In 2003, the EPA ranked cadmium seventh on the top 20 hazardous substances. Human health risks associated with cadmium exposure include kidney damage, respiratory diseases, and various cancers. To reduce cadmium-induced damage, cells increase stress-response proteins needed for cellular repair, detoxification, or export of cadmium. Metallothioneins (MT-1 and MT-2) function in humans to chelate cadmium to remove it from the cell. Regulation of MT-1 and MT-2 is coordinated by the metallothionein transcription factor, MTF-1. Previous work determined that C. elegans has two metallothionein genes (
mtl-1 and
mtl-2) but lacks a MTF-1 homolog. Previously, cadmium-inducible genes were identified by differential display analysis (
cdr-1) and by microarray analysis (see abstract by Yuxia Cui). We hypothesize that C. elegans has a unique mechanism for regulating cadmium-inducible genes (
mtl-1,
mtl-2, and
cdr-1) independent of MTF-1. The transcription of
mtl-1,
mtl-2, and
cdr-1 may be regulated by similar transcription factors. To identify conserved promoter motifs, the European Molecular Biology Open Source Software (EMBOSS) suite was used to compare the 300 bp upstream region from the transcription start site of
mtl-1,
mtl-2, and
cdr-1 for C. elegans and C. briggsae. Both strands were examined for exact alignments of 6 bp in length or longer. This comparison resulted in 983 matches. This list was refined by only including alignments present in at least 3 promoters and resulted in 162 motifs. To determine the motifs needed for proper gene expression, transgenic strains will be created in which the promoter of C. elegans
mtl-1,
mtl-2, or
cdr-1 controls GFP expression. PCR site-directed mutagenesis will be used to mutate the conserved motifs within these promoter regions. After exposure to cadmium, GFP expression levels and location will be compared between the mutant and wild-type promoter motif(s). These experiments will determine conserved promoter motifs and may suggest proteins that are essential for proper gene expression. To identify genes that regulate these cadmium-inducible genes, two screens are being conducted. First, feeding RNAi is being used to create a reduction of gene function in transgenic
mtl-1:gfp,
mtl-2:gfp, and
cdr-1:gfp strains with and without cadmium. To identify gain-of-function mutations and mutations in genes resistant to RNAi, a forward genetic screen is being conducted. The integrated promoter:gfp transgenic strains are mutagenized with EMS and ENU and exposed to cadmium. Mutants are selected that show an increase or decrease GFP expression after cadmium exposure. Using these strategies, genes regulating
mtl-1,
mtl-2, and
cdr-1 will be identified.