Eβ inactivation preferentially impairs Dβ1 rearrangement. (A) Schematic diagrams of germ-line Dβ and Jβ gene segments and their rearrangement intermediates and products. Dβ and Jβ gene segments are shown as open and filled boxes, respectively. RSSs are shown as either open or filled triangles. Arrows indicate directions of PCR primers used for assaying germline Dβ gene segment, CJ, SEs, and SJ. Double lines indicate oligonucleotide linker for LM-PCR assay. (B) Flow cytometry reanalysis of purified DN and DP thymocytes from wild-type and Eβ^R/R mice. CD4 and CD8 staining profiles are shown for live cells. Numbers indicate the percentages of cells in the gated areas. (C) PCR assays for Dβ1-Jβ1, Dβ1-Jβ2, and Dβ2-Jβ2 CJ in DN and DP thymocytes. Various joints in purified DN and DP thymocytes of wild-type (+/+) and Eβ^R/R mice were measured by a semiquantitative PCR. DNA was either undiluted (undil) or serially diluted every 5-fold into RAG2^-/- kidney DNA and then amplified. JAK3 was amplified to verify DNA quality and relative amount. PCR products were separated on agarose gel and hybridized with specific Dβ probes. Rearrangements to different Jβ are labeled. G.L., germ-line. Representative data from one of the four experiments are shown. (D) PCR assays for Dβ-Jβ SJ in DN and DP thymocytes. The levels of Dβ1-Jβ1, Dβ1-Jβ2.7, and Dβ2-Jβ2.7 SJ in DN and DP thymocyte DNA were assayed by PCR followed by hybridization with Dβ-specific oligonucelotide probes. Half of the PCR products were digested with ApaL1 before electrophoresis. Representative data from one of the four experiments are shown.

Eβ inactivation preferentially impairs Dβ and Jβ cleavages in DN thymocytes. (A) LM-PCR assays for Dβ and Jβ SEs. DNA from DN and DP thymocytes of wild-type and Eβ^R/R mice was diluted and used to assay for SEs derived from cleavages at 3′ Dβ1, 3′ Dβ2, 5′ Jβ1(Jβ1.1, Jβ1.2, Jβ1.3, and Jβ1.4), 5′ Jβ2(Jβ2.1, Jβ2.2, Jβ2.3, Jβ2.4, and Jβ2.5), 5′ Dβ1, and 5′ Dβ2 by LM-PCR. PCR products were separated by agarose gels and hybridized with specific Dβ or Jβ probes. For Jβ1 and Jβ2 SEs, only the most abundant Jβ1.1 and Jβ1.2 or Jβ2.1 and Jβ2.2 are shown (marked). The numbers indicate the band intensities normalized to JAK3 (C), with the level of SEs in wild-type DN thymocytes as 1. Representative data from one of the four experiments are shown. (B) PCR assays for Dβ1-Dβ2 CJ. The levels of Dβ1-Dβ2 CJ in DN and DP thymocytes of wild-type and Eβ^R/R mice were assayed by PCR followed by hybridization with a Dβ1 oligonucleotide probe. Representative data from one of the four experiments are shown. (C) Controls for PCR assays. Efficiencies of the LM-PCR were controlled by assaying for Jα50 SEs. The relative amounts of DNA in different samples were estimated by PCR assays for JAK3. The linear range values (25-fold dilution) were used for normalization. Representative data from one of the four experiments are shown.

Most of the rare Dβ1-Jβ2 joints in Eβ-mutant mice are actually Dβ1-Dβ2-Jβ2 rearrangements. PCR assays for Dβ-Jβ rearrangements in thymocytes of wild-type (+/+), Eβ^-/-, Eβ^R/R, and Eβ^R/Rp53^-/- mice were carried out as in Fig. 1C. Rearrangement products were cloned and sequenced. Sequences of Dβ1-Jβ2.7 joints are shown, and the rest is shown in Fig. 6. Germ-line sequences are shown for comparison. N-nucleotides are indicated, and possible P-nucleotides are underlined.

Most of Dβ1 and Dβ2 gene segments are in germ-line configuration in Eβ^R/R thymocytes. (A) Southern blotting assays for germ-line Dβ1 levels. Ten micrograms of DNA from DN and DP thymocytes of wild-type and Eβ^R/R mice were digested with HindIII followed by hybridization with a Dβ1-Jβ1 intronic probe and then a β-actin probe. The numbers indicate the relative levels of germ-line Dβ1 after normalization to the β-actin levels. (B) The levels of germ-line Dβ1 and Dβ2 in DN and DP thymocyte DNA were assayed by PCR and compared with those in kidney DNA of RAG2^-/- mice. DNA samples were either undiluted (undil) or diluted every 5-fold before PCR assay. JAK3 amplification was as in Fig. 1. The PCR assays were performed twice with the same results. One set of data is shown.