Green fluorescent protein and epitope tag fusion vectors for Dictyostelium discoideum

Plasmid. 2000 Nov;44(3):231-8. doi: 10.1006/plas.2000.1487.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 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*

Substances

  • Bacterial Proteins
  • Escherichia coli Proteins
  • Luminescent Proteins
  • Oligopeptides
  • Peptides
  • Recombinant Fusion Proteins
  • pdxA protein, E coli
  • Green Fluorescent Proteins
  • FLAG peptide
  • Oxidoreductases

Associated data

  • GENBANK/AF269225
  • GENBANK/AF269226
  • GENBANK/AF269235
  • GENBANK/AF269237
  • GENBANK/AF269238
  • GENBANK/AF270470