Too big to rig

TBTR

?

Molecular and biochemical properties of TbTR. (A) TRAP assay with 0.15 μg T. brucei PF cell extract using TS primer as substrate. The TS oligo, reverse primer, or the cell extract was omitted in lanes 2, 3, or 4, respectively. Lane 6 represents an RNase-treated sample. (B) TRAP assay to compare the telomerase activities in WT (lanes 2 and 3), TbTR-DKO (lanes 4 and 5), and TbTR-DKO cells with a complementation TbTR allele (lanes 6 and 7) with or without RNase A treatment. 0.25 μg (lanes 2 and 3), 0.28 μg (lanes 4 and 5) or 0.43 μg (lanes 6 and 7) of cell extract was used in each TRAP reaction, and 20 μl (lanes 2-5) or 40 μl (lanes 6 and 7) of final products were loaded in each lane, respectively. (C) Detecting spliced leader (SL) sequence at the 5′ end of immunopurified TbTR. RT-PCR was done with (+) or without (−) RT using a primer specific to TbTR and a primer specific to the SL sequence. Lanes 2 and 4, RT with random primer (RP) and Oligo dT primer (OP), respectively. Lane 3 represents control reaction without RT. (D) Determination of 5′ cap status of TbTR from total RNA and TbTERT-IP RNA. Total or TbTERT antibody-immunopurified RNA fraction from WT cells were immunoprecipitated with TMG antibody, and the IP products were treated with (+) or without (−) RT followed by amplification with TbTR-specific primers. The supernatant fraction of TbTERT-IP (sup) and the total RNA extract from TbTR-DKO cells were examined the same way. Genomic DNA (gDNA) was amplified with the same TbTR primers as a control. (E) PF T. brucei cells were treated with RNAP III inhibitor Indazolo-sulfonamide for 24 h followed by RNA extraction. Northern blotting was done with TbTR- or U2 snRNA-specific oligonucleotide probes (top). Densitometry-quantified TbTR and U2 snRNA levels were normalized against untreated samples, and the relative changes are shown at the bottom.

https://www.researchgate.net/figure/Molecular-and-biochemical-properties-of-TbTR-A-TRAP-assay-with-015-mg-T-brucei-PF_fig6_235905273