FIG. 6.

Overexpression of p130 represses Pol I transcription in vitro and in vivo. (A) NIH 3T3 cells (4 × 10⁵) were cotransfected with 10 μg of an rDNA minigene (pMr1930-BH), together with 2 μg (lane 2), 5 μg (lane 3), or 10 μg (lane 4) of pCMV-p130 expression plasmid. RNA was isolated after 44 h, and transcripts from the reporter construct were detected on a Northern blot with a ³²P-labeled riboprobe complementary to pUC-derived sequences present in pMr1930-BH. Cytochrome oxidase 1 (cox1) mRNA was used as an internal control. (Bottom panel) To monitor p130 expression in the transfected cells, equal amounts of cell extracts (10 μg) were subjected to Western blot analysis with anti-p130 antibodies. (B) p130 represses Pol I transcription in vitro. Increasing amounts of GST-pRb (lanes 1 to 3), GST-p130 (lanes 4 to 6), GST-p107 (lanes 7 to 9), or GST (lanes 10 to 12) were added to a reconstituted transcription system containing 15 ng of linearized template pMrWT, partially purified TIF-IA/IC, TIF-IB, Pol I, and 15 ng of UBF.