The Lymphoid V(D)J Recombination Activity: Studies of Exogenous Plasmid Substrates in Whole Cells

Abstract

The V(D)J recombination activity recombines V, D and J segments at the Ig and TCR loci to generate the variable domain exons of the genes encoding the antigen receptors for humoral and cell-mediated immunity. We have been interested in understanding the mechanism of this DNA recombination reaction and how the activity which mediates it is targeted to the loci and regulated during B and T-cell differentiation. For our investigations, we developed an assay that allows the recombination activity in cells to be measured and the products of the reaction to be readily recovered. The’assay uses DNA substrates that remain extrachromosomal after transfection into eukaryotic cells and are recombined by the V(D)J recombination activity (Hesse et al. 1987). These substrates contain V (D) J “joining signals”, the characteristic DNA sequence motifs that are found adjacent to each V, D and J segment at the Ig and TCR loci and that serve as recombination signals. Each signal sequence consists of a heptamer and an A/T-rich nonamer separated by a nonconserved spacer sequence. A recombination event is directed by two signals, one with a 12-base spacer (a 12-signal) and the other with a 23-base spacer (a 23-signal). Standard V(D)J recombination generates two recombinant junctions; coding elements are fused in a “coding joint”, generating a region of continuous coding sequence, and joining signals are fused at their heptamers in a “signal joint”. Typically a few nucleotides are lost from one or both coding ends in a coding joint. Non-template directed nucleotide addition is also usual. This base loss and addition adds to the diversity generated by coding segment joining.