FIGURE 1.

Influence of uracil on the expression of the EGFP reporter gene in transiently transfected HeLa cells. A, positions of uracil (underlined) paired with adenine (U:A) or mispaired with guanine (U:G) within the TS and NTS DNA strands of the EGFP gene. The cut positions of the nicking endonucleases used for insertion of the synthetic oligonucleotides are indicated (▿, Nt.Bpu10I; ▴, Nb.Bpu10I). Possible mRNA and protein sequences arising from transcription of templates containing uracil in the TS are on the basis of the specificity of transcriptional mutagenesis reported in Ref. 17. B, verification of uracil incorporation into vector DNA by excision with the E. coli UDG and incision of the resultant AP site by endonuclease IV (E IV). C, time course analyses of the effects of the unique uracils on EGFP expression, measured by flow cytometry as the specific fluorescence in single cells. Reported values are relative to those of the control constructs, obtained by incorporation of the respective unmodified oligonucleotides (T:A and C:G). Shown is a summary of transfection experiments performed with three independent preparations of each type of vector DNA. Data are mean ± S.D.