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. 1999 Sep 14;96(19):10788–10793. doi: 10.1073/pnas.96.19.10788

Figure 1.

Figure 1

Recombination efficiency is affected by the chromatin structure of the substrate. (A) Recombination substrate pSC1 contained a 12 RSS (black triangle) embedded in the MMTV LTR. The 23 RSS (white triangle) is downstream of the luciferase reporter gene. The Epstein–Barr origin of replication (Orip) is included to allow plasmid maintenance. After V(D)J recombination, signal-joint products could be amplified by PCR (arrows) and detected in a Southern blot probed with an oligonucleotide that spanned the newly formed signal joint (rectangle). The other product of the recombination reaction (the coding joint) is shown but not assayed. (B) Drug treatment does not increase the efficiency of recombination of transiently introduced substrates. pSC1 (lanes 1–4) or methylated pSC1, pSC1 ME (lanes 6–9) were cotransfected with Rag-1 and Rag-2 expression vectors (RAG1/2) into 293 cells, and the drugs butyrate (But) or dexamethasone (Dex) were added as indicated. Assays were performed as described in A. Fourfold less pSC1 DNA than in lanes 1–4 was transfected to generate lane 5. Relative activities were determined by using the titration series in which transient pSC1 plus RAG1/2 was defined as 1. (C) Stably maintained episomes are inhibited by ≈100-fold. Tenfold dilutions of transiently transfected pSC1 and RAG1/2 extract into empty extract generates a titration curve (lanes 1–4). Stably maintained pSC1 cells transfected with RAG1/2 (lane 6) or without RAG1/2 (lane 5) were amplified in parallel. (D) Stably maintained substrates are refractory to recombination, and methylation further decreases the efficiency of recombination. Cell lines stably maintaining pSC1 (lanes 1–4) or methylated pSC1, pSC1 ME (lanes 7–10) were transfected with RAG1/2 vectors and were treated with drugs as indicated. Note: the absolute amount of recombination in D, lane 2 is ≈100-fold less than in B, lane 2. Relative activities were calculated as in B except stable pSC1 plus RAG1/2 was defined as 1. (E) The levels of Rag proteins are unaffected by drug treatments. Expression vectors for Rag-1 and Rag-2 were cotransfected into 293 cells and were treated with dexamethasone or butyrate as indicated. Bands labeled control hybridized nonspecifically with the immunodetection reagents used.