Proc. Natl. Acad. Sci. USA Vol. 85, pp. 1615-1619, March 1988 Immunology Exon-intron organization and sequence comparison of human and murine Til (CD2) genes (genomic maps/molecular cloning/T-cell receptors/DNA sequencing) DON J. DIAMOND*t, LINDA K. CLAYTON*t, PETER H. SAYRE*, AND ELLIS L. REINHERZ*t *Laboratory of Immunobiology, Dana-Farber Cancer Institute and Departments of tPathology and tMedicine, Harvard Medical School, 44 Binney Street, Boston, MA 02115 Communicated by Barry R. Bloom, November 2, 1987 (receivedfor review September 23, 1987) ABSTRACT Genomic DNA clones containing the human mic domain rich in prolines and basic residues. cDNAs and murine genes coding for the 50-kDa T11 (CD2) T-cell encoding the murine and rat equivalents of T11 have been surface glycoprotein were characterized. The human T11 gene cloned and shown to define a homologous structure bearing is =12 kilobases long and comprised of five exons. A leader a 50% overall identity at the amino acid level (12-14). In exon (L) contains the 5'-untranslated region and most of the contrast to immunoglobulins whose variable and constant nucleotides defining the signal peptide [amino acids (aa) -24 region domains consist of antiparallel (3-sheets, protein mod- to -5]. Two exons encode the extracellular segment; exon Exi eling of murine and human T11 external segments indicates is 321 base pairs (bp) long and codes for four residues of the that these structures most likely belong to an a/,B-protein leader peptide and aa 1-103 of the mature protein, and exon folding class (12). Furthermore, the cytoplasmic region of Ex2 is 231 bp long and encodes aa 104-180. Exon TM is 123 bp T11 in each species is predicted to have a nonglobular long and codes for the single transmembrane region of the conformation and is sufficiently elongated to allow for po- molecule (aa 181-221). Exon C is a large 765-bp exon encoding tential interactions with multiple other intracellular proteins. virtually the entire cytoplas'mic domain (aa 222-327) and the It is thus likely that the cytoplasmic region subserves a signal 3'-untranslated region. The murine T11 gene has a similar transduction function. organization with exon-ntron boundaries essentially identical Here we report on the isolation and characterization of to the human gene. Substantial conservation of nucleotide human and murine T11 genes. DNA sequence analysis sequences between species in both 5'- and 3'-gene flanking demonstrates that the human and murine exon-intron orga- regions equivalent to that among homologous exons suggests nization is virtually identical.§ Of note, the extracellular that murine and human genes may be regulated in a similar segment of the mature T11 protein is encoded by two exons, fashion. The probable relationship of the individual T11 exons whereas almost the entire intracellular segment and 3'- to functional and structural protein domains is discussed. untranslated region is encoded on a single exon. The 50-kDa T11 (CD2) surface glycoprotein plays a major role in T-lymphocyte function (1-5). Monoclonal antibodies MATERIALS AND METHODS directed against an epitope on the external segment of the Genomic Library Screening and Restriction Mapping. A human T11 molecule (T111) block T-lymphocyte activation, human peripheral blood lymphocyte genomic library (kindly including that mediated through the T-cell receptor for provided by S. Orkin, Children's Hospital, Boston, MA) antigen and major histocompatibility complex (Ti-T3) (6). In containing DNA partially digested with Sau3aI and ligated contrast, antibodies defining two other extracellular segment into the EMBL3A vector (15) was screened with a 335-base- epitopes (T112 and T113) in concert give rise to antigen- independent human T-cell activation (1-3). Thus, interaction pair (bp) 5' fragment of the human T11 cDNA (PB2), ending of specific monoclonal antibodies with the T11 molecule at the unique EcoRV site (positions 8-335), and with a produces either profound antagonistic or agonistic effects on 401-bp 3' fragment ending at the second internal Taq I site T lymphocytes in vitro. The ability of anti-T111 monoclonal (positions 617-1017) (9). Genomic fragments containing re- antibodies to inhibit T-lymphocyte activation is predicated, gions that hybridized to PB2 cDNA were cloned into pUC18 at least in part, on their capacity to abrogate T11-mediated for further restriction analysis. Exons were identified within cell-cell contact (7). In fact, the spontaneous interaction the human Til gene through hybridization with kinase- between human T lymphocytes and sheep erythrocytes is treated 17-base oligonucleotides whose sequences were de- but one manifestation of the role of T11 in facilitating rived from the human cDNA. All mapping distances are cell-cell adhesion (8). accurate to within 100 bp. Oligonucleotide synthesis em- To more precisely define the biochemistry of this func- ployed standard cyanoethyl phosphoramidite chemistry on tionally important molecule, we and others have elucidated an Applied Biosystems model 381A (Applied Biosystems, the complete primary structure of the human T11 molecule Foster City, CA). by protein microsequencing and cDNA cloning (9) or by BALB/c mouse liver DNA was partially digested with antibody selection of expressed cDNA clones (10, 11). The Mbo I, and the size-selected 15- to 20-kilobase (kb) frag- following three segments of the protein have been defined: ments were ligated into the EMBL3B vector and propagated (i) an extracellular segment comprising more than half of the in the host bacterium LE392 to produce a murine genomic molecule and bearing only limited homology to members of library. The 819-bp 5' EcoRI fragment of the murine cDNA the immunoglobulin gene superfamily including the T4 T-cell XB2 (12) was used to screen the library for murine T11 surface protein (9, 10); (ii) a single hydrophobic transmem- brane segment; and (iii) a lengthy 117-amino acid cytoplas- Abbreviation: aa, amino acid(s). §These sequences reported in this paper are being deposited in the EMBL/GenBank data base (Bolt, Beranek, and Newman Labora- The publication costs of this article were defrayed in part by page charge tories, Cambridge, MA, and Eur. Mol. Biol. Lab., Heidelberg) payment. This article must therefore be hereby marked "advertisement" (accession no. J03622 for the human T11 gene and J03623 for the in accordance with 18 U.S.C. §1734 solely to indicate this fact. murine T11 gene). 1615 Downloaded by guest on September 26, 2021 1616 Immunology: Diamond et al. Proc. Natl. Acad. Sci. USA 85 (1988) sequences. Restriction analysis of the phage DNA and the coding segments of T11 overlapped the derived phage subcloning of hybridizing fragments was performed as clones such that the 5' and 3' ends of the corresponding above. cDNA were located on separate phage inserts (Fig. 1). Sequence Analysis. Fragments obtained by electroelution Results of restriction analysis and DNA sequencing of from agarose gels of bacteriophage A DNA digests were human and murine T11 genes indicate that, in each case, five subcloned into bacteriophage M13mpl8 or -mpl9 sequenc- exons are encoded within a gene -12 kb long (Fig. 1). The ing vectors by standard procedures (16). Sequence analysis exons are separated by introns that vary from 0.1-3.9 kb was performed on these clones by the dideoxy chain- long. Both the sizes and positions of the exons and introns termination procedure of Sanger et al. (17) with either are extremely similar between the human and murine T11 [a-32P]dATP or adenosine 5'[a-35S]thiotriphosphate (dATP genes (see Table 1). This conservation of the length of the [35S]) as a single radioactive nucleotide. In some cases, individual exons and introns and their relative positions sequence information was obtained from fragments cloned suggests that both genes evolved similarly to encode pro- into pUC18 by utilizing avian myeloblastosis virus reverse teins with homologous function. Comparison of the murine transcriptase (New England Nuclear) on double-stranded and human genomic copies of interleukin 2 (19), the 6 DNA as described by Chen and Seeburg (18). All 5'- and subunit of T3 (20), the a subunit of the T-cell receptor Ti (21, 3'-flanking sequences and coding regions were sequenced on 22), and the f8 subunit of the T-cell receptor Ti (23, 24) also both strands. revealed similar exon-intron organizations. These genes presumably have similar functions in both species. The AND DISCUSSION T-cell lineage restricted distribution of the human and mu- RESULTS rine T11 mRNA, shown in RNA gel blot analysis (9, 10), Isolation of Recombinant Phage Containing the Human and provides further evidence for the similarity of function of Mouse Tl1 (CD2) Gene Segments. A genomic library con- these two genes. Chromosomal locations for the murine and structed in the A vector EMBL3A (15) by partial Sau3aI human T11 genes (ref. 13 and L.K.C. and E.L.R., unpub- restriction endonuclease digestion of human peripheral lished results) have been shown to be on chromosomes 3 and blood lymphocyte DNA was screened with 5'- and 3'- 1, respectively. These chromosomes are known to be syn- specific fragments from the human T11 cDNA. Three types tenic, which further implies a common origin and evolution of phage were recovered from the library that encompassed for the murine and human T11 genes. all of the coding segments from the known cDNA sequence Nucleotide Sequence Analysis. By using a series of 17-base (9) as well as extensive 5'- and 3'-flanking regions (Fig. 1). oligonucleotide primers generated from the sequence of the The 5' fragment detected two classes of phage, such as murine and human T11 cDNAs, the exon-intron structure of ABT2D2 and ABT2; whereas the 3' fragment detected ABT2 the corresponding T11 genes was determined (Fig. 2). The and ABT1 (see Fig.