MRB8620 is essential for RNA editing in BS T. brucei. (A) Scheme depicting how MRB8620 double knockout was generated. Arrows indicate the locations of PCR primer pairs I and II, just flanking the knockout loci and amplifying the MRB8620 ORF, respectively, used to identify the single and double knockouts generated by homologous recombination in the cell line in which one of the ATP synthase γ-subunit alleles bears a L262P site mutation. (B) Agarose gel-resolved PCR products of primer pair I flanking the genomic integration site of the constructs from genomic DNA isolated from the parental hemizygous L262P-mutant BS T. brucei, single knockout (SKO) and MRB8620 double knockout (DKO). The size of each amplicon is indicated by the arrow. Primer pairs for identification are depicted in A. (C) As in B, but showing the PCR amplicon from primer pair II amplifying sequence from within the MRB8620 ORF. (D) Relative abundance of mitochondrial mRNAs in MRB8620 double knockout cells compared with the parental hemizygous ATP synthase L262P γ-subunit mutant cell line. Whiskers denote range of obtained relative abundances within technical triplicates. Labeled as in Figure 3A.
