IDENTIFICATION OF V(D)J RECOMBINATION CODING END INTERMEDIATES IN NORMAL THYMOCYTES

Diversity of vertebrate antigen receptors is accomplished in large par t by a somatic gene rearrangement process known as V(D)J recombination . The first step of the reaction appears to be the creation of a doubl e strand break immediately between the recombination signal sequence ( RSS) and the coding gene segment to generate a signal end and a coding end. Signal ends have been shown, both in vitro and in vivo, to be pr ecise and blunt, while coding ends generated in vitro are covalently s ealed hairpins. It has been difficult to document the existence of cod ing ends in vivo in normal lymphoid precursors, presumably because of their low abundance. To date, they have been identified in vivo only i n a transformed pre-B cell line and in cells from the mutant scid mous e, where they largely conform to the hairpin structure found in vitro. Here, we identify T cell receptor J alpha gene coding ends in normal murine thymocytes. We demonstrate that these ends are processed, not b lunt, and that most are not hairpin terminated, in sharp contrast to p revious in vivo and in vitro observations. These results provide the f irst direct demonstration of this important intermediate of V(D)J reco mbination in normal lymphoid precursors and have implications for the mechanism of coding joint formation in vivo. (C) 1997 Academic Press L imited.