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 generate GFP insertions in frame with the amino acid sequence of the protein of 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 fusions 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 suppressor tRNA gene; the supF portion of the GS transposon can be utilized as a selectable marker in bacteria. Its sequence contains a fortuitous open reading frame, and thus it can be translated continuously with the gfp amino acid sequence. As a target for GFP insertions, we used a plasmid carrying the native Ty1 retrotransposon of the yeast Sacharomyces cerevisiae. The resulting multiple GFP fusions to Ty1 capsid protein Gag and Ty1 integrase were useful in determining the cellular localization of these proteins. Libraries of GFP fusions generated by transposition in vitro represent a novel and potentially powerful method to study the cell distribution and cellular localization signals of proteins.
Merkulov
G V
GV
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Boeke
J D
JD
D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D004251:DNA Transposable Elements; D004352:Drug Resistance, Microbial / Q000235:genetics; D015723:Gene Library; D005798:Genes, Bacterial; D005819:Genetic Markers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013347:Subcellular Fractions / Q000378:metabolism; D014295:Trimethoprim / Q000494:pharmacology
D004251:DNA Transposable Elements; D005819:Genetic Markers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D014295:Trimethoprim