Journal: Nucleic Acids Research
Article Title: A TFIIB-like protein is indispensable for spliced leader RNA gene transcription in Trypanosoma brucei
Figure Lengend Snippet: Silencing of TFIIB like expression affects SL RNA synthesis in vivo. ( A ) Total RNAs prepared from RNAi-induced cells were analyzed by primer extension assays using 5′- 32 P-end labeled oligonucleotides specific for SL RNA and U2 snRNA. The lower of the two extension products for the SL RNA is a result of the hyper-methylated SL RNA cap, which prematurely terminates reverse transcription. DNA marker (M) sizes are indicated on the left. ( B ) Nascent RNAs were labeled with [α- 32 P]UTP in permeabilized cells in which expression of TFIIB like dsRNA was induced for the times specified. The RNAs were separated on a 6% polyacrylamide/50% urea gel and visualized by autoradiography. SL RNA and tRNA are indicated on the right and DNA marker (M) sizes on the left. ( C ) Labeled, nascent RNA was used to probe dot blots containing the complete coding regions of the SL RNA, GPEET procyclin, α-tubulin, heat shock protein 70 (HSP70), 18S ribosomal RNA, U2 snRNA and U6 snRNA. The vector pTZ18U served as a control. Shown are low exposures of experiments I–III for the SL RNA and a long exposure of experiment I for all other RNAs. ( D ) Quantification of the dot blot signal strengths from three independent experiments. The signal of non-induced cells was set to 100%.
Article Snippet: Briefly, reactions of 40 µl containing 8 µl of extract, 20 mM potassium l -glutamate, 20 mM KCl, 3 mM MgCl2 , 20 mM HEPES–KOH, pH 7.7, 0.5 mM of each nucleoside triphosphate (NTP), 20 mM creatine phosphate, 0.48 mg/ml of creatine kinase, 2.5% polyethylene glycol, 0.2 mM EDTA, 0.5 mM EGTA, 4 mM DTT, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 12.5 µg/ml vector DNA, 20 µg/ml GPEET-trm template and 7.5 µg/ml SLins19 template were incubated for 1 h at 27°C.
Techniques: Expressing, In Vivo, Labeling, Methylation, Marker, Autoradiography, Plasmid Preparation, Dot Blot