Cloning and Sequencing of a Partial CYPlA Gene in Creek Chub (Semotilus a/romaculatus): The first step in the development of a biomarker of exposure to AhR Ligand

Abstract

Biomarkers are powerful tools that allow scientists to assess exposure of biota to environmental chemicals. In studying the hazardous waste site surrounding the former 3M/Dynacolor plant in Brockport, NY, a biomarker capable of assessing combined exposure to polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) was needed. These compounds, along with polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), mediate toxicity through the arylhydrocarbon receptor, resulting in carcinogenicity and endocrine disruption with obvious human health and environmental risks. The goal of my study was to develop a biomarker of exposure that would allow for the evaluation of the combined exposure to PCBs, PAHs, PCDFs and PCDDs of the creek chub (Semoti!us atromacularus), a common fish in Brockport Creek adjacent to the hazardous waste site. The first step in the preparation of such a biomarker is the sequencing of a portion of the CYP1A gene, a gene induced by the aryl-hydrocarbon receptor. The objectives of my study were to ( l) isolate DNA from creek chub tissue, (2) amplify a segment of the CYP1A gene from genomic DNA using degenerate primers, (3) clone the fragment using the TA cloning technique, and ( 4) sequence the fragment and to compare it phylogenetically with known CYP1A sequences. The sequence of the isolated fragment was homologous with CYP1A genes of teleost fishes, showing high percent identity with portions of the CYP1A gene oft wo members of the same taxonomic family (Cyprinidae), the common carp ( Cyprinus carpio) and the zebrafish (Danio rerio). The isolation of this sequence offers the possibility of developing a biomarker of exposure that measures the amount of CYP1A mRNA induced in various tissues of S. atromaculatus.