Figure 2.
CAF1, DHH1 and NOT1 are essential for bloodstream trypanosome growth. (A) Trypanosomes were transfected with plasmids designed for tetracycline-inducible RNAi targeting CAF1, DHH1 or NOT1 and three independent cell lines were selected. Growth without tetracycline (filled symbols, continuous lines, CAF1+, NOT1+, DHH1+ cells) is illustrated as a cumulative exponential curve (log scale, initial number divided by 105) and the division time is indicated. Growth in the presence of tetracycline (added at time = 0) is shown using open symbols and dashed lines; the depletion of CAF1, DHH1 or NOT1 is indicated by the downward arrow. The three different symbols represent three different cell lines in each case. (B) Effect of CAF1 depletion on the levels of various RNAs in procyclic trypanosomes. The CAF1 mRNA was reduced to about 10% of normal after RNAi induction. Total glyoxal-treated RNA was separated on agarose gels, blotted and hybridized with the probes shown on the left. PC: procyclic; BS: bloodstream; tet: tetracycline. Cell lines are: W-cells expressing tet repressor and T7 polymerase; C-cells with inducible CAF1 RNAi. The experiments were performed in duplicate. Quantitation using the rRNA as a loading control revealed no reproducible effects of CAF1 depletion on any mRNA, apart from EP which increased 1.2–3.1-fold. (C) Effect of CAF1 depletion on the levels of various RNAs in bloodstream-form trypanosomes. Details as for (B) except that formaldehyde-agarose gels were used.