TFIIIB Subunit Bdp1 Participates in RNA Polymerase III Transcription in the Protozoan Parasite Leishmania major
Figure 5
Nuclear run-on analyses with the LmBdp1 double-knockout +1 cell line. (a) Labeled nascent RNA from isolated nuclei from the LmBdp1 double-knockout +1 cell line (DKO+1) and wild-type (WT) parasites was hybridized to dot blots of double-stranded DNAs (2 μg) cloned into pGEM-T Easy. The 18S rRNA gene (18S) was tested as a representative Pol I gene. The Pol II genes analyzed were LmjF.06.0200 (200), LmjF.06.0210 (210), LmjF.06.0370 (370), and α-tubulin (TUB). The Pol III genes examined were tRNA-Phe (PHE), tRNA-Tyr (TYR), 5S rRNA (5S), and the U2 snRNA (U2), which was examined in duplicate dots. The tRNA-Sec (SEC), transcribed by both Pol II and Pol III, was also analyzed. As control, an empty vector was assessed (PG). (b) The signals obtained for each gene in panel (a) and from two more independent experiments were quantified and plotted, considering as 100% the signal obtained with wild-type promastigotes. Values represent means of the three experiments. Standard deviation bars are shown. RNA levels were normalized to the level of α-tubulin. (c) Nuclear run-on analysis performed as indicated in panel (a). The Pol I genes analyzed were 24Sβ rRNA (24Sβ) and the 18S rRNA (18S). The Pol II genes examined were SL RNA (SL) and α-tubulin (TUB). As control, an empty vector was also analyzed (PG). (d) The signals obtained for each gene in panel (c) and from two more independent experiments were quantified and plotted, as indicated in panel (b).