RESUMO
Checkpoint kinase 2 (CHK2) plays a central and conserved role in the eukaryotic DNA damage response. Few cell cycle checkpoint proteins have been examined in aquatic organisms, and this study is the first to characterize CHK2 expression in a fish species. CHK2 was cloned from Oncorhynchus mykiss, the rainbow trout. The coding region extends over 5741 nucleotides in the genome, including 13 introns, and specifies a predicted 533 amino acid protein. Southern blot analysis revealed that CHK2 exists as a single copy in the rainbow trout genome. Recombinant protein representing the FHA domain was used to generate polyclonal anti-CHK2 antibodies. While CHK2 transcript levels were relatively low in gill and high in brain, the opposite was true for protein levels. Both gill and brain cell cultures were treated with bleomycin, which induces double-strand DNA breaks. There was no effect on levels of CHK2 in gill cells, suggesting that the protein is constitutively active in this tissue. In contrast, brain cells upregulated CHK2 in a dose-dependent manner. The tissue specific expression of CHK2 and its ability to respond to bleomycin treatment suggests that some checkpoint proteins may serve as suitable biomarkers for DNA damage in rainbow trout and other fish species.
