Identification of Two Nickel Ion-Induced Genes, NCI16 and Pc GST1 , in Paramecium caudatum

Abstract

dHere, we describe the isolation of two nickel-induced genes in Paramecium caudatum, NCI16 and PcGST1, by subtractive hybridization. NCI16 encoded a predicted four-transmembrane domain protein (ϳ16 kDa) of unknown function, and PcGST1 encoded glutathione S-transferase (GST; ϳ25 kDa) with GST and glutathione peroxidase (GPx) activities. Exposing cells to cobalt chloride also caused the moderate upregulation of NCI16 and PcGST1 mRNAs. Both nickel sulfate and cobalt chloride dose dependently induced NCI16 and PcGST1 mRNAs, but with different profiles. Nickel treatment caused a continuous increase in PcGST1 and NCI16 mRNA levels for up to 3 and 6 days, respectively, and a notable increase in H 2 O 2 concentrations in P. caudatum. NCI16 expression was significantly enhanced by incubating cells with H 2 O 2 , implying that NCI16 induction in the presence of nickel ions is caused by reactive oxygen species (ROS). On the other hand, PcGST1 was highly induced by the antioxidant tertbutylhydroquinone (tBHQ) but not by H 2 O 2 , suggesting that different mechanisms mediate the induction of NCI16 and PcGST1. We introduced a luciferase reporter vector with an ϳ0.42-kb putative PcGST1 promoter into cells and then exposed the transformants to nickel sulfate. This resulted in significant luciferase upregulation, indicating that the putative PcGST1 promoter contains a nickel-responsive element. Our nickel-inducible system also may be applicable to the efficient expression of proteins that are toxic to host cells or require temporal control. N ickel is used extensively for electroplating metals, alloys such as cupronickel, and rechargeable batteries. Occupational exposure to nickel occurs in industrial workers, in particular those involved in mining, smelting, and refining, the production of steel and other metals, and electronic devices (1). Nickel compounds are released into the environment from power plants that burn oil, trash incinerators, wastewater from nickel mines, and industries that manufacture nickel products for industrial and consumer use. Nickel has toxic and carcinogenic effects on most microorganisms and animals and is considered to impose an industrial health hazard (2, 3). Nickel compounds such as nickel subsulfide (Ni 3 S 2 ) are potent carcinogens, but soluble nickel salts such as nickel chloride (NiCl 2 ) exert weaker effects. The molecular mechanisms involved in the cytotoxicity and carcinogenicity of nickel compounds are not fully understood, but nickel might be associated with the intracellular production of reactive oxygen species (ROS), including superoxide, H 2 O 2 , singlet oxygen, and hydroxyl radicals (4-8). Nickel also increases lipid peroxide (LPO) levels, resulting in the generation of peroxyl radicals, lipid hydroperoxides, and alkoxyl radicals (9-11). Carcinogenesis related to nickel is explained by several types of DNA damage, such as cleavage, depurination, cross-linking, and DNA base damage caused by ROS (12). Nickel inhibits processes in the DNA repair system, such as DNA ligation and DNA p...

Immaturin-Nuclease as a Model System for a Gene-Programmed Sexual Development and Rejuvenescence in Paramecium Life History

“…The nucleotide sequence of the immaturin gene indicates that it originates in the GST cytosol family (GST C family) of proteobacteria. The gene encoding GST is found in all organisms, including prokaryotes and eukaryotes, and the GST proteins are grouped into three superfamilies known as cytosolic, mitochondrial, and microsomal proteins [ 21 , 22 , 23 ]. The differences in the amino acid sequence of these superfamilies represent complex gene evolution, suggesting their importance in physiological functions, such as detoxification of endogenous compounds and degradation of xenobiotic compounds [ 24 , 25 , 26 , 27 ].…”

Micromanipulation in Paramecium: From non‐mendelian inheritance to the outlook for versatile micromachines

“…The nickel-responsive element is involved in the putative PcGST1 (P. caudatum glutathione S-transferase gene) promoter. The nickel-inducible system may be applicable to the efficient expression of proteins that require temporal control (Takenaka et al, 2014).…”