Figure 2. Screening for functional amber suppression with mCherry.

(A) The extrachromosomal array unc-54p∷mCherryTAG156∷unc-54 3'UTR showed fluorescence in muscle cells in the absence of any amber suppressor tRNA/RS. (B) A single integrated copy of the same mCherry amber reporter in strain LWA1560 (wlSi151[unc54∷mCherryTAG156 cb-unc-119(+)]) had no detectable fluorescence in muscle cells. The observed autofluorescence was from intestinal granules, which are distinct from muscle cells in morphology and location. (C) LWA1560 crossed with an endogenous amber suppressor (sup-7(st5); wlSi151) showed red fluorescence in muscle cells. Because the sup-7 suppressor tRNA is expressed in all muscle cells, all of the cells uniformly displayed red fluorescence. (D) LWA1561 (LWA1560 with wlEx1[unc-54∷LeuRS_rpr-1∷ ${\text{tRNA}}_{\text{CUA}}^{\text{Leu}}$ + pRF4(rol-6)]) showed strong fluorescence in some body wall muscles. Only a subset of muscles were fluorescent due to the mosaic nature of the extrachromosomal array expressing the E. coli ${\text{tRNA}}_{\text{CUA}}^{\text{Leu}}$. (E) LWA1560 with rpr-1p: ${\text{tRNA}}_{\text{CUA}}^{\text{Leu}}$ + pRF4(rol-6) had no muscle fluorescence. The observed autofluorescence was from intestinal granules. (F) LWA1562 (LWA1560 with wlEx22[unc-54∷TyrRS_rpr-1∷ ${\text{tRNA}}_{\text{CUA}}^{\text{Tyr}}$ + pRF4(rol-6)]) showed red fluorescence in some body wall muscles. Because the ${\text{tRNA}}_{\text{CUA}}^{\text{Tyr}}∕\text{TyrRS}$ was expressed in an extrachromosomal array, the red fluorescence was mosaic. Dashed lines indicate worm outline. All images are confocal Z stacks and representative of each genotype at young adulthood.