INSERTIONAL MUTAGENESIS BY A MODIFIED IN-VITRO TY1 TRANSPOSITION SYSTEM

Transposable elements are useful tools for insertional mutagenesis and have many potential applications in the characterization of complex g enomes. Here we describe a system which facilitates the construction o f large transposon insertion libraries useful for genome sequencing an d functional genomic analysis. We developed two transposons, TyK and T yK'GFP+, which can be introduced into target DNAs by Ty1-mediated tran sposition in vitro, and several modifications which decrease the frequ ency of false transposition events and direct the recovery of transpos itions into Passenger rather than vector DNA. Insertions of TyK'GFP+ a dditionally may yield fusions to the Aequorea green fluorescent protei n (GFP), useful in studies of gene expression and protein targeting. T ransposition in vitro was obtained into target DNAs of up to 50 kb in size, restriction mapping showed insertion to be relatively random, an d the sequence of 55 insertion sites showed neither strong site nor ba se compositional preference. Our data suggest that TyK-based artificia l transposons will be suitable for a variety of genetic applications i n many organisms. (C) 1997 Elsevier Science B.V.