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. 2023 Oct 2;12:e79725. doi: 10.7554/eLife.79725

Figure 1. Analysis of the impact of mtDNAuaDf5 on fitness parameters.

(A) Diagram of C. elegans mtDNA. Black bars with arrows indicate the locations of genes and direction of transcription. Black lines with letters indicate the locations of tRNAs. Green bars show the locations of the mtDNAuaDf5 deletion as well as the linked w47 insertion that was identified via Illumina sequencing. (B) Brood size analysis of mtDNAuaDf5 compared to laboratory wildtype N2. (C) Embryonic lethality analysis of uaDf5 compared to laboratory wildtype N2. (D) Developmental rate analysis of mtDNAuaDf5 compared to laboratory wildtype N2, counting how many hours it takes for starved L1s to reach gravidity once plated on food. (E) Developmental rate analysis of mtDNAuaDf5 compared to laboratory wildtype N2, staging worms 60 hr after synchronized, starved L1s are plated on food. (F) Survival curve analysis of mtDNAuaDf5 compared to laboratory wildtype N2, day 1 is defined as the day starved L1s are plated on food. Median lifespan and statistics are presented in Figure 1—figure supplement 2. For (B–E), box plots show median and IQR (Interquartile Range), and the diamond indicates the mean. Statistical analysis was performed using the Mann–Whitney test (***p < 0.001, n.s. not significant).

Figure 1.

Figure 1—figure supplement 1. Characterization of the uaDf5 allele.

Figure 1—figure supplement 1.

(A) The percentage of reads that mapped to the w47 insertion in uaDf5 samples (red) and wildtype samples (blue). Error bars represent standard deviation of the mean (SEM). (B) Table outlining the percentage of reads that mapped to the w47 and uaDf5 mutations in uaDf5 samples. *The number of variant reads was determined by averaging the mapped reads across the deleted region and subtracting that from the average number of reads mapped to the rest of the mtDNA genome (total reads). (C) Diagram showing the mitochondrial respiratory chain (MRC) machinery subunits. Blue indicates nuclear DNA (nDNA)-encoded subunits, orange and green indicate mtDNA-encoded subunits. Green indicates those subunits that are knocked out in the uaDf5 allele (including NADH dehydrogenase 4 [ND4] which is knocked out by the linked w47 mutation). (D) Diagram showing the likely effect of the w47 mutation on ND4 protein translation. ND4 is a 409-aa long transmembrane subunit that spans the inner mitochondrial membrane 13 times. The w47 mutation results in a premature stop codon at position 89, eliminating 10 of the 13 alpha-helix membrane domains. (E) Diagram showing the likely effect of the uaDf5 mutation on ND1 protein translation. ND1 is a 291-aa long transmembrane subunit that spans the inner mitochondrial membrane 8 times and CYTB is a 370-aa long transmembrane subunit that spans the inner mitochondrial membrane 8 times. The uaDf5 mutation results in a 266 amino acid long fusion protein that connects the first 185 amino acids (and 5 subunits) of ND1 with the last 81 amino acids (and 3 subunits) of CYTB.
Figure 1—figure supplement 1—source data 1. ddPCR reads of mitochondrial DNA.
Figure 1—figure supplement 2. Lifespan analysis of the impact of uaDf5.

Figure 1—figure supplement 2.

Lifespan analysis of N2-bearing uaDf5 compared to wildtype N2. Day 1 is defined as the day starved L1s are plated on food. Box plots show median and IQR, and the diamond indicates the mean. Statistical analysis was performed using the Mann–Whitney test (n.s., not significant).