Expression of ADA mRNA and ADA proteins.
(A) Ethidium bromide–stained agarose gel of fragment c.1099-1290 amplified from uncloned ADA cDNA. A 191-bp product is expected from both MutAΔ11 and wild-type ADA cDNA; MutA-derived cDNA gives a 204-bp product. Control lanes: 1, wild-type ADA cDNA; 8, equal mixture of wild-type and MutA ADA cDNAs. Sib B (lanes 2-4): 2, B LCL; 3, posttreatment T LCL; 4, pretreatment T cells. Sib A (lanes 5-7): 5, B LCL; 6, T LCL; 7, T cells. (B) Western blot of ADA in extracts of lymphoid cells. Lanes 1-5, T cells and T LCLs: 1, Sib B pretreatment T cells; 2, Sib A pretreatment T cells; 3, Sib B posttreatment T LCL; 4, Sib A posttreatment T LCL; 5 control T LCL. Lanes 6-8, B LCLs: 6, Sib B; 7, Sib A; 8, control. (C-E) Expression of recombinant MutA-derived ADA protein. (C) SDS-PAGE of 35S in vitro translation products resulting from the wild-type (WT) and MutA cDNA. (D) In situ assay for ADA activity of the translation products shown in panel C after nondenaturing electrophoresis. Arrow indicates position of human ADA. Dark bands at the top and bottom are, respectively, rabbit ADA and rabbit hemoglobin. (E) Western blot of ADA proteins expressed in E coli SØ3834. Lanes: 1, wild type; 2, MutA; 3, G360X; 4, 360-363EPTS; 5, 360-363EPTS+20 (see “Materials and methods” for description).
