Mechanisms that direct ordered assembly of T cell receptor β locus V, D, and J gene segments

T cell receptor (TCR) beta variable region genes are assembled in progenitor T cells from germ-line V beta, D beta, and J beta segments via an ordered two-step process in which D beta to J beta rearrangements occur on both alleles before appendage of a V beta to a preexisting DJ beta complex. Direct joining of V beta segments to nonrearranged D beta or J beta segments, while compatible with known restrictions on the V(D)J recombination mechanism, are infrequent within the endogenous TCR beta locus. We have analyzed mechanisms that mediate ordered V beta, D beta, and J beta assembly via an approach in which TCR beta minilocus recombination substrates were introduced into embryonic stem cells and then analyzed for rearrangement in normal thymocytes by recombinase-activating gene 2-deficient blastocyst complementation. These analyses demonstrated that V beta segments are preferentially targeted for rearrangement to D beta as opposed to J beta segments. In addition, we further demonstrated that V beta segments can be appended to nonrearranged endogenous D beta segments in which we have eliminated the ability of D beta segments to join to J beta segments. Our findings are discussed in the context of the mechanisms that regulate the ordered assembly and utilization of V, D, and J segments.