Yeast - useful tools

mips


Für GFP Vektoren

A second generation of GFP-vectors for subcellular localization
studies in budding yeast

U. Güldener and J. H. Hegemann

Heinrich-Heine-Universität, Institut für Mikrobiologie, Universitätsstr.1, 40225 Düsseldorf, Germany, e-mail: hegemann@uni-duesseldorf.de,

Tel.: +49-211-81-13733, Fax.: +49-211-81-15370

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has proven to be an extremely useful tool as a marker for gene expression and as a tag to study protein localization. Previously we developed the vectors pGFP-N-FUS and pGFP-C-FUS which allow the N-terminal or C-terminal in-frame fusion of the gfp wildtype gene to the gene of interest and subsequent determination of the subcellular location of the fusion protein .

Based on this first set of vectors we have now constructed a new series of GFP vectors. The new plasmids are equipped with the so-called yEGFP version constructed by Cormack and coworkers . In this yEGFP version all codons of the original gfp gene were exchanged by optimal codons for Candida albicans. In addition 2 amino acid changes were incorporated near the chromophore that had been shown to increase GFP fluorescence . The yEGFP3 not only proved to exhibit stronger fluorescence, but the maturation from the non-fluorescent preform to the mature fluorescence form occurs much faster .

The gene encoding the yEGFP3 protein was inserted in the polylinker of the CEN/ARS vectors p413MET25 (HIS3 marker) and p416MET25 (URA3 marker) . Eight (nine in the case of the N-FUS vectors) different restriction sites can be used to insert the gene of interest in frame N-terminal (plasmids pUG23 and pUG35) or C-terminal (plasmids pUG34 and pUG36) to the yEGFP3 encoding open reading frame. The expression level of the fusion protein can be regulated by adding methionine to the media.

Marker

[C-FUS vectors]

[N-FUS vectors]

HIS3

pUG23
[map]*[sequence]

pUG34
[map]* [sequence]

URA3

pUG35
[map]* [sequence]

pUG36
[map]* [sequence]


* Plasmid maps with enlarged MCS showing unique sites in bold type.

order a plasmid hegemann@uni-duesseldorf.de

Literature:

Cormack, B. P., Bertram, G., Egerton, M., Gow, N. A., Falkow, S. and Brown, A. J. (1997). Yeast- enhanced green fluorescent protein (yEGFP)a reporter of gene expression in Candida albicans. Microbiology 143, 303-11.

Cormack, B. P., Valdivia, R. H. and Falkow, S. (1996). FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33-8

Mumberg, D., Muller, R. and Funk, M. (1994). Regulatable promoters of Saccharomyces c erevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucleic Acids Res 22, 5767-8.

Niedenthal, R. K., Riles, L., Johnston, M. and Hegemann, J. H. (1996). Green Fluorescent Protein As a Marker For Gene Expression and Subcellular Localization in Budding Yeast. Yeast 12 (8), 773-786.