Journal: The Journal of Experimental Medicine
Article Title: Defects in coding joint formation in vivo in developing ATM-deficient B and T lymphocytes
Figure Lengend Snippet: Increased TCRα sJ locus CE accumulation in Atm −/− thymocytes. (A) Southern analysis strategy showing the relative positions of the NsiI (N) and StuI (S) sites, PS6, and the CαI probe. Fragment sizes generated by germline TCRα sJ alleles and Jα56 and Jα61 CEs are indicated. The schematics are not drawn to scale. (B) Southern blot analyses of total thymocyte DNA and kidney DNA (K) digested with either NsiI or HincII and hybridized to the CαI probe or PS6. Bands corresponding to Jα61 and Jα56 CEs are indicated (arrows). The band corresponding to the germline fragment is indicated by an asterisk. Hybridization of StuI and NsiI digested DNA to PR2 is shown as a DNA loading control. The molecular mass markers (in kb) are indicated. (C) LMPCR analysis of Jα56 and Jα61 CEs, as described in Materials and methods. Shown are fivefold serial dilutions of linker-ligated samples. IL-2 gene PCR is also shown as a DNA loading control. (D and E) Exonuclease V sensitivity of Jα56 CEs was assayed by hybridizing the membrane from Fig. 3D to the CαI probe. Bands corresponding to the Jα61 and Jα56 CEs are indicated (arrows). The band corresponding to the germline TCRα sJ allele is indicated by an asterisk. The positions of the molecular mass markers (in kb) are indicated (D). Retention of the Jα56 CE band in exonuclease V–treated samples was quantified as described in Materials and methods (E). (F and G) TCRα sJ/sJ :Atm − / − thymocyte DNA was treated with mung bean nuclease followed by exonuclease V before digestion with StuI and hybridization to the CαI probe, as described in Materials and methods. Fragments corresponding to Jα61 and Jα56 CEs are indicated (arrows). The fragment corresponding to the germline TCRα sJ allele is indicated by an asterisk (F). Retention of the Jα56 CE band in samples treated with mung bean nuclease and exonuclease V was quantified as described in Materials and methods (G).
Article Snippet: For exonuclease V digestion, 15 μg of thymocyte DNA was treated with increasing concentrations of exonuclease V, using the manufacturer's buffer conditions (USB Corporation), for 1 h at 37°C before restriction enzyme digestion and Southern blot analyses.
Techniques: Generated, Southern Blot, Hybridization, Polymerase Chain Reaction