Reference - Detail
|Author||Levi S, Polyakov M, Egelhoff TT.|
|Title||Green fluorescent protein and epitope tag fusion vectors for Dictyostelium discoideum.|
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.
|MeSH||Amino Acid Sequence Animals Bacterial Proteins / genetics* Bacterial Proteins / metabolism Base Sequence Cloning, Molecular / methods* Dictyostelium / genetics* Dictyostelium / metabolism Escherichia coli Proteins* Genetic Vectors / genetics* Green Fluorescent Proteins Luminescent Proteins / biosynthesis Luminescent Proteins / genetics* Molecular Sequence Data Oligopeptides Oxidoreductases* Peptides / genetics* Peptides / metabolism Plasmids / genetics* Recombinant Fusion Proteins / biosynthesis Recombinant Fusion Proteins / genetics*|
|WOS Category||MICROBIOLOGY GENETICS & HEREDITY|
|Cellular slime molds||G90010|