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.

Identification of Mutations that Delay Somatic or Reproductive Aging ofCaenorhabditis elegans

“…First, we used a low-density, genome-wide map of single nucleotide polymorphisms (SNPs) that differ between the N2 (Bristol) and the CB4856 (Hawaiian) strains as described by Bruinsma et al (2008). am117 males were mated to CB4856 hermaphrodites, F 1 cross-progeny were isolated, and F 2 self-progeny were picked as eggs to individual Petri dishes.…”

“…To determine the chromosomal linkage of am117, we mated homozygous am117 mutants to the polymorphic CB4856 strain isolated in Hawaii and selected F 2 worms that displayed the scrawny phenotype. We used the method of DNA pyrosequncing to analyze single nucleotide polymorphisms (SNPs) that differ between the am117 strain and the CB4856 strain (Bruinsma et al 2008). am117 displayed highest linkage to a SNP marker positioned on the right arm of chromosome I (Materials and Methods).…”

The Cation Diffusion Facilitator Gene cdf-2 Mediates Zinc Metabolism in Caenorhabditis elegans

“…The use of C. elegans to study metal biology has included analyses of heme, magnesium, zinc, and cadmium (Bruinsma et al 2002;Bruinsma et al 2008;Dong et al 2008a;Rajagopal et al 2008;Kemp et al 2009). The fully sequenced C. elegans genome contains 14 predicted genes that encode CDF proteins (Kambe et al 2004;K.…”

Rapid identification of homologous recombinants and determination of gene copy number with reference/query pyrosequencing (RQPS)

“…PCR conditions were 8 min at 95°C followed by 40 cycles at 95°C (30 sec), 60°C (30 sec), and 72°C (30 sec). Single-strand PCR products were purified as described (Bruinsma et al 2008). Briefly, single-stranded biotinylated PCR products were immobilized on streptavidin-coated Sepharose beads in a vacuum prep tool (Biotage).…”