Analysis of secondary Tcra locus recombination in Atm^−/− thymocytes. (A) Quantitation of Ja rearrangements by Southern blot. The Ja probes were 5′ (Jα19330.11), middle (Jα42417.4), and 3′ (Jα4.1). Ja hybridization to purified splenic B-cell DNA was used as a denominator to calculate the loss of Ja signals from thymocytes, shown below each lane. The schematic (not to scale; using the experimental strategy of Petrie et al¹⁴ ) represents the Tcra locus; arrows indicate relevant restriction sites, and the size of the genomic fragments to which each Ja probe hybridizes. A second experiment was performed with a similar outcome. (B) Tcra repertoire analysis in Atm^−/− mice reveals unbiased Ja region usage. Thymic cDNA from two 3-week-old Atm^−/− mice and 2 Atm^+/+ littermates was amplified with a Va3-specific primer and a Ca antisense primer. The PCR products were cloned and sequenced. The stacked histograms represent the percentages of Tcra mRNAs using Ja segments found in the 20-kb (kilobase) intervals that span the proximal (5′), middle, and distal (3′) regions of the Ja locus in Atm^−/− thymocytes and controls. The results represent 15 independent Atm^−/− and 15 Atm^+/+ thymus cDNA clones.