Volume 18, Issue 1, Pages 65-74 (January 2003) Extrachromosomal Recombination Substrates Recapitulate beyond 12/23 Restricted V(D)J Recombination in Nonlymphoid Cells  David Jung, Craig H. Bassing, Sebastian D. Fugmann, Hwei-Ling Cheng, David G. Schatz, Frederick W. Alt  Immunity  Volume 18, Issue 1, Pages 65-74 (January 2003) DOI: 10.1016/S1074-7613(02)00507-1

Figure 1 Experimental Strategy (A) Diagram of the TCRβ locus highlighting different RS spacer lengths found in the locus. 23-RSs are shaded in black and 12-RSs are white. (B) General format of the constructs used in this study. See Experimental Procedures for details. (C) The Vβ14 23-RS rearranges equally to two identical 5′Dβ1 12-RSs in the substrate. Immunity 2003 18, 65-74DOI: (10.1016/S1074-7613(02)00507-1)

Figure 2 Beyond 12/23 Restriction of V(D)J Recombination in Nonlymphoid Cells PCR reactions were carried out in 10-fold serial dilutions, on total recovered plasmid populations following cotransfection with RAG expression constructs. After separation of the products through a 1.2% agarose gel, Southern blotting was carried out and an oligonucleotide probe was used to detect the PCR products. (A) The position of the 5′Dβ1 12-RS was varied in the substrates. RSs in the substrates are listed from top to bottom, which corresponds to 5′→3′ in Figure 1B. (B) All six Jβ1 12-RSs were assessed for their ability to compete with the 5′Dβ1 12-RS for the Vβ14 23-RS. The first lane is a size marker for the expected rearrangement products. (C) The 3′Dβ1 23-RS was analyzed for rearrangement with either the 5′Dβ1 12-RS or the Jβ1.4 12-RS (substrate 1), and for rearrangement with either the 5′Dβ2 12-RS or the Jβ1.4 12-RS (substrate 2). The Vβ14 23-RS was analyzed for rearrangement with either the 5′Dβ1 12-RS or the Jβ1.4 12-RS (substrate 3) and for rearrangement with either the 5′Dβ2 12-RS or the Jβ1.4 12-RS (substrate 4). Immunity 2003 18, 65-74DOI: (10.1016/S1074-7613(02)00507-1)

Figure 3 Different Vβ 23-RSs Do Not Rearrange with Jβ 12-RSs, while the 3′Dβ1 23-RS Rearranges with All Jβ 12-RSs RSs in the substrates are listed from top to bottom, which corresponds to 5′→3′ in Figure 1B. (A) Vβ2, Vβ4, Vβ5.2, Vβ18, and Vβ14 were assayed for preference of rearrangement with the 5′Dβ1 12-RS or the Jβ1.4 12-RS. The bottom panel is a longer exposure of the top panel. (B) All six Jβ1 12-RSs were tested for their ability to compete with the 5′Dβ1 12-RS for the 3′Dβ1 23-RS. Immunity 2003 18, 65-74DOI: (10.1016/S1074-7613(02)00507-1)

Figure 4 Relative Contributions of RS Sequences and Coding Flanks to Beyond 12/23 Restriction (A) Different hybrid 12-RSs in competition with the 5′Dβ1 12-RS for the Vβ14 23-RS. Substrate 1 contains the wild-type Jβ1.4 12-RS with its native coding flank, while substrate 2 contains a 5′Dβ1 12-RS attached to the Jβ1.4 coding flank. Substrate 3 replaces the Jβ1.4 12 bp spacer with the 5′Dβ1 12 bp spacer, and substrate 4 replaces the Jβ1.4 heptamer/nonamer with the 5′Dβ1 heptamer/nonamer. (B) Rearrangement by hybrid Vβ14 23-RSs with the 5′Dβ1 12-RS and the Jβ1.4 12-RS. Substrate 1 contains the wild-type Vβ14 23-RS with its native coding flank, while substrate 2 contains a 3′Dβ1 23-RS attached to the Vβ14 coding flank. Substrate 3 replaces the Vβ14 heptamer/nonamer with the 3′Dβ1 heptamer/nonamer, and substrate 4 replaces the Vβ14 23-spacer with the 3′Dβ1 23-spacer. (C) Multiple different trials for rearrangement by the 3′Dβ1 23-RS attached to the Vβ14 coding flank (substrate 2 in Figure 4B). The left two control panels show rearrangement by a 3′Dβ1 23-RS attached to its native coding flank. Immunity 2003 18, 65-74DOI: (10.1016/S1074-7613(02)00507-1)

Figure 5 Beyond 12/23 Restriction in a Cell-Free System (A) Recombinant MBP-RAG1 and GST-RAG2 fusion proteins were expressed and purified. Body-labeled substrates were amplified from the respective plasmids by PCR, and the cleavage reactions were performed using 1.5 mM Mg2+. Cleavage products were analyzed on a 4% polyacrylamide gel. The identity of the bands is indicated to the right. Substrates 1–5 are the same as substrates 1–5 in Figure 5B. (B) MBP-RAG1 and GST-RAG2 were cotransfected with the respective substrates into CHO cells and analyzed as in Figures 2–4. RSs in the substrates are listed from top to bottom, which corresponds to 5′→3′ in Figure 1B. Immunity 2003 18, 65-74DOI: (10.1016/S1074-7613(02)00507-1)