Isolation of viroid-RNA-binding proteins from an expression library with nonradioactive-labeled RNA probes.
The detection and isolation of cDNAs of tomato proteins that are able to bind to viroid RNA molecules are described. They were found by screening of a lambda gt11 cDNA expression library using a modification of the previously established ligand-blotting procedure to detect DNA- and RNA-binding proteins. The essentials of our modifications are the use of (i) digoxigenin-labeled viroid RNA, (ii) low concentration of the labeled probes and (iii) an expression library that allows the direct isolation of cDNA clones. The analysis of various isolated clones showed that this method is reliable for RNA-ligand screening and North-Western blotting. Applied to viroid RNA, these experimental tools provide the precondition for further studies on the specificity of the isolated proteins.
Universität Hamburg, Institut für Allgemeine Botanik, Germany.
R
R
Werner
H P
HP
Mühlbach
M C
MC
Guitton
D015723:Gene Library; D005822:Genetic Vectors; D018551:Solanum lycopersicum / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D015347:RNA Probes; D012367:RNA, Viral / Q000302:isolation & purification*; D016601:RNA-Binding Proteins / Q000302:isolation & purification*; D014772:Viroids / Q000235:genetics*
D010940:Plant Proteins; D015347:RNA Probes; D012367:RNA, Viral; D016601:RNA-Binding Proteins