by Kunkel et al. [T. A. Kunkel, J. D. Roberts, R. A. Zakour, Methods Enzymol. 154, 367 (1987)]. The template DNA for mutagenesis was prepared from M13 mp19 containing the cDNA encoding Drosophila TFIIB, harboring an Nde site at the translation initiation site. For NH2-terminal deletion mutants, deoxyoligonucleotides that introduce an Nde site (CATATG) at the deletion points were used; mutants were confirmed by digestion of replicative form (RF) DNA with Nde I. The COOH- terminal deletion mutants were made with deoxy- oligonucleotides that create the termination codons and Hind Ill restriction sites (TAAGCTT) at the indicated positions; mutants were detected by digestion of RF DNA with Hind IlIl. For amino acid substitutions, deoxyoligonucleotides that in- troduce the desired amino acid changes were used, and mutants were confirmed by DNA se- quencing. For bacterial expression, RF DNA was digested by Nde and Eco RI and subcloned into Nde I- and Eco RI-digested 6His-pET-5a [(16); F. W. Studier, A. H. Rosenberg, J. J. Dunn, J. W. Dubendorff, ibid. 185, 60 (1990)]. 9. Escherichia coli BL21 harboring the TFIIB expres- sion plasmid were grown in TBG medium to an OD at 600 nm of 0.8; protein synthesis was then induced with 0.5 mM isopropyl P-D-thiogalacto- side at 370C for 3 hours. Cells were collected by centrifugation (6000g, 10 min) and resuspended in 1/10 culture volume of buffer B [20 mM tris-HCI (pH 7.9), 0.2 mM EDTA, 10% glycerol, 2 mM dithiothreitol (DTT), 0. 1 mM phenylmethylsulfonyl fluoride] containing 1 mM imidazole, 0.1% Noni- det P-40, and 500 mM KCI. Cell suspensions were incubated on ice for 15 min and then disrupted by sonication. After centrifugation (8000g, 20 min), supernatant was applied onto a Ni-agarose col- umn (Qiagen, Studio City, CA), equilibrated with buffer B containing 10 mM imidazole and 100 mM KCI. After washing with the same buffer, protein was eluted with buffer B containing 100 mM imidazole and 100 mM KCI. The COOH-terminal deletion and the second basic repeat mutants, which are mostly insoluble under these condi- tions, were prepared as follows: The cell pellet was suspended in 1/10 culture volume of buffer B containing 6 M guanidine HCI and 500 mM KCI, and disrupted by a short pulse of sonication. After centrifugation (8000g, 20 min), supernatant was applied onto a Ni-agarose column. After washing with buffer B containing 6 M guanidinium-HCI, 500 mM KCI, and 10 mM imidazole, protein was eluted with the same buffer containing 100 mM imidaz- ole. The eluate was then dialyzed against buffer B containing 2 M guanidine-HCI and 500 mM KCI for 2 hours at 4°C and then dialyzed against buffer B containing 1 M guanidine HCI and 100 mM KCI overnight at 4°C. 10. In vitro transcription was carried out as described (6), except that the native human TFIID fraction was used. The template plasmids used in each experiment are described in the appropriate fig- ure legends. 11. 32P-end-labeled double-stranded DNA with the adenovirus major late promoter sequence (-40 to +10 of the transcription initiation site) was used as a probe. Each complete system contained 5 x 105 dpm of probe (about 50 fmol), poly(dGdC) (10 sLg/ml) (Pharmacia), 35 mM Hepes-KOH buff- er (pH 8.0), 7.5 mM MgCI2, 6% (vN) glycerol, 60 mM KCI, 6 mM DTT, 60 pM EDTA, Drosophila TFIIDr (0.5 pg/mI), and TFIIB (1.2 Wg/mI) in a total volume of 25 pi. After incubation at 30°C for 40 min, products were analyzed on a 4% polyacryl- amide gel (59:1) containing TBE buffer [89 mM tris, 89 mM boric acid, and 2 mM EDTA (pH 8.0)] and 3% (v/v) glycerol at 100 V with the use of TBE buffer as a running buffer. 12. T. Yamamoto et al., Proc. Nat!. Acad. Sci. U.S.A. 89, 2844 (1992). 13. S. Buratowski, S. Hahn, L. Guarante, P. A. Sharp, Cell 56, 549 (1989). 14. E. Maldonado et al., Mo!. Cell. Biol. 10, 6335 (1 990). 15. J. Colgan, S. Wampler, J. L. Manley, Nature 362, 549 (1993). 466 16. D. B. Nikolov etal., ibid. 360, 40 (1992). 17. A. Hoffmann etal., ibid. 346, 387 (1990). 18. We thank M. Brenner and H. A. Nash for critical reading of the manuscript and D. Schoenberg for editing the manuscript. Supported by Nippon Su- isan Kaisha Ltd. (S.Y.) and Toyobo Biotechnology Foundation (K.H.). M.H. was an Alexandrine and Alexander Sinsheimer Scholar. A portion of this study was supported by National Institutes of Health grants CA42567 and A127397 (to R.G.R.) and GM45258 (to M.H.), by Grant for Special Project Research from the Ministry of Education, Science, and Culture of Japan (to M.H.), by funds from Sankyo Co. Ltd. and Sagawa Foundation for Cancer Research (to M.H.), and by general support from the Pew Trusts to The Rockefeller University. 10 September 1992; accepted 1 June 1993 Direct Association of Adenosine Deaminase with a T Cell Activation Antigen, CD26 Junichi Kameoka, Toshiaki Tanaka, Yoshihisa Nojima, Stuart F. Schlossman, Chikao Morimoto* CD26, the T cell activation molecule dipeptidyl peptidase IV (DPPIV), associates with a 43-kilodalton protein. Amino acid sequence analysis and immunoprecipitation studies demonstrated that this 43-kilodalton protein was adenosine deaminase (ADA). ADA was coexpressed with CD26 on the Jurkat T cell lines, and an in vitro binding assay showed that the binding was through the extracellular domain of CD26. ADA deficiency causes severe combined immunodeficiency disease (SCID) in humans. Thus, ADA and CD26 (DPPIV) interact on the T cell surface, and this interaction may provide a clue to the pathophysiology of SCID caused by ADA deficiency. CD26, a T cell activation molecule (1, 2), is a 1 10-kD glycoprotein that is also present on epithelial cells of various tissues, includ- ing the liver, kidney, and intestine. CD26 is identical with dipeptidyl peptidase IV (DPPIV) (3), which can cleave NH2-termi- nal dipeptides from polypeptides with either L-proline or L-alanine at the penultimate position. No physiological substrates have yet been identified. We isolated cDNA encoding human CD26 and established CD26-transfected Jurkat T cell lines (4). Functional analysis of these Jurkat transfec- tants showed that cross-linking of the CD26 and CD3 antigens with their respec- tive antibodies (Abs) resulted in enhanced intracellular calcium mobilization and in- terleukin-2 production, providing direct ev- idence that the CD26 antigen plays an integral role in T cell activation. The cDNA sequence of CD26 predicted a type II membrane protein with only six amino acids in the cytoplasmic region (4, 5), suggesting that other association molecules are involved in CD26-mediated signal transduction. CD26 associates with CD45 (6), which might be involved in regulat- ing the p56`ck activity through its protein phosphatase activity. Another candidate for the signal transduction molecule was a 43-kD protein, p43, which can be copre- cipitated by antibody to CD26 (anti- CD26) from 1251-labeled T cells, phyto- hemagglutinin (PHA) blast cells, and Division of Tumor Immunology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medi- cal School, Boston, MA 02115. *To whom correspondence should be addressed. SCIENCE * VOL. 261 * 23 JULY 1993 from CD26-transfected Jurkat cell lines (4). To identify p43, we purified the protein by immunoaffinity chromatography from CD26-transfected Jurkat cells. The purified protein was separated by SDS-polyacryl- amide gel electrophoresis (PAGE), trans- ferred to a nitrocellulose membrane, and stained by Ponceau S, resulting in a single band of 43 kD in addition to the CD26 1 10-kD protein (Fig. 1). The 43-kD protein was then digested with trypsin, separated by reversed-phase high-pressure liquid chro- matography (rpHPLC), and subjected to amino acid sequencing. According to the homology search, the amino acid sequences of the two peptides derived from p43 were completely identical to those of residues 35 to 64 and 172 to 206 of the human adeno- sine deaminase (ADA) (7). ADA is a 41-kD protein, expressed in all tissues (highest expression in lymphocytes), that catalyzes the conversion of adenosine and deoxyadenosine to inosine and deoxy- inosine, respectively. ADA is present on the cell surface, as well as in the cytoplasm, of human fibroblasts, rabbit renal tubular cells, and human mononuclear blood cells (8). ADA deficiency causes severe com- bined immunodeficiency disease (SCID) in humans (9), yet no direct interaction be- tween ADA and T cell surface molecules has been identified. The possibility that CD26 is associated with ADA was investigated by biochemical analysis with polyclonal rabbit Ab to ADA (anti-ADA) (10). Immunoblotting with anti-ADA after immunoprecipitation from CD26 transfectants by various Abs demon- M. i.i ----- MM ~ ~. l ...l... 1.