The use of a drug resistance cartridge for in vitro insertion and deletion mutagenesis of a cosmid clone

Abstract

A drug-resistant cartridge was employed in the construction of families of insertion mutants of a cosmid clone. The cartridge contains a cml gene and has identical restriction enzyme sites, EcoRI, BamHI, SalI, andPstI, on both ends. The families of mutants were made by ligation of the cartridge to the cosmid, which was linearized or partially digested, followed by in vitro packaging and transduction. From these families we selected cosmid derivatives which either have a unique BamHI site at a predetermined site in the cosmid or have deletions covering different portions of the original clone. The extent of a large gene cluster cloned into the original cosmid was identified by confirming the gene function in some of the deletion mutants. The possibility for further and various uses of this cartridge is discussed.