Grantee Research Project Results
Regulation of Metallothionein Gene Expression in the Nematode Caenorhabditis elegans
EPA Grant Number: U915011Title: Regulation of Metallothionein Gene Expression in the Nematode Caenorhabditis elegans
Investigators: Moilanen, Lorita H.
Institution: Duke University
EPA Project Officer: Packard, Benjamin H
Project Period: January 1, 1996 through July 30, 1999
Project Amount: $102,000
RFA: STAR Graduate Fellowships (1996) RFA Text | Recipients Lists
Research Category: Fellowship - Toxicology , Academic Fellowships , Human Health
Objective:
The objective of this research project is to identify molecular mechanisms responsible for the regulation of metal-inducible metallothionein (MT) gene expression in the soil nematode Caenorhabditis elegans.
Approach:
The overall strategy is to employ a well-characterized soil organism to probe the regulation of a key gene in metal detoxification and homeostasis. The aim of the research is to identify metal regulatory elements (MREs) within the promoters of two C. elegans MT genes, and isolate and characterize proteins that interact with the candidate MREs. Candidate MREs will be identified by site-directed mutagenesis and deletion experiments. The functional significance of each modification will be determined in vivo (inside the organism) by creating transgenic nematodes containing a ß-galactosidase reporter gene. Changes in the level of pattern of reporter gene activity in cadmium-exposed nematodes will be used to identity functional promoter elements. Metal-dependent interaction of crude extract proteins with candidate MREs will be evaluated by gel mobility shift assay and DNaseI footprinting. Regulatory proteins that interact with candidate MREs will be isolated from extracts prepared from control and cadmium-exposed nematodes using conventional protein isolation techniques and affinity chromatography. Binding characteristics of the isolated proteins will be characterized by gel mobility shift assay, DNaseI footprinting, and Southwestern blotting assays. Each binding protein will be subjected to partial amino acid sequencing. Complementary DNAs (cDNAs) encoding metal-regulatory proteins will be obtained by screening C. elegans expression libraries with oligonucleotides based on the partial amino acid sequence.