Figure 7.

Modulatory effect of Tat on antisense transcription. (A) Jurkat cells were transfected with 10 μg of circular or BamHI-digested pAsLTRXLuc plasmid in combination with 5 μg of pCMV-tat or empty pCMV vector. (B) 293T cells were transfected with 400 ng of circular or BstZ17I-digested pNL4.3ΔBstAsLucR-E-, 200 ng of pActin-LacZ and 200 ng of pCMV-tat or empty vector. (C) 293T cell clones and a pooled population stably transfected with pNL4.3ΔBstAsLucR-E- were transfected with 200 ng pCMV-tat or empty vector. Results are presented as fold induction compared to empty vector. (D) A pool of 293T cells stably transfected for pNL4.3ΔNar1 was transfected with 5 μg of pCMV-tat or the empty pCMV5 vector. cDNA synthesis was performed with random primers. PCR amplifications were performed to detect the presence of the HIV antisense transcript (lane 3-4-7-8) using 24-6/25-3 primers. β-actin amplification was performed as control (lane 1-2-5-6). Lanes 1, 2, 3 and 4 represent control for DNA contamination to which RNA was directly added for PCR amplification. (E) RNA from the transfected 293T cells described in D were also used for amplification of antisense transcripts from 24-6F-synthesized cDNA using 30-20 (anchor) primer in combination with 25-3 (lane 1, 3, 5 and 7) or 26-5 (lanes 2, 4, 6 and 8) primers. Samples were tested for cDNA cleanup efficiency (lanes 1, 2, 5 and 6). Tat expression in transfected cells is indicated above the gel for both latter panels. Luciferase activities in A, B and C represent the mean value of three measured samples ± S.D.