XIA, M; FENG, G; PITTMAN, C; BIESIOT, P; WANG, S*; Univ of Southern Mississippi, Hattiesburg; Univ of Southern Mississippi, Hattiesburg; Mississippi State Univ, Starkville; Univ of Southern Mississippi, Hattiesburg; Univ of Southern Mississippi, Hattiesburg: Isolation of Fish Genes Whose Level of Expression Is Altered in Response to Pollutant Exposure

The goal of the project is to isolate genes in the killifish Fundulus grandis that are differentially expressed in response to polycyclic aromatic hydrocarbon (PAH) exposure. The purpose for isolating such genes is to develop the use of gene expression profile monitoring as a tool to evaluate the toxicity of complex mixtures of chemicals produced by solvated electron reduction of PAHs. Using mRNA isolated from the livers of fish exposed to phenanthrene and control fish, we constructed two subtractive hybridization libraries enriched for differentially expressed genes. A total of 1,330 cDNA clones were then screened by reverse Northern dot blots which identified 27 and 10 cDNAs of genes that appeared to be up- and down-regulated, respectively. Up-regulated genes include those for complement C3, fibronectin, transferrin, two egg envelope proteins and vitellogenin. Down-regulated genes include those for lysozyme, serum amyloid A and trypsin. Thirteen cDNAs encode genes of unknown function. Changes in expression among these genes are currently being verified using real-time quantitative RT-PCR. The difference in the level of expression between exposed and control fish for genes measured thus far varied between 2.2 fold depression (lysozyme) and 8.9 fold elevation (choriogenin H). The efficiency of isolating genes that are truly differentially expressed using the subtractive hybridization/reverse Northern blot approach we used was lower than expected. The yield can perhaps be increased in the future by compensating for the inefficiency with an increase in the scale of the search process using microarray technology.