The objective of this project is to introduce students to the role of trace metals and metalloproteins in early embryonic development. In particular, the research will focus on the isolation and characterization of metallothionein(MT)-like proteins from the brine shrimp Artemia salina and the factors which influence its expression during development. Metallothioneins are a class of low molecular weight metal binding proteins found in a variety of organisms, including crustacean. They are characterized by their highly conserved primary structure and high affinity for essential trace metals such as copper and zinc, both of which are required for normal cell growth and differentiation. The involvement of MT in metal ion homeostasis and human health is well documented. The fact that MT is inducible by heavy metals and a number of other extracellular stress factors also makes this an attractive system for the study of eukaryotic gene expression. A class of low molecular weight metal binding proteins, referred to as ZnBPII (zinc binding proteins, group II), has been detected in cytosolic extracts from Artemia. The mixture is composed of at least four discrete MT-like zinc metalloproteins one of more of which are developmentally regulated. The molecules will be purified to homogeneity using gel filtration, ion-exchange chromatography, and C18 reverse phase HPLC chromatography. Purity of the product will be checked by SDS-Page. The amino acid composition will be analyzed for each of the proteins to determine their similarity to mammalian metallothioneins. A developmental profile for the expression of each of the four proteins will be determined. Subsequently, embryos will be exposed to non-lethal concentrations of copper and cadmium to determine the effect of metal on the expression of the individual proteins. Finally, a bacterial cDNA library generated from Artemia will be screened to isolate the potential gene for this protein. Southern blot analysis of Artemia genomic DNA using a human MT gene probe has revealed the presence of MT-lime gene sequences in Artemia. Positive clones will be isolated and the inserts sequenced by the Sanger method for DNA sequencing. Initially, students will be exposed to the basic biochemical and molecular biology techniques will be to impress on them the importance of fundamental chemical principles as they apply to the study of living organisms. Thereafter, they will be encouraged to design and implement original experimental protocols to answer a specific question regarding metal ion homeostasis in embryonic development.
