Identification of mutations in Caenorhabditis elegans that cause resistance to high levels of dietary zinc and analysis using a genomewide map of single nucleotide polymorphisms scored by pyrosequencing

Abstract

Zinc plays many critical roles in biological systems: zinc bound to proteins has structural and catalytic functions, and zinc is proposed to act as a signaling molecule. Because zinc deficiency and excess result in toxicity, animals have evolved sophisticated mechanisms for zinc metabolism and homeostasis. However, these mechanisms remain poorly defined. To identify genes involved in zinc metabolism, we conducted a forward genetic screen for chemically induced mutations that cause Caenorhabditis elegans to be resistant to high levels of dietary zinc. Nineteen mutations that confer significant resistance to supplemental dietary zinc were identified. To determine the map positions of these mutations, we developed a genomewide map of single nucleotide polymorphisms (SNPs) that can be scored by the high-throughput method of DNA pyrosequencing. This map was used to determine the approximate chromosomal position of each mutation, and the accuracy of this approach was verified by conducting three-factor mapping experiments with mutations that cause visible phenotypes. This is a generally applicable mapping approach that can be used to position a wide variety of C. elegans mutations. The mapping experiments demonstrate that the 19 mutations identify at least three genes that, when mutated, confer resistance to toxicity caused by supplemental dietary zinc. These genes are likely to be involved in zinc metabolism, and the analysis of these genes will provide insights into mechanisms of excess zinc toxicity.

Figure 1.—

Supplemental dietary zinc caused dose-dependent development delays and lethality. Wild-type animals (open diamonds) and cdf-1(n2527) mutants (solid circles) were cultured on NAMM with supplemental zinc sulfate starting at the egg stage, and development was monitored daily. (A) Animals that did not progress to the adult stage within 4 days were defined as not displaying a normal developmental rate. Each data point represents ∼100 animals. (B) Animals that did not progress to the adult stage within 10 days were defined as not surviving to adulthood. Most animals that did not survive to adulthood died as L1 larvae. The concentration of supplemental zinc that caused ∼50% lethality (LC₅₀) is indicated.

Figure 2.—

Phenotypic analysis of zinc-resistant mutants. (A) For wild-type and mutant strains (designated am118-am140), ∼100 eggs were placed individually on petri dishes and development was monitored for 10 days. About 25% of wild-type animals matured to adulthood in this time, and mutant values were normalized by setting the wild-type value to 1.0. (B) Wild-type animals (open diamonds) and am132 mutants (solid squares) were cultured on NAMM with supplemental zinc sulfate starting at the egg stage, and development was monitored daily. Animals that did not progress to the adult stage within 4 days were defined as not displaying a normal developmental rate. Each data point represents ∼100 animals.

Figure 3.—

DNA pyrosequencing is a quantitative method for genotyping C. elegans SNPs. (Top, A–F) Representative pyrograms depicting the analysis of DNA from N2 homozygotes (A and D), CB4856 homozygotes (B and E), and N2/CB4856 heterozygotes (C and F). The nucleotides listed below the pyrograms were added to the reaction sequentially, and the height of a peak indicates the amount of light emitted, which is proportional to the number of nucleotides incorporated into DNA. The polymorphic nucleotides are underlined (Bottom, A–F) Histograms in which the pyrogram peak heights were normalized by setting the average signal for incorporation of a single nucleotide equal to 1.0 [the values for dATP incorporation were not included in this calculation, since dATP can act as a substrate for luciferase and might contribute some false-positive signal (Ronaghi 2001)]. (A–C) The analysis of polymorphism pkP6155: the N2 sequence is CGA, the CB4856 sequence is TGA, and the N2/CB4856 heterozygote sequence is (C/T)GA. (D–F) The analysis of polymorphism pkP1057: the N2 sequence is TTGG, the CB4856 sequence is ATGG, and the N2/CB4856 heterozygote sequence is (A/T)TGG.

Figure 4.—

Genetic map of SNP markers that can be scored by pyrosequencing. Horizontal lines represent the six chromosomes (numbered at the left), which are aligned at genetic map position 0, approximately the center. Polymorphisms between the N2 strain and the CB4856 strain are named above using the standard C. elegans nomenclature (two-letter laboratory designation, P for polymorphism, and a number); the position in map units is shown below. Each polymorphism is described in Table 1.

Figure 5.—

A genetic map showing the positions of mutations that cause zinc resistance. Horizontal lines represent chromosomes I, V, and X (numbered at the left). Genes that can be mutated to cause a visible phenotype and polymorphisms between the N2 strain and the CB4856 strain are named above, and the position in map units is shown below. Mutations that cause zinc resistance are shown above the polymorphism (large font and boldface type) that displayed the highest linkage, except for the mutations am128 and am136, which displayed equally high linkage to pkP6152 and pkP6155, and the mutation am134, which displayed slightly higher linkage to pkP5066 (Table 3). Lines with two-headed arrows indicate intervals that contain mutations that cause zinc resistance determined by three-factor mapping experiments with genes that can be mutated to cause a visible phenotype (see materials and methods for three-factor mapping data).