Analysis of intermediate excision circles or signal joints.

Analysis of intermediate excision circles or signal joints illustrates that E2A and HEB induce single-step TCRD recombinations in nonlymphoid cells. (A) In the example of a Vδ2-Dδ3 rearrangement, the coding and signal joints formed during direct coupling of the Vδ2 and Dδ3 segments are shown. (B,C) Analysis of signal joints in BOSC 23 cells transfected with E47, HEB, RAG1, and RAG2 expression vectors. Genomic DNA (200 ng) of the various transfectants was used for PCR amplification by means of Dδ2-lower plus Dδ3-upper (panel B) or Vδ2-sj 3′ plus Dδ3-5′ S (panel C) primers that specifically detect Dδ2-Dδ3 and Vδ2-Dδ3 signal joints, respectively. Thymus DNA was used as positive control, and GFP- or mock-transfected BOSC 23 DNA as nonspecific template control. PCR products were run on a 2% agarose gel and stained with ethidium bromide. Similar to the coding joints shown in Figure 1, Dδ2-Dδ3 and Vδ2-Dδ3 signal joints were observed upon combined transfection of either E47 or HEB together with RR. In line with the data on Dδ2-Dδ3 PCR products, Dδ2-Dδ3 signal joints were also detectable in genomic DNA derived from transfectants expressing RR only (lane RR). Importantly, transfection with 2 μg RAG expression vectors (lane RR2) did not result in detectable signal joints; this amount of RAG1/RAG2 vector leads to RAG activity levels that are more in line with the RAG activity levels in those cases in which 6 μg RAG1/RAG2 is cotransfected with E2A or HEB (W. J. Romanow, personal communication, 2001).