FIG. 3.

Analysis of RNA synthesized from p72 promoter mutants. Preconfluent monolayers of Vero cells (1.5 × 10⁵) were transfected with 16 μg of Lipofectamine reagent and 1 μg of plasmids containing different mutations of the p72 promoter regulating the luciferase gene. The primer for the luciferase gene was complementary to nucleotides between bp 35 and 64 of the noncoding strand of the gene. The primer for the D117R gene has been previously described (32). Total RNA was obtained at 18 h postinfection, hybridized with end-labeled primer, and extended with avian myeloblastosis virus reverse transcriptase. The sizes (in nucleotides) of the major bands, calculated by using products of an irrelevant DNA sequencing reaction for markers, are indicated. The lower panel shows the quantitated values of the transcripts produced by each mutant normalized first to the internal D117R transcript value and then to the p72.6 value. A diagram showing the relative promoter lengths and sequences of the promoters used is shown on the left. Mutant p72.19C has a point mutation in which the A residue at position +3 was replaced by C.