Intermolecular V(D)J Recombination Is Prohibited Specifically at the Joining Step  Jung-Ok Han, Sharri B Steen, David B Roth  Molecular Cell  Volume 3, Issue 3, Pages 331-338 (March 1999) DOI: 10.1016/S1097-2765(00)80460-8

Figure 1 Efficient and Accurate Cleavage in trans (A) Extrachromosomal V(D)J recombination substrates and expected intermediates. Double-strand breaks are introduced precisely between the RSS (open triangle, 12-RSS; shaded triangle, 23-RSS) and the coding segments (squares). Cleavage of p12/23 produces signal ends on an excised molecule, whereas cleavage of p12 and p23 produces a 12- and a 23-signal end on each plasmid. (B) Detection of signal ends by ligation-mediated PCR (LMPCR). Blunt-signal ends were detected by ligating a double-stranded oligonucleotide (DR20) prior to PCR amplification (Roth et al. 1993). LMPCR products derived from p12/23 are of the expected sizes. DNA amounts added to ligation reactions are indicated above each lane. 1× corresponds to 2% of the DNA recovered from each transfection. 1:10, 1:100, etc., represent further dilutions. -R represents the absence of both RAG-1 and RAG-2 vectors for the transfection. Marker lanes contain radiolabeled 1 kb ladder (GIBCO-BRL). The blot for signal ends was probed with the DR55 oligonucleotide. The filled arrow and a pair of heavy lines represent primers for PCR and a double-stranded oligonucleotide for ligation, respectively. The open arrow denotes the DR55 primer. Similar results were obtained from the LMPCR for 23-spacer signal ends (data not shown). The extra band at 154 bp in lane 5 was also observed in the absence of RAG1 and RAG2 for several transfections. (C) Analysis of the structure of signal ends. A full-length, blunt signal end generates an ApaL1 restriction site due to ligation with a double-stranded oligonucleotide, DR20, described previously (Roth et al. 1993). Nonintact signal ends containing deletion or addition of nucleotides would not be cut by ApaL1. LMPCR products for 12-RSS were subjected to digestion with ApaL1 restriction enzyme at 37°C overnight. Uncut (-Apa) and cut (+Apa) samples were loaded in adjacent lanes. 1:10 dilution of 1/50th of the transfection was used for LMPCR. Blots were probed with a PvuII fragment from p12/23 described previously (Steen et al. 1996). Similar results were obtained for 23-RSS (data not shown). Molecular Cell 1999 3, 331-338DOI: (10.1016/S1097-2765(00)80460-8)

Figure 2 Intermolecular Signal Joints Form Efficiently (A) A diagram for signal joints derived from p12/23 or p12 and p23. In this and subsequent figures, open arrows denote the DR55 primer and filled arrows indicate the ML68 primer. (B) Signal joints were detected by PCR using DR55 and ML68 primers. The blot was hybridized with an end-labeled oligonucleotide probe (DR55). Similar results were obtained with a junction-specific oligonucleotide probe or the PvuII fragment from p12/23 (data not shown). (C) ApaL1 restriction digestion. PCR products were digested with ApaL1 as described above. Uncut (-Apa) and cut (+Apa) samples were loaded in adjacent lanes. 1:10 dilution of 1/50th from the transfection was used for LMPCR. The blot was hybridized with the PvuII fragment from p12/23. Molecular Cell 1999 3, 331-338DOI: (10.1016/S1097-2765(00)80460-8)

Figure 3 Intermolecular Coding Joints Are Rare (A) A diagram for coding joints derived from p12/23 or p12 and p23. In this and subsequent figures, single arrowheads denote the DR99 primer; double arrowheads indicate the DR100 primer. (B) Coding joints were detected by PCR using DR99 and DR100 primers (Han et al. 1997). The blot was hybridized with an end-labeled oligonucleotide probe (DR100). Lanes 7 and 8 are from independent transfections. Molecular Cell 1999 3, 331-338DOI: (10.1016/S1097-2765(00)80460-8)

Figure 4 Efficient Intermolecular End Joining p12 and p23 were digested with SalI or BamHI, respectively, and cotransfected into RMP41 cells. Vertical arrows indicate sites for SalI (S) and BamHI (B). Digestion removes the RSS, as there is a site on each side of the RSS. Junctions formed by end joining were analyzed by PCR (24 cycles) using the same primers employed to detect signal and coding joints. Products were detected by Southern blotting probed with DR100 (left panel) or DR55 (right panel). Molecular Cell 1999 3, 331-338DOI: (10.1016/S1097-2765(00)80460-8)

Figure 5 Detection of Signal Joints and Coding Ends on Linear Monomer and Dimer Plasmids (A) Assay for intermolecular signal joints. Signal joints in each gel fraction were amplified by PCR using DR55 and ML68 primers. The blot was hybridized with DR55. The triangle indicates decreasing molecular weight. Lower fraction numbers (shown above lanes) indicate higher molecular weights. SJ, signal joints. (B and C) Assay for coding ends. DNA was pretreated with mung bean nuclease to open hairpins. Coding ends were amplified by LMPCR using DR100 and DR20. The blot was probed with DR100. Similar results were obtained for 12- coding ends (data not shown). CE, coding ends. Molecular Cell 1999 3, 331-338DOI: (10.1016/S1097-2765(00)80460-8)

Figure 6 Intermolecular Hybrid Joints Form Efficiently (A) A diagram for hybrid joints derived from p12/23 or p12 and p23. (B and C) Hybrid joints were amplified using ML68 and DR99 for 23-hybrids (B) or DR55 and DR100 for 12-hybrids (C). ML68 and DR55 oligonucleotides were used as probes, respectively. Similar results were obtained with junction-specific probes (data not shown). Molecular Cell 1999 3, 331-338DOI: (10.1016/S1097-2765(00)80460-8)

Figure 7 Formation of Intermolecular Coding Joints Is Specifically Disfavored in Lymphoid Cells (A) Signal joints were amplified as described in Figure 2 (24 cycles) and detected by Southern blotting using an oligonucleotide probe. (B) Coding joints were amplified as described in Figure 3 (24 cycles) and detected by Southern blotting using an oligonucleotide probe. CJ, coding joints. Molecular Cell 1999 3, 331-338DOI: (10.1016/S1097-2765(00)80460-8)