Figure 1.
Diagram of nhr-80, fat-5, fat-6, and fat-7 Genes and Mutations
(A) nhr-80 is composed of a zinc finger domain (green boxes) and a ligand-binding domain (light blue boxes). nhr-80(tm1011) contains a 446-bp deletion (light grey bar).
(B) fat-5, fat-6, and fat-7 all contain four trans-membrane domains (dark blue boxes) and three histidine boxes (red boxes). fat-5(tm420) consists of a 779-bp deletion (light grey bar). fat-6(tm331) contains a 1,232-bp deletion (light grey bar), and a 428-bp insertion (purple bar). The fat-7 alleles are point mutations with fat-7(wa36), creating a premature stop codon and fat-7(wa37) changing a conserved histidine into a tyrosine.
Figure 2.
Fatty Acid Composition of nhr-80
Relative abundance of selected fatty acid species expressed as percentage of total fatty acid as determined by gas chromatography analysis. The nhr-80 mutants have significantly higher levels of 18:0 and lower levels of 16:0 and 18:1 Δ9 than wild type. Error bars represent the standard error. *significant differences between wild type and nhr-80 mutant, p < 0.01.
Figure 3.
Expression of the Δ9 Desaturase Genes in nhr-80
(A) Gene expression by QPCR in the nhr-80 mutant reveals a decrease in expression of the Δ9 desaturase genes relative to wild type. Error bars represent standard error.
(B) Transformed lines expressing Δ9 desaturase gene GFP fusions grown to adulthood on empty vector control bacteria or nhr-80(RNAi) bacteria. Exposure times for photographs were adjusted due to different GFP expression in the three genes, although the exposure time for the two treatments was kept the same for each genotype. The exposure time for the fat-5::GFP worms was 1/4 s, for the fat-6::GFP worms was 1/30 s, and for the fat-7::GFP worms was 1/8 s. After 4 d, there is a dramatic reduction in Δ9 desaturase gene expression in the intestine for fat-5::GFP and fat-7::GFP lines grown on nhr-80(RNAi).
Figure 4.
Aging of Adult Mutant Populations
(A) Life span of nhr-49, nhr-80, and wild type at 25 °C. The nhr-80 and wild type display a considerably longer life than the nhr-49 mutants. All lifespan data are presented as mean lifespan ± standard error (total number of animals scored). Wild type: 13.9 ± 0.4 (83); nhr-49: 8.2 ± 0.2 (80); nhr-80: 12.5 ± 0.5 (70).
(B) Life span of fat-5, fat-6, fat-7, and wild type at 25 °C. The fat-5, fat-6, and fat-7 mutants exhibit a lifespan similar to wild type. Wild type: 13.9 ± 0.4 (83); fat-5: 15.9 ± 0.6 (82); fat-6: 14.2 ± 0.5 (82); fat-7: 15.0 ± 0.5 (75).
Figure 5.
Fatty Acid Composition of the Δ9 Desaturase Mutants
(A−C) There is little change in fatty acid composition for fat-5 (A), fat-6 (B), and fat-7 (C) mutants compared to wild-type worms when grown under standard growth conditions with OP50 E. coli as the sole food source.
(D−F) Axenic growth conditions for wild-type worms and fat-5 (D), fat-6 (E), and fat-7 (F) mutants reveal major changes in fatty acid composition for fat-5 and fat-6 mutants compared to wild-type worms. In all graphs relative abundance of selected fatty acid species is expressed as percentage of total fatty acid as determined by gas chromatography analysis. Error bars represent the standard error. *significant difference from wild type, p < 0.01
Figure 6.
Expression of the Δ9 Desaturase Genes in the fat-5, fat-6, and fat-7 Mutants
Gene expression by QPCR in the fat-5, fat-6, and fat-7 mutants relative to wild type reveals an increase in Δ9 desaturase gene expression in the fat-6 and fat-7 mutants, relative to wild type. Error bars represent standard error of 7–12 experiments.
Figure 7.
Effects of nhr-80(RNAi) in the Δ9 Desaturase Mutant Background
(A) Photographs showing adult worms after 4 d of growth on nhr-80(RNAi) and empty vector control bacteria. The fat-6 mutants grown on nhr-80(RNAi) are thin, pale, and produce no viable progeny.
(B) Relative abundance of 18:0 expressed as a percentage of total fatty acid as determined by gas chromatography analysis. The fat-6 mutants grown on nhr-80(RNAi) (n = 5) accumulate much higher levels than fat-6 mutants grown on control (n = 7) and wild type grown on nhr-80(RNAi) (n = 6). *significant differences from growth on control bacteria, p < 0.01.
(C) Effects of nhr-80 on Δ9 desaturase gene expression in fat-5, fat-6, and fat-7 mutants. QPCR in fat-5, fat-6, fat-7, and wild type for worms grown on empty vector control bacteria (ev) and nhr-80(RNAi) (80i) (n = 6). Values are expressed relative to fat-6 expression in wild-type worms grown on control bacteria. For all graphs error bars represent standard error.