Expression of in situ biomarkers in striped bass
The proposed project has the following objectives : 1) Identify and sequence the striped bass (BB) glutathione Stransferase(s) (GSTs) gene(s); 2) Use the identified sequences as a probe to compare GST mRNA levels in control versus laboratory and/or selected field exposed SB.
The goal of this research is to develop a biomonitoring model for specific California surface waters utilizing SB, the popular game fish which is present in this environment. An approach based on the increased/decreased expression of GSTs will be used to screen the effect of experimental exposures of SB to toxic chemicals including polycyclic aromatic hydrocarbons, rice field herbicides and pesticides for their hepatoxicity. GSTs are a multigene family of enzymes catalyzing the conjugation of numerous electrophiles with reduced glutathione (GSH) by formation of the thioether bond. These reactive electrophiles include metaboli tes and endogenous compounds, drugs and pollutants. For the majority of substrates, the GSH conjugate is less toxic than the parent compound providing the organism protection against chemical insult. The level of expression of GST isoforms is extremely important in determining exposure to pollutant xenobiotics.
We have already isolated 3 GST proteins from SB following standard protein purification techniques and a GSH affinity column. These proteins were digested with CNBr, purified with HPLC and N-terminal sequenced. We plan to utilize the oligos derived from the back translated peptide sequences as well as conserved sequences (obtained from the literature) to screen a SB cDNA library for SB GSTs. Initially, hepatic mRNA will be isolated from control SB and cDNA synthesized using standard protocols. A cDNA library will be prepared in a lambda gtlO bacteriophage (Stratagene gigapack II cloning kit) and probed for the presence of glutathione (GST) clones using the previously described probes. positive clones will be isolated and the data used to further PCR amplify specific sequences of striped bass GST cDNA of exposed and control fish. It should be possible to determine biomarkers of host mediated protection (Phase II, GSTs) resulting from specific chemicals.