Libraries of green fluorescent protein fusions generated by transposition in vitro

Two artificial transposons have been constructed that carry a gene encoding Green Fluorescent Protein and can be used for generating libraries of GFP fusions in a gene of interest. One such element, AT2GFP, can be used to gen erate GFP insertions in frame with the amino acid sequence of the protein o f interest, with a stop codon at the end of the GFP coding sequence; AT2GFP also contains a selectable marker that confers trimethoprim resistance in bacteria. The second element, GS, can be used to generate tribrid GFP fusio ns because there is no stop codon in the GFP transposon, and the resulting fusion proteins contain the entire amino acid sequence encoded by the gene. The GS element consists of a gfp open reading frame and a supF amber suppr essor tRNA gene; the supF portion of the GS transposon can be utilized as a selectable marker in bacteria. Its sequence contains a fortuitous open rea ding frame, and thus it can be translated continuously with the gfp amino a cid sequence. As a target for GFP insertions, we used a plasmid carrying th e native Ty1 retrotransposon of the yeast Sacharomyces cerevisiae. The resu lting multiple GFP fusions to Ty1 capsid protein Gag and Ty1 integrase were useful in determining the cellular localization of these proteins. Librari es of GFP fusions generated by transposition in vitro represent a novel and potentially powerful method to study the cell distribution and cellular lo calization signals of proteins. (C) 1998 Elsevier Science B.V. All rights r eserved.