V(D)J recombination generates a high frequency of nonstandard TCR D delta-associated rearrangements in thymocytes

The standard products of V(D)J recombination are coding junctions, which encode Ag receptor polypeptides, and their commonly excised reciprocal products, signal junctions. Additional nonstandard products also have been detected, mostly in artificial recombination substrate studies. The occurrence of nonstandard products, including pseudonormal, hybrid, and open/shut junctions, indicates significant indeterminacy of the V(D)J recombinase. However, the incidence of nonstandard products for endogenous Ag receptor genes in vivo has not been specifically addressed. The data presented here show that for the TCR-delta locus, D element-associated recombination in mouse thymocytes results in a high incidence of nonstandard recombination products. D delta 1-D delta 2 rearrangements, both chromosome retained and excised episomal products, were studied by polymerase chain reaction amplification, cloning, and sequence analysis. The proximity of D delta 1 and D delta 2 elements, and the fact that both are flanked by 5' and 3' recombination signal sequences with 12-bp and 23-bp spacers, respectively, results in frequent pseudonormal joining. The resulting products are signal junctions retained on the chromosome. Excised episomal products include coding junctions, hybrid junctions formed in apparent violation of the 12/23 spacer rule, and standard signal junctions; some signal junctions show evidence of imprecise cleavage. Evidence for open/shut and/or oligonucleotide capture events was also seen. Similar rearrangements were detectable in thymocytes of mutant scid mice. These findings indicate a high degree of indeterminacy of V(D)J recombinase-mediated D delta 1-D delta 2 rearrangement in both wild-type and scid thymocytes. This indeterminacy affects the productive potential of TCR-delta loci.