Characterization of terminal deoxynucleotidyl transferase and polymerase μ in zebrafish

Terminal deoxynucleotidyl transferase (TdT) contributes to the junctional diversity of immunoglobulin and T-cell receptors by incorporating nucleotides in a template-independent manner. A closely related enzyme, polymerase mu (pol mu), a template-directed polymerase, plays a role in general end-joining double-strand break repair. We cloned zebrafish TdT and pol mu and found them to be 43% identical in amino acid sequence. Comparisons with sequences of other species revealed conserved residues typical for TdT in the zebrafish sequence that support the template independence of this enzyme. Some but not all of these features were identified in zebrafish pol mu. In adult fish, TdT expression was most prominent in thymus, pro- and mesonephros, the primary lymphoid organs in teleost fish and in spleen, intestine, and the tissue around the intestine. Pol mu expression was detected not only in pro- and mesonephros, the major sites for B-lymphocyte development, but also in ovary and testis and in all tissue preparations to a low extent. TdT expression starts at 4 dpf and increases thereafter. Pol mu is expressed at all times to a similar extent. In situ studies showed a strong expression of TdT and pol mu in the thymic cortex of 8-week-old fish. The characterization of zebrafish TdT and pol mu provide new insights in fish lymphopoiesis and addresses the importance and evolution of TdT and pol mu themselves.