title: Green fluorescent protein and epitope tag fusion vectors for Dictyostelium discoideum.
abstract: We have constructed expression vectors for Dictyostelium discoideum which encode a green fluorescent protein (GFP) sequence upstream of a multicloning site for introduction of sequences of interest. Insertion of cDNAs into the multicloning site results in expression of fusion protein bearing an amino- or carboxyl-terminal GFP tag which can be used for fluorescent localization studies in Dictyostelium cells. A parallel construct fuses a FLAG epitope tag at the amino terminus of expressed protein. Each fusion cartridge was placed either in a G418-resistance vector allowing transactivated Ddp2-based extrachromosomal replication or in a vector allowing autonomous Ddp1-based replication. Distinct differences in expression stability were observed in the two vector types. When GFP-expressing cells were analyzed by fluorescence microscopy, significant cell-to-cell variability in expression level was observed when expression was based on the Ddp2 vector, while less cell-to-cell variation in expression level was observed when the Ddp1 backbone was used for expression.
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve School of Medicine, Cleveland, Ohio 44106-4970, USA.
authors.forename: S
authors.initials: S
authors.lastname: Levi
authors.forename: M
authors.initials: M
authors.lastname: Polyakov
authors.forename: T T
authors.initials: TT
authors.lastname: Egelhoff
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004023:Dictyostelium / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010088:Oxidoreductases; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; C060787:pdxA protein, E coli; D049452:Green Fluorescent Proteins; C085706:FLAG peptide; D010088:Oxidoreductases