[
RNA,
2010]
The two codon-specific eubacterial release factors (RF1: UAA/UAG and RF2: UAA/UGA) have specific tripeptide motifs (PXT/SPF) within an exposed recognition loop shown in recent structures to interact with stop codons during protein synthesis termination. The motifs have been inferred to be critical for codon specificity, but this study shows that they are insufficient to determine specificity alone. Swapping the motifs or the entire loop between factors resulted in a loss of codon recognition rather than a switch of codon specificity. From a study of chimeric eubacterial RF1/RF2 recognition loops and an atypical shorter variant in Caenorhabditis elegans mitochondrial RF1 that lacks the classical tripeptide motif PXT, key determinants throughout the whole loop have been defined. It reveals that more than one configuration of the recognition loop based on specific sequence and size can achieve the same desired codon specificity. This study has provided unexpected insight into why a combination of the two factors is necessary in eubacteria to exclude recognition of UGG as stop.
[
Biochem Mol Biol Int,
1993]
Oligonucleotides related to parts of a globin-like sequence in the genome of Caenorhabditis elegans were used to probe a cDNA library from the same species. A complete globin-like sequence was found in the cDNA, showing that a globin gene appears to be expressed. The hypothetical protein was compatible with the conventional globin fold but may be truncated in the B helix, as in Chironomus globin III. An intron in the codon for residue E3 in the E helix was removed in expression. An initiation codon preceded the globin but the sequence upstream (extending for 30 nucleotides to the vector ligation site) had characteristics both of the code for a protein hydrophobic leader and of a trans-spliced RNA leader. The evidence indicates that C. elegans globin has a single domain, unlike some nematodes that express two tandem globin domains in a continuous translation product, and from its sequence may be predicted to have a high affinity for oxygen.
[
microPublication Biology,
2020]
The purpose of this study was to determine the utility of the wMicroTracker as a screening platform to assess the motility of various parasites. We tested three species of parasites: the adult and larval stages of the filarial nematode Brugia pahangi, the schistosomula stage of the trematode Schistosoma mansoni, and the epimastigote stage of the protozoan parasite Trypanosoma cruzi. We optimized the assay for the number of parasites per well, plate type and media volume using the wMicroTracker and compared those readouts to readouts from the WormAssay (Marcellino et al. 2012) when possible. The WormAssay has been used in phenotypic drug screens to identify new compounds for the treatment of lymphatic filariasis, onchocerciasis and schistosomiasis (Storey et al. 2014; Bulman et al. 2015; Weeks et al. 2018; Tyagi et al. 2019). The original WormAssay was developed by Marcellino et al. 2012 and was subsequently modified to the Worminator by Storey et al. 2014 to observe smaller worms with an inverted microscope. wMicroTracker (InVivo Biosystems) protocols optimized for C. elegans adults (1 mm in length by 80 m in width, highly motile) were used to optimize parasite assays based on the size and motility of each of the parasite species as compared to C. elegans adults.To use the wMicroTracker, two factors need to be considered: the size of the parasite of interest and how active they are. The wMicroTracker detects movement when an organism crosses the stationary LED beam at the center of the well. Hence, parasites that do not travel throughout the well should be assayed in a U-bottom plate to ensure movement is detected. For parasites that do travel throughout the well, a flat bottom plate is sufficient. To determine the number of parasites to use per well, we compared their size to that of C. elegans adults. Following the InVivo Biosystems protocol (https://invivobiosystems.com/wp-content/uploads/2018/12/Protocol_toxicity-in-c-elegans_122018.pdf), C. elegans adults were screened in the wMicroTracker with 40-50 adults per well in 100 L of M9 buffer (Stiernagle 2006). This produced results in the range of 25-35 mean movement units per well (Figure 1A). To optimize screening methods in the wMicroTracker for other parasites, the size and motility of parasites of interest were compared to C. elegans adults to ensure control wells gave mean movement units around this range.B. pahangi females (34.7 mm in length by 139 m in width (Mutafchiev et al. 2014), highly motile) were screened with one worm per well in 500 L of RPMI in a 24-well flat bottom plate with 8-9 replicate wells. B. pahangi males (18.0 mm in length by 77 m in width (Mutafchiev et al. 2014), high motility) were screened with four worms per well in 500 L of RPMI in a 24-well flat bottom plate with 4-7 replicate wells. B. pahangi females and males were assessed in both screening platforms. Both platforms showed similar motility profiles in response to 50 M ivermectin or 1% DMSO as a negative control (Figure 1B