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. 2005 Apr;25(8):3276–3285. doi: 10.1128/MCB.25.8.3276-3285.2005

FIG. 2.

FIG. 2.

Identification of the minimal requirements for efficient termination in the Gγ gene. (a) The efficiency of termination was assessed by the amount of read round into the HIV promoter, as measured by analysis of nuclear RNA extracted from transfected cells. A riboprobe spanning the HIV promoter is protected by transcripts which initiate at the HIV promoter (80-nt product) and also by transcripts which fail to terminate and read round the transfected plasmid and across the HIV promoter (241-nt protected fragment). (b) Analysis of the efficiency of termination in Gγ deletion clones. The final exon of the Gγ-globin gene from +1394 relative to the transcription start site and various amounts of 3′ flank were cloned downstream of the HIV LTR promoter (−161 to +80) and exons 1 to 3 (position +35 to +1373) of the Aγ-globin gene. The resultant clones were cotransfected into HeLa cells with the Tat expression plasmid by calcium phosphate precipitation. After 24 h nuclear RNA was extracted and analyzed using the riboprobe described for panel a. Clones examined were as follows: lane 1, HγG14 (3.1-kb Gγ 3′ flank); lane 2, HγG9 (1.9 kb); lane 3, HγG7 (1.1 kb); lane 4, HγG9Δ5-7 [contains the 3′ flank regions up to 0.4 kb and 1.1 to 1.9 kb downstream of the poly(A) site]; lane 5, HγG4 (0.4-kb 3′ flank).Lane U represents the untransfected control. Lane M contains markers (sizes from top to bottom: 517, 394, 344, 285, 244, 210, 190, 148, 130, and 75 nt), while lane P contains the undigested riboprobe. The protected fragments corresponding to HIV initiation (filled arrow) and read round (open arrow) are indicated. (c) Fine deletions in the Gγ 3′ flank were generated and analyzed as described above. The clone HγG7 showing no read round (lane 1) was used as a base to delete the internal fragments. Deletions (relative to the Gγ transcription start site) were of nt +2004 to +2409 (HγG7Δ5; lane 2), +2004 to +2722 (HγG4; lane 3), +1714 to +2004 (HγG7Δ4; lane 4), +1714 to +2722 (HγG3; lane 5), and +2394 to +2620 (HγG7Δ6; lane 6). The Gγ poly(A) site is at position +1592. Deletion clones were cotransfected with the Tat plasmid and assayed as described above. Markers (lane M) of 244, 210, 190, 179, 148, 130, and 106 nt (top to bottom) are shown. Each clone was analyzed on at least two separate occasions, giving the same result each time.