FIG. 2.

Impact of the mutation Y165C on the transactivation of cellular genes involved in dNTP biosynthesis. (A) Transactivation of the RNR promoter P^R2. (B) Transactivation of the TS promoter P^TS. DLR assays were performed with growth-arrested NIH 3T3 cells transiently cotransfected with an IE1-encoding donor plasmid, the respective firefly luciferase-encoding reporter plasmid, and the Renilla luciferase-encoding plasmid pRL-TK for standardization of the transfection efficacy. Vector pUC19 was used as an empty donor plasmid for control. Plasmid maps (not drawn to scale) are illustrated. The donor plasmids were pIE100/1 (here referred to as pWT), specifying the authentic IE1 protein; pMut, specifying the mutated protein IE1-Y165C; and pRev, specifying the rescued protein IE1-C165Y. MIEPE, MIE promoter and enhancer; Ex, exon. The arrows indicate the direction of transcription; the arrowheads mark the location of the mutation in exon 4. Correct expression of the IE1 protein was inspected for all donor plasmids by IE1-specific immunofluorescence (not depicted). Luminescence data are expressed as RLU and are normalized for transfection efficacy by forming the quotient of firefly luciferase activity (RLU^FL) and Renilla luciferase activity (RLU^RL). The solid circles and gray-shaded bars represent data from triplicate transfection cultures and their median value, respectively, for each experiment, with a total of three independent experiments performed for each type of donor plasmid tested. P values (two-sided), determined by the distribution-free (nonparametric) Wilcoxon-Mann-Whitney rank sum test (n1 = n2 = 9), are indicated.