Wild-type, haly-1(am130) and haly-1(am132) hermaphrodites were synchronized at the L1 stage and cultured on NAMM supplemented with zinc (mM). The fraction of worms that grew to adulthood over seven days was monitored; the data were normalized by setting the value at 0 mM supplemental zinc equal to 1.0. Each point indicates mean value ± SE (n = 4 replicates with 50 worms per replicate). The fraction adult for haly-1(am130) and haly-1(am132) was significantly higher than wild type at 0.15 mM and higher concentrations of supplemental zinc (p<0.05).

(A) The haly-1 mRNA structure – boxes represent exons, shaded regions are untranslated, lines indicate introns, and AAA indicates the poly A addition site. (B) The predicted C. elegans HALY-1 protein is aligned with HAL proteins from Homo sapien, the zebra fish Danio rerio and the bacteria Cupriavidus metallidurans. Shading indicates residues identical to C. elegans HALY-1. The locations of the haly-1(am132) nonsense mutation and haly-1(am130) missense mutation are shown. (C) Histidine ammonia lyase causes the reductive deamination of L-histidine to urocanic acid.

Populations of wild-type and haly-1(am130) animals consisting of a mixture of developmental stages were cultured in CeMM with the indicated levels of zinc. Total zinc content was determined by ICP-MS and calculated in parts per million (ppm). Bars indicate mean values ± SE (n = 2 independent replicates). Values for wild-type and haly-1(am130) animals were not significantly different at 0.075 mM (p = 0.6) and 1 mM (p = 0.3).

(A) Wild-type, haly-1(am130), and haly-1(am132) hermaphrodites were synchronized at the L1 stage and cultured on NAMM supplemented with nickel (mM). The fraction of worms that grew to adulthood over seven days was monitored; the data were normalized by setting the value at 0 mM supplemental nickel equal to 1.0. Each point indicates mean value ± SE (n = 6 replicates with 50 worms per replicate). The fraction adult for haly-1(am130) and haly-1(am132) was significantly higher than wild type at 0.02–0.06 mM supplemental nickel (p<0.01). (B) Wild-type animals were cultured with no histidine (closed circles) or with 0.1 mM L-histidine (open circles). Each point indicates mean value ± SE (n = 6 replicates with 50 worms per replicate). The fraction adult for WT + His was significantly higher than WT at 0.02 mM and higher concentrations of supplemental nickel (p<0.01).

(A) A genetic map of chromosome X. Loci defined by mutations that cause visible phenotypes and SNPs are indicated above, and map units are shown below. Three-factor mapping experiments positioned am132 between unc-115 and egl-15. Multi-factor mapping experiments positioned am132 between the SNPs CE6-177 and CE6-1202, an interval of 270 kb. (B) Lines indicate DNA, boxes represent exons, and shading represents untranslated regions. The fosmid WRM0624bE06 includes eight predicted ORFs shown as arrows indicating orientation of transcription, including haly-1 (F47B10.2). Plasmid pJM1 included only the wild-type haly-1 ORF, whereas pJM2 has a deletion mutation and pJM3 has a nonsense change identical to haly-1(am132). Rescue indicates the number of independently derived transgenic strains that displayed wild-type zinc tolerance over the total number of transgenic strains analyzed.

(A) Wild-type, haly-1(am132), cdf-1(n2527) and cdf-1(n2527) haly-1(am132) hermaphrodites were synchronized at the L1 stage and cultured on NAMM supplemented with zinc. The fraction of worms that grew to adulthood over seven days was monitored; the data were normalized by setting the value at 0 mM supplemental zinc equal to 1.0. Each point indicates mean value ± SE (n = 3 replicates with 50 worms per replicate). The fraction adult for cdf-1(n2527) haly-1(am132) was significantly different than cdf-1(n2527) and haly-1(am132) at 0.06 mM supplemental zinc (p<0.05). (B) The data from panel A at 0.04 mM supplemental zinc are displayed as a bar graph. The fraction adult for cdf-1(n2527) haly-1(am132) was significantly higher than cdf-1(n2527) (p = 0.0009). (C) Wild-type, haly-1(am132), sur-7(ku119) and haly-1(am132) sur-7(ku119) hermaphrodites were analyzed as described above at 0.04 mM supplemental zinc. Bars indicate mean values ± SE. The fraction adult for haly-1(am132) sur-7(ku119) was significantly different than sur-7(ku119) and haly-1(am132) (p<0.02). (D) Wild-type, haly-1(am132), cdf-2(tm788) and haly-1(am132) cdf-2(tm788) hermaphrodites were analyzed as described above at 0.1 mM supplemental zinc. Bars indicate mean values ± SE. The fraction adult for haly-1(am132) cdf-2(tm788) was significantly different than cdf-2(tm788) and haly-1(am132) (p<0.003).

Wild-type (A), haly-1(am130) (B) and haly-1(am132) (C) hermaphrodites were synchronized at the L1 stage and cultured on NAMM supplemented with zinc (mM). Closed symbols indicate culture without histidine, and open symbols indicate culture with 0.1 mM histidine. The fraction of worms that grew to adulthood over seven days was monitored; the data were normalized by setting the value at 0 mM supplemental zinc equal to 1.0. Each point indicates mean value ± SE (n = 4 replicates with 50 worms per replicate). The fraction adult for WT + His was significantly higher than WT at 0.15 mM and higher concentrations of supplemental zinc (p<0.0005). The fraction adult for haly-1(am130) + His was significantly higher than haly-1(am130) at 0.25 mM and higher concentrations of supplemental zinc (p<0.03). The fraction adult for haly-1(am132) + His was significantly higher than haly-1(am132) at 0.25 mM and higher concentrations of supplemental zinc (p<0.005). (D) Populations of animals consisting of a mixture of developmental stages were cultured in CeMM with either 0, 0.075 or 1.5 mM zinc. Total histidine content was determined by amino acid analyzer and calculated in nmoles L-histidine per µg protein. The histidine values for wild-type and mutant strains were not significantly affected by the level of dietary zinc (data not shown), and bars indicate mean values ± SE of six independent experiments comprised of two samples at each concentration of dietary zinc. Values for wild type were significantly different than values for haly-1(am130) (p = 0.0009) and haly-1(am132) (p = 0.019), whereas values for the two mutant strains were not significantly different (p = 0.22). (E) Wild-type and haly-1(am132) hermaphrodites were synchronized at the L1 stage, cultured on NAMM supplemented with 1 mM L-histidine (+) or 0 mM L-histidine (−) and no supplemental zinc for 24 hours, washed extensively, and then cultured on NAMM supplemented with 0 mM or 0.4 mM zinc and 0 mM L-histidine for six days. The fraction of worms that grew to adulthood over seven days was monitored. Bars indicate mean value ± SE (n = 4 replicates with 50 worms per replicate). At 0.4 mM supplemental zinc, values for wild-type worms pre-treated with L-histidine were significantly higher than values for worms that were not pre-treated (p = 0.022). Values for haly-1(am132) animals pre-treated with L-histidine were higher than values for animals that were not pre-treated (p = 0.06).