FIG. 4.
HIV-1 LTR is transcriptionally most responsive in the G2 phase of the cell cycle. (A) A total of 109 Jurkat-LTR-luc cells were separated by counterflow centrifugal elutriation into different phases of the cell cycle. The relative DNA content of each elutriated fraction was determined by PI staining of nuclei, and the proportion of cells in each phase of the cell cycle (G1, S, and G2/M phases) is indicated (right-hand y axis). Luciferase activity for each elutriated fraction determined from lysates of 106 cells from each fraction, expressed in relative light units (RLU), is represented by the histograms (left-hand y axis). (B) RPA of luciferase RNA from Jurkat-LTR-luc cells that were elutriated into different phases of the cell cycle. Total RNA isolated from 5 × 106 cells of each elutriated fraction was used for RPA. Each RPA reaction contained either 8 μg of total RNA hybridized to 2 × 105 cpm of the single-stranded luciferase RNA probe (top panel) or 2 μg of total RNA hybridized to 2 × 105 cpm of the single-stranded GAPDH RNA probe (bottom panel). The numbers on top of the lanes indicate the elutriated fraction number. MW, molecular weight standards. Sizes (in base pairs) are shown on the left.