Rearrangements in RAG1-S723C thymocytes and RAG1-S723C/p53 double-mutant thymic lymphomas. (A) TCRγV3S1 (chr 13) to TCRβJ2S7 (chr 6) interchromosomal trans-rearrangements were PCR amplified from genomic thymocyte DNA isolated from mice of the indicated genotypes (top panel). S723C indicates RAG1^S723C/S723C; Kid, kidney. Products corresponding to trans-rearrangements were detected by Southern blot analysis. Levels of rearrangements were normalized to PCR amplification of a nonrearranging locus (middle panel). Representative results are shown. Vertical lines have been inserted to indicate repositioned gel lanes. PCR products corresponding to TCRγV3-TCRβJ2 rearrangements in RAG1-S723C thymocytes were subcloned and sequenced (bottom panel). Coding sequences for TCRγV3 and TCRβJ2 are shown in boxes (bold); nucleotides added (center column) include P nucleotides (underlined) and N nucleotides; number of clones of each sequence are indicated. (B) Genomic DNA isolated from RAG1-S723C/p53 double-mutant lymphomas was digested with EcoRI then analyzed by Southern blot. Dβ1 to Jβ1 rearrangements were detected using a probe located 5′ of the Dβ1 segment (diagram). Individual tumors are indicated, top. C indicates wild-type thymus DNA; Kid, nonrearranging tissues; GL, unrearranged, germline band; and R, D-Jβ1 rearrangements. Amounts of input DNA were normalized to a nonrearranging locus (LR8). (C) PCR amplification of Dβ1 to Jβ1, Dβ2 to Jβ2, and Vβ8 to DJβ1 rearrangements were performed as described in Figure 2A using genomic DNA (250 ng) isolated from RAG1-S723C/p53 double-mutant lymphomas (tumor numbers indicated, top), wild-type thymocytes (C), and a nonrearranging tissue (tail). Input DNA levels were normalized to a nonrearranging locus (ATM). Vertical lines have been inserted to indicate repositioned gel lanes.