Dicistronic regulation of fluorescent proteins in the budding yeast Saccharomyces cerevisiae

Abstract

Fluorescent proteins are convenient tools for measuring protein expression levels in the budding yeast Saccharomyces cerevisiae. Co-expression of proteins from distinct vectors has been seen by fluorescence microscopy; however, the expression of two fluorescent proteins on the same vector would allow for monitoring of linked events. We engineered constructs to allow dicistronic expression of red and green fluorescent proteins and found that expression levels of the proteins correlated with their order in the DNA sequence, with the protein encoded by the 5'-gene more highly expressed. To increase expression levels of the second gene, we tested four regulatory elements inserted between the two genes: the IRES sequences for the YAP1 and p150 genes, and the promoters for the TEF1 gene from both S. cerevisiae and Ashbya gossypii. We generated constructs encoding the truncated ADH1 promoter driving expression of the red protein, yeast-enhanced Cherry, followed by a regulatory element driving expression of the green protein, yeast-enhanced GFP. Three of the four regulatory elements successfully enhanced expression of the second gene in our dicistronic construct. We have developed a method to express two genes simultaneously from one vector. Both genes are codon-optimized to produce high protein levels in yeast, and the protein products can be visualized by microscopy or flow cytometry. With this method of regulation, the two genes can be driven in a dicistronic manner, with one protein marking cells harbouring the vector and the other protein free to mark any event of interest.

Figure 1

Sequences of the promoter and IRES regulatory elements. (A) pTEF1 promoter from Ashbya gossypii. (B) pTEF1 promoter from Saccharomyces cerevisiae. (C) p150 leader sequence/IRES; the TIF4631 locus in yeast. (D) YAP1 leader sequence/IRES.

Figure 2

Orientation of the genes affects their expression levels. (A)-(B) show raw flow cytometry data in the form of dot plots. The x-axis displays fluorescence in the green channel and the y-axis displays fluorescence in the red channel. (A) The yeGFP gene the 5'-position and Cherry in the 3'-position. (B) Cherry gene in the 5'-position and yeGFP in the 3'-position. (C) Quantification of the mean fluorescence intensities of yeGFP and Cherry in both orientations, with green bars representing yeGFP and red bars representing Cherry. Note that this Cherry gene is not the yeast-enhanced version. Experiment 1 is yeGFP, experiment 2 is a yeGFP-DHFR fusion, and experiment 3 is the reverse orientation (DHFR-yeGFP) fusion.

Figure 3

(A) Schematic of the dicistronic DNA construct. The yeCherry gene is in the 5’-position driven by the 700-nt ADH1 promoter. The “X” represents one of four regulatory elements (see Figure 1) to enhance expression of the 3’-gene yeGFP. The ADH1 termination sequence follows the yeGFP gene. (B)-(E) Fluorescence micrographs of SC252a cells that harbor vectors carrying one of the four dicistronic constructs. In all cases yeCherry is the 5’-gene driven by the 700-nt ADH1 promoter. The yeGFP gene is driven by the (B) pTEF1 promoter from Ashbya gossypii, (C) pTEF1 promoter from Saccharomyces cerevisiae, (D) p150 IRES or (E) YAP1 IRES. All exposures are 0.5 seconds.

Figure 4

Mean fluorescence intensities measured by flow cytometry. The yeCherry gene is in the 5’-position driven by the 700-nt ADH1 promoter. The 3’-yeGFP gene is driven by: no additional regulatory element, designated “null”, the pTEF1 promoter from Ashbya gossypii, the pTEF1 promoter from Saccharomyces cerevisiae, p150 IRES (or p150 leader) and YAP1 IRES (or YAP1 leader). Red bars show yeCherry levels and the green bars correspond to yeGFP levels. Each bar represents the average of two fluorescence measurements.