Figure 3.
Analysis of vector expression by dual luciferase assay (DLR). (A) Luciferase assay for cells containing CEN/ARS and 2 μ URA3-marked plasmids. PPGK1-Rluc reporter in plasmids pXP118 and pXP218 were transformed into yeast strain yFF1683 and cells were grown in SD – ura medium to logarithmic phase. Cell number was determined by OD at A600 and the indicated number of cells was used to assay plasmid-borne Rluc and chromosomal Fluc activities. Data represent the mean of three independent assays and the bars show one standard deviation (SD). The plasmids without the Rluc reporter served as negative controls. Upper panel, cells contain pXP118-Rluc (CEN/ARS plasmid with URA3 marker). Lower panel, cells contain pXP218-Rluc (2 μ plasmid with URA3 marker). Left panels, RLuc : FLuc ratios; right panels, scatter plots of Rluc vs. Fluc for the same number of cells as shown in left panels. (B) Comparison of expression from differently marked pXP vectors. PPGK1-Rluc-TCYC1 reporters in the CEN/ARS and 2 μ plasmids with MET15, TRP1, HIS3, LEU2-d8 and URA3 were transformed individually into yeast strain yFF1683. Activities were analysed as described in (A). (C) Comparison of expression from pXP CEN/ARS and 2 μ vectors using different promoters. Cells containing URA3-marked vectors with PPGK1, PTEF1 and PHXT7–391 controlling expression of Rluc were analysed as described in (A)