Figure 4

No evidence for stop codon suppression in Trichomonas vaginalis . (A) Bar diagram of the frequency of annotated gene pairs and their distances in base triplets (light grey). Dark grey bars indicate the gene pairs, for which expression evidence exists. Note that the most abundant distances originate from highly conserved and large gene families. (B) Illustration of four selected candidates, in which two adjacent genes share the same reading frame and in combination match to a single BLAST hit. (C) RT-PCR demonstrates the full-length transcription of the gene pairs including the C-terminal HA-tag. RNA was isolated from transfected trichomonads, transcribed into complementary DNA and served as template for the PCR using specific forward and HA-reverse primers (+). RNA served as a negative control (−). (D) Multiplex western blot analysis of the same candidates demonstrates only candidate #1 is translated. 50 μg of protein extract loaded, anti-HA in blue, anti-SCS (succinyl-coenzyme A synthetase subunit alpha; TVAG_047890; 33 kDa) in pink as a loading control, and TVAG_337240::HA served as a positive control. For SCS the double bands are routinely observed [64], and the two additional bands migrating below the 44 kDa TVAG_337240::HA fusion protein likely represent degradation products.