Academic journal article Genetics

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

Academic journal article Genetics

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

Article excerpt

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.

METALS such as zinc, iron, and copper play essential roles in biological systems. Here we focus on zinc, since it is one of the most abundant metals in animals and it has a wide range of functions. Zinc is a divalent cation that is not redox active in biological systems. Zinc is an essential catalytic component of >300 enzymes (in all six major classes) and a critical component of structural motifs such as zinc fingers (VALLEE and FALCHUK 1993). The analyses of several eukaryotic genomes have led to the estimate that zincmay be required for the function of >3% of all proteins (LANDER et al. 2001). Zinc has also been implicated in signaling processes and may be a signaling molecule: zinc is concentrated in some synaptic vesicles and then released into the synapse where it might modulate neurotransmission (FREDERICKSON and BUSH 2001; COLVIN et al. 2003; WALL 2005; YAMASAKI et al. 2007). Zinc affects epidermal growth factor receptor/Ras-mediated signal transduction, thus playing a role in cell fate determination (WU et al. 1999; BRUINSMA et al. 2002; SAMET et al. 2003; YODER et al. 2004). The importance of the processes that involve zinc is demonstrated by the observation that severe zinc deficiency is incompatible with growth and survival. Although zinc is essential, excess zinc can be deleterious. The mechanisms of excess zinc toxicity have not been well defined, but a plausible model is that excess zinc binds inappropriate sites in proteins or cofactors, perhaps replacing the physiologically relevant metals (ZHAO and EIDE 1997).

Because zinc is essential but also potentially toxic, organisms must have systems for efficient zinc uptake and distribution but also systems for zinc excretion or detoxification. These systems must involve mechanisms that sense zinc levels and trigger a regulatory response to achieve zinc homeostasis (TAPIERO and TEW 2003). Important progress has been made in characterizing proteins involved in zinc metabolism and mechanisms of zinc homeostasis. However, the understanding of these processes remains incomplete. The best-characterized model systems of zinc metabolism and homeostasis are single-celled organisms, such as bacteria and yeast (reviewed by GAITHER and EIDE 2001; EIDE 2003; HANTKE 2005). Some mechanisms of zinc metabolism defined in yeast appear to be conserved in vertebrates (LIUZZI and COUSINS 2004). Because zinc is a hydrophilic ion that cannot diffuse passively across membranes, specific transport mechanisms are required for it to enter and exit cells and organisms. …

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