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

T cell receptor (TCR) beta variable region genes are assembled in progenito r 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 al leles 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, whi le compatible with known restrictions on the V(D)J recombination mechanism, are infrequent within the endogenous TCR beta locus. We have analyzed mech anisms that mediate ordered V beta, D beta, and J beta assembly via an appr oach in which TCR beta minilocus recombination substrates were introduced i nto embryonic stem cells and then analyzed for rearrangement in normal thym ocytes by recombinase-activating gene 2-deficient blastocyst complementatio n. These analyses demonstrated that V beta segments are preferentially targ eted for rearrangement to D beta as opposed to J beta segments. In addition , we further demonstrated that V beta segments can be appended to nonrearra nged 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 t he context of the mechanisms that regulate the ordered assembly and utiliza tion of V, D, and J segments.