Zero background yeast reporter plasmids.
UAS-less reporter plasmids are widespread and powerful tools for the identification and analysis of binding sites for transcriptional activators. The common reporter plasmids for the yeast Saccharomyces cerevisiae are multicopy (2mu) vectors with the CYC1 core promoter upstream of the lacZ gene. Insertion of putative or known activator binding sites upstream of the core promoter puts lacZ (beta-galactosidase) expression under the control of the corresponding activator. Although these constructs have proved to work well for most purposes, they have certain limitations: (1) they give significant and carbon-source-dependent lacZ background expression; (2) unlike most other yeast promoters, the CYC1 upstream region has a partially open chromatin structure with an accessible TATA box; (3) they use only a single, moderately sensitive reporter; and (4) the use of multicopy vectors can result in activator titration. Here, we introduce novel reporter plasmids based on the yeast MEL1 (alpha-galactosidase) gene that can overcome all of these limitations. It is also shown that background expression is due to fortuitous activator binding sites within the plasmid backbones that are insufficiently shielded from the core promoters in the common CYC1 reporter plasmids.
Melcher
K
K
Institute for Microbiology, Biocenter, University of Frankfurt, 60439, Frankfurt, Germany. k.melcher@em.uni-frankfurt.de
Sharma
B
B
Ding
W V
WV
Nolden
M
M
D001665:Binding Sites; D003574:Cytochrome c Group / Q000235:genetics; D045304:Cytochromes c; D005690:Galactose / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics*; D005947:Glucose / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D007763:Lac Operon / Q000235:genetics; D008320:Maltose / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D012680:Sensitivity and Specificity; D015534:Trans-Activators / Q000378:metabolism; D000519:alpha-Galactosidase / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D008320:Maltose; D045304:Cytochromes c; D000519:alpha-Galactosidase; D001616:beta-Galactosidase; D005947:Glucose; D005990:Glycerol; D005690:Galactose