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 Sound by scre ening of a lambda gt11 cDNA expression library using a modification of the previously established ligand-blotting procedure to detect DNA- a nd RNA-binding proteins. The essentials of our modifications are the u se of (i) digoxigenin-labeled viroid RNA, (ii) low concentration of th e labeled probes and (iii) an expression library that allows the direc t isolation of cDNA clones. The analysis of various isolated clones sh owed that this method is reliable for RNA-ligand screening and North-W estern blotting. Applied to viroid RNA, these experimental tools provi de the precondition for further studies on the specificity of the isol ated proteins.