In vitro genetic and functional evaluation of the Rag 1 defect from Subject P. (A) Sequence analysis of the patient (middle) and his father (top) and mother (bottom), showing the novel mutation 1621delC and a previously reported 1566G > T, missense mutation (A). Both of these mutations are located within the catalytic core of RAG1 (B). (B) The effect of patient mutations on Rag1 protein. Both mutations are predicted to disrupt the catalytic domain of Rag1 with a missense W522C and the novel deletion L541CfsX30 with the resulting truncated open reading frame as indicated. (C) Mouse pro-B Abelson line deficient for Rag1 was infected with inverted substrate. (D) Subclones 1, 2, and 3 with single copy stable integrants were transduced with wild-type, G1566T, or 1621delC, then treated with STI-571 (an abl kinase inhibitor) to promote cell differentiation and induction of Rag activity. The level of GFP expression (x-axis) indicated the recombinase activity level, and only minimal GFP level was detected in the absence of STI-571.