Figure 1. Putative germline TFAM overexpression modulates mtDNA copy number and heteroplasmy:(A) Schematic of strategies to increase germline TFAM levels via transgenic expression at the endogenous
glh-1 locus. Strategy (1) utilizes the viral T2A ribosomal skipping sequence: sequence encoding T2A::TFAM was inserted in frame with the endogenous
glh-1 coding sequence, resulting in the production of a single mRNA that is translated into two independent polypeptides via ribosomal skipping of the T2A sequence. Strategy (2) utilizes the C. elegans SL2 trans-splicing recognition sequence of the gene
rla-1: sequence encoding SL2::TFAM was inserted immediately following the endogenous
glh-1 stop codon, resulting in the production of two independent mRNAs that could then be translated into two polypeptides independently of each other. Endogenous
glh-1 sequence (maroon), TFAM(
hmg-5) sequence (green), T2A sequence (cyan), SL2 trans-splicing recognition sequence [black line between
glh-1 and TFAM(
hmg-5)]. (B) Quantification of total mtDNA copy number from whole L4 larvae by qPCR in wild-type, TFAM-O/E (T2A), TFAM-O/E (SL2), TFAM-GFP (rf), TFAM-O/E (T2A); TFAM-GFP (rf), and TFAM-O/E (SL2); TFAM-GFP (rf) animals. (C) Quantification of uaDf5 heteroplasmy in uaDf5 and TFAM-O/E (T2A); uaDf5 adults. Small dots are data points from individual L4 worms in (B), and technical ddPCR replicates in (C), from each of three color-coded biological replicates; the mean from each replicate is shown as a larger circle, the mean of means as a horizonal line, and the S.E.M as error bars. n.s., not significant (p> 0.05), **p ≤ 0.01, **** p ≤ 0.0001, unpaired two-tailed Student's t-test.