Physiological variable (V), diversity (D) and joining (J) recombination and analogous recombination substrates. (a) V and J segments on opposite strands (as found in the human Igκ locus) are joined by inversion between the recombination signal sequence (RSS)12 (filled triangle) and RSS23 (open triangle) motifs to generate a linked signal joint and coding joint (the VJ rearrangement). (b) V and J segments located on the same strand (as found in the human Igκ and λ loci) are recombined by deletion of the intervening DNA, leaving the coding segment on the chromosome and the signal joint on an excised circle of DNA. (c) In the inversion substrate, the DsRed gene and the EGFP gene are located on opposite stands, flanked by RSS12 and RSS23 motifs. V(D)J recombinase activity flips the segment allowing DsRed to be replaced by EGFP. (d) In the deletion substrate, the RSS motifs are in opposite orientations and flank the DsRed gene. On recombination, the DsRed gene is deleted, placing EGFP adjacent to the promoter. A single promoter is present in both constructs (curved arrow). The positions of the primer sequences F1 and R1, which were used to analyse recombination at the DNA level and for RT-PCR analysis, are shown.