Green fluorescent protein and epitope tag fusion vectors for Dictyosteliumdiscoideum

We have considered expression vectors for Dictyostelium discoideum which en code 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 localizati on studies in Dictyostelium cells. A parallel construct fuses a FLAG epitop e tag at the amino terminus of expressed protein. Each fusion cartridge was placed either in a G418-resistance vector allowing transactivated Ddp2-bas ed extrachromosomal replication or in a vector allowing autonomous Ddp1-bas ed replication. Distinct differences in expression stability were observed in the two vector types. When GFP-expressing cells were analyzed by fluores cence 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 w as used for expression. (C) 2000 Academic Press.