The application of mammalian models suggests that the ratio of reduced to oxidized glutathione may be an important indicator of oxidative stress and DNA damage in our model system. In addition, a reduction in total GSH level is often associated with oysters from contaminated sites suggesting a possible disruption in the biosynthetic pathway. In addition, although metallothioneins are best known for their role in the detoxification of metals, they play an important role in cellular metals homeostasis. Therefore, metal contamination can potentially disrupt cellular homeostasis by inhibiting the induction of constitutively expressed metallothioneins or interfering in their various functions. We are currently attempting to identify nucleotide sequences, through the generation of cDNA libraries, of both constitutively expressed and inducible isoforms of metallothionein.
Cells respond to a variety of stimuli resulting in the expression of a protein.
The combination of proteomics and genomics allows the researcher to address the regulatory aspects of gene expression. The successful development of the cDNA libraries will provide us with the probes necessary to perform northern hybridizations on induced mRNA in various oyster tissues. In addition, cDNA clones can be ligated into a plasmid for expression of the respective protein. In unison with northern blotting, the expressed protein can be used to develop antibodies for in situ immunocytochemistry or western blotting. Furthermore, successful completion of these libraries will allow us to develop probes to various oxidative stress genes and compare the induction of these genes to the in vivo activity of their metalloprotein products.