Simultaneous deletion of the intervening sequences from the human interferon-gamma gene by oligodeoxynucleotide-directed mutagenesis.

ABSTRACT

Oligodeoxynucleotide (oligo)-directed mutagenesis was used to simultaneously delete the three intervening sequences (IVS) from the human interferon-gamma (IFN-gamma) gene. Prior to the deletion experiment, the two largest IVS of the native gene were shortened by restriction enzyme digestion and subsequent ligation (IVS-1 was reduced from 1239 bp to 399 bp and IVS-3 from 2425 bp to 183 bp). This modified gene was cloned into vector M13mp9. Three oligos [21-25 nucleotides (nt) long] were synthesized, one to bridge each of the three introns. A different set of three oligos was made for use as hybridization probes to identify colonies containing the correct deletion. A study was made of enzymatic reaction and primer annealing conditions needed to optimize intron deletions. The efficiency of the simultaneous deletions was higher than the product of the efficiencies of the deletions of the individual IVS, suggesting that the deletion events are not independent. The gene created by the simultaneous deletion was cloned into an Escherichia coli plasmid expression vector and IFN-gamma activity was produced.