Figure 1.

Differences in the steady-state levels of Queuosine in tRNA between developmental stages of T. brucei cannot be explained by changes in general nutrients. (A) total RNA from procyclic form (PF, insect stage) and bloodstream form (BF, mammal stage) T. brucei was analyzed by APB-gel northern blot, using a radioactive oligonucleotide probe specific for tRNA^Tyr, to determine the levels of Q-containing (Q34) vs. unmodified tRNA (G34) (left panel) and quantification of the bands in (right panel), where percent Q-tRNA was calculated by dividing the intensity of the Q34 band by the sum of Q34 and G34 and multiplied by 100. (B) shows a growth curve of PF T. brucei comparing complete media to similar media lacking glucose, hemin or fetal bovine serum (FBS). The graph shows the log of cells per ml of media from cumulative cell counts over 6 days as indicated. (C) APB-gel northern blot as in (A) to assess Q levels in tRNA^Tyr, where ‘comp.’ denotes complete media as compared to media lacking glucose (-glucose), hemin (-hemin) or fetal bovine serum (–FBS) (left panel) and quantification of the band intensities from the left panel to determine Q levels as in. In all blots, ‘ox’ refers to an oxidized control (negative control); oxidation of the cis-diols from Q prevents the observed band shift and indicates the presence of Q on the shifted untreated samples. All graphs are representative of at least 3 different independent experiments.