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. 2014 Dec 30;43(2):803–816. doi: 10.1093/nar/gku1360

Figure 1.

Figure 1.

Identification of C. elegans HZA element. (A) Wild-type animals were cultured with 0, 100 or 200 μM supplemental zinc. RNA was extracted from a synchronized population at L4/adult stages, and mRNA levels of indicated genes were determined by qRT-PCR. The bars indicate mRNA levels at 0, 100 and 200μM supplemental zinc; mRNA levels at 0 μM supplemental zinc were set equal to 1.0 for each gene. Values are the average ± SEM of four independent experiments. All four genes displayed statistically significant increases at 100 and 200 μM supplemental zinc (*P < 0.05, **P < 0.01, ***P < 0.005). (B) Diagrams (not to scale) show the extent of DNA from the ttm-1b gene (black line) upstream of the translation start site fused to the coding region of GFP (green box). Images show transgenic animals containing these plasmids that were cultured with 0 or 100 μM supplemental zinc at L4/adult stage and imaged by fluorescence microscopy. GFP fluorescence signals (green) were captured with the identical settings and exposure times and overlaid with bright field images. (C) Transgenic animals containing reporter constructs shown in panel B were cultured with 0 or 200 μM supplemental zinc, and GFP mRNA levels were analyzed by qRT-PCR. The bars indicate the fold change of GFP mRNA levels as the ratio between the level at 200 and 0 μM supplemental zinc. A positive value indicates induction in high zinc. Values are the average ± SEM of three independent experiments. The −2.4 kb promoter displayed significantly reduced induction compared to the −6-kb promoter (*P < 0.05). (D) A sequence logo (position weight matrix) of the HZA element identified by MEME using the promoter regions of C. elegans mtl-1, mtl-2, cdf-2 and ttm-1b. The height of the nucleotide at each position represents the frequency in the four DNA sequences scaled in bits. (E) The sequence of the HZA element in mtl-1, mtl-2, cdf-2 and ttm-1b. The location is the first nucleotide of the DNA motif relative to the translation start site (ATG); (+) and (−) indicate the same and opposite orientation relative to the direction of transcription, respectively.