. By thermodynamic calculations the shift in the gel electrophoresis due to a mutation can be calculated depending on the position of the mutation. The theoretical results were tested with the mutations known so far.
Riesner
D
D
Heinrich-Heine-Universität Düsseldorf, Germany.
Kellings
K
K
Wiese
U
U
Wulfert
M
M
Mirenda
C
C
Prusiner
S B
SB
D000818:Animals; D006224:Cricetinae; D004252:DNA Mutational Analysis / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008647:Mesocricetus / Q000235:genetics; D009691:Nucleic Acid Denaturation; D009696:Nucleic Acids / Q000032:analysis*; D009841:Oligonucleotides / Q000302:isolation & purification; D016366:Open Reading Frames; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011328:Prions / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011489:Protein Denaturation; D012608:Scrapie / Q000378:metabolism; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013696:Temperature
D009696:Nucleic Acids; D009841:Oligonucleotides; D011328:Prions