SIMPLE PROCEDURE FOR CREATION OF IN-FRAME DELETION MUTATIONS THROUGHOUT AN OPEN READING FRAME

A general method is presented for randomly mutagenizing open reading f rames (ORF) to generate in-frame deletions and insertions. The protoco l requires expression of the ORF of interest as a hybrid ORF-beta-gala ctosidase fusion protein. This allows colorimetric screening for beta- galactosidase activity during subsequent mutagenesis steps. Consequent ly, proteins with no suitable phenotypic selection or screening proper ties can be readily screened for mutations that disrupt and subsequent ly rescore the reading frame of the hybrid protein. In addition, this system provides gene expression Sor subsequent biochemical analysis of the mutant proteins. The bovine papillomavirus type 1 (BPV-1) E1 ORF has been mutagenized using this method as an example.