FIG. 3.

RT-PCR analysis of wild-type and mutant tat genes. Total RNA was obtained from uninfected 293 cells (lanes 1), 293 cells stably transfected with HIV-1 wild-type (lanes 2) or HIV-1 Δtat (lanes 3), and 293 cells containing both HIV-1 Δtat and wild type tat (lanes 4) or the mutated tat genes corresponding to [E2G, D5G, E9G], P3L, P[6, 10]L, P[10, 14]L, C27S, K41A, and K/R[50-57]G (lanes 5 to 11, respectively). Primers specific for plasmid-derived tat mRNA or cellular β-actin mRNA were annealed to RNA obtained from each of the 293 cell lines, and a reverse transcription reaction was performed in the presence (A and B) or absence (C and D) of M-MLV RT. PCR was performed on each cDNA reaction mixture to detect either the tat (A and C) or β-actin (B and D) gene. PCR products were resolved on a 1.5% agarose gel. Molecular mass markers are shown for each gel (lanes M). PCRs with a plasmid containing the tat gene (panel A, lanes 12 and 13) (equivalent to 0.1 and 0.5 pg) or serially diluted β-globin cDNA (panel B, lanes 12 and 13) are shown.