Gene 198 (1997) 27–35 Insertional mutagenesis by a modified in vitro Ty1 transposition system Levi A. Garraway, Luiz R.O. Tosi, Yixin Wang, Jeffrey B. Moore, Deborah E. Dobson, Stephen M. Beverley * Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Received 24 January 1997; accepted 18 April 1997; Received by C.M. Kane Abstract Transposable elements are useful tools for insertional mutagenesis and have many potential applications in the characterization of complex genomes. Here we describe a system which facilitates the construction of large transposon insertion libraries useful for genome sequencing and functional genomic analysis. We developed two transposons, TyK and TyK∞GFP+, which can be introduced into target DNAs by Ty1-mediated transposition in vitro, and several modifications which decrease the frequency of false transposition events and direct the recovery of transpositions into passenger rather than vector DNA. Insertions of TyK∞GFP+ additionally may yield fusions to the Aequorea green fluorescent protein (GFP), useful in studies of gene expression and protein targeting. Transposition in vitro was obtained into target DNAs of up to 50 kb in size, restriction mapping showed insertion to be relatively random, and the sequence of 55 insertion sites showed neither strong site nor base compositional preference. Our data suggest that TyK-based artificial transposons will be suitable for a variety of genetic applications in many organisms. © 1997 Elsevier Science B.V. Keywords: Transposon; DNA sequencing; Green fluorescent protein; Gene fusions 1. Introduction et al., 1986). Burns et al. (1994) used this approach to generate nearly 3000 independent Saccharomyces cere- visiae protein fusions to Escherichia coli b-galactosidase, The characterization of complex eukaryotic genomes facilitating studies of cellular localization and function. by determination of their complete sequence has resulted Similarly, Chun and Goebl (1996) used a related in a shift in emphasis by genome scientists (Johnston, approach to identify essential genes affecting bud mor- 1996; Pennisi, 1996). Now, the major task is to gain an phogenesis in S. cerevisiae. Both of these studies required understanding of the cellular function of genes and their the generation of large, random transposon insertion utilization under different environmental or developmen- libraries, and here we describe a convenient system tal conditions. Transposable elements offer many poten- which facilitates their construction for use in any tial advantages suitable for incorporation into genome- organism. wide studies, as modified transposons are popular tools In selecting transposition systems, important factors for insertional mutagenesis, ‘‘primer island’’ sequencing are the randomness of insertion site and the ability to and the construction of gene fusions (Casadaban et al., design transposons for specific applications. Often, spe- 1983; Silhavy et al., 1984; Seifert et al., 1986; Berg et al., cialized bacterial strains or manipulations are required 1989; Burns et al., 1994; Sherratt, 1995; Morgan et al., for transposon delivery, which can prove daunting to 1996). One powerful approach for genome scientists is novices. A yeast Ty1-based transposition system was the method of shuttle transposon mutagenesis (Seifert recently described which offers several advantages (Devine and Boeke, 1994). The transposition reaction * Corresponding author. Present address: Department of Molecular Microbiology, Washington University School of Medicine, 660 S. is performed in vitro, employing only a linear Ty1 donor Euclid Avenue, St. Louis, MO 63105, USA. Tel.: +1 314 7472630; fragment, a target plasmid and transposase in the form Fax: +1 314 747-2634; e-mail: beverley@borcim.wustl.edu of Ty1 VLPs which are readily prepared (Boeke et al., 1985). Although endogenous Ty1 itself shows a prefer- Abbreviations: GFP, green fluorescent protein; LTR, long terminal ence for certain locations within the yeast genome such repeat; PEG, poly(ethylene glycol ); RT, reverse transcriptase; VLP, virus-like particle. as tRNA loci or other transposon LTRs (Ji et al., 1993), 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0378-1119(97)00288-6