Organization and sequences of the diversity , joining , and constant region genes of the human T-cell receptor 13 chain ( gene rearrangement / immune genes / generation of diversity )

The organization and sequences of the human (3-chain T-cell receptor diversity, joining, and constant region segments are described. The (3 chain of the human T-cell receptor, analogous to the mouse counterpart, consists of two distinct constant region genes =10 kilobases apart. The two constant region genes, Cpu and Cp.2, are very similar not only in sequence but also in genomic organization. The coding sequences of each of these Cp constant region genes are divided into four exons. The first two exons encode most of the extracellular constant domain. The third exon encodes a major part of the presumed transmembrane portion, and the last exon contains the cytoplasmic coding sequence as well as 3' untranslated sequences. Except for a stretch of =95 highly conserved nucleotides extending 3' of the first exon of the C region genes, little homology can be found between the intron sequences of Cpg and Cp2. A small cluster of joining region (Jp) gene segments is located ""5 kilobases upstream of each of these two constant regions. The first cluster, Jp1, contains six functional J gene segments while the second, Jp2, contains seven functional J gene segments. In addition, diversity region (Dp) gene segments are located ==600 base pairs upstream of each Jp. Recombinational signals containing highly conserved heptamer and nonamer sequences separated by 12 or 23 bases are found adjacent to all of these Dp and Jp gene segments. These signal sequences are thought to be involved in the somatic recombination processes. These results indicate that what appears to be a gene duplication event giving rise to these two distinct regions must have arisen a long time ago in the evolution of this gene locus. An essential property of the immune system is its ability to generate enormous diversity in antibody and T-cell responses. During the past several years, the genetic and molecular mechanisms responsible for the generation of antibody diversity have been elucidated (1, 2). Functional antibody genes are formed by DNA recombination during B-cell development. The recombination event joins together one member of each of the dispersed clusters of variable (V), diversity (D) (for heavy chains only), and joining (J) gene segments to give a unique and functional antibody gene. Recognition structures composed ofconserved heptamer and nonamer sequences separated by nonconserved spaces of 12 or 23 nucleotides adjacent to the V, D, and J gene segments are believed to be essential in the recombinational process (1, 2). Recently, the structure and diversity generation of the T-cell receptor (TcR) have also been studied (3, 4). The cloning of both the aand /-chain TcR genes (5-10) has allowed direct comparisons with immunoglobulin genes to be made. Both chromosomal location (11, 12) and partial DNA/protein sequence homologies (5-10) verify that the TcR and immunoglobulin gene families are distinct. The organization of the P-chain locus in humans is similar to that of immunoglobulin (13-17). Like immunoglobulin, the TcR /3 chain is composed of noncontiguous segments of V, J, and constant (C) region genes (13-17). Furthermore, recognition structures similar to those involved in the recombinational process in immunoglobulin are found to be adjacent to the Vp, Do, and Jo gene segments. Recently, similar findings were reported on the determination of the genomic organization of the human TcR a chain (18). In this paper, we report the genomic organization and sequences of the D, J, and C regions of the human TcR /3 chain. MATERIALS AND METHODS Human Germ-Line Genomic Clones. The Maniatis library containing human genomic sequences in X phage Charon 4A was used (19). Phages (5 x 106) were plated and screened for recombination ofphage-containing sequences homologous to the human TcR P-chain region sequences (5, 20). A plaque hybridization method was used to isolate these clones. The inserts were characterized by analysis of single and double restriction enzyme digests as well as restriction fragment hybridization to various DNA probes (Jr and Cp-specific) by Southern transfer methods. DNA Sequencing. Two strategies were used to obtain the sequences of the TcR 3-chain inserts. The resultant M13 subclones were sequenced by the dideoxy-chain termination method of Sanger et al. (21). (i) Oligo-directed sequencing. Large fragments [1-4 kilobases (kb)] of the TcR p-chain gene were purified by electrophoresis through 1% low melting point agarose and were subcloned into M13 vectors. The initial 350-400 base pairs (bp) of insert sequences were obtained by using a universal M13 primer. Synthetic oligonucleotides (15-mer) complementary to the 3' end of the most recently determined sequences were synthesized and used to prime the subsequent sequencing reaction. (it) Shotgun sequencing (22). Fragments ofthe TcR p-chain gene were purified by electrophoresis through 1% low melting point agarose and further fragmented by restriction enzyme digestion using Sau3A, Hpa II, HinpI, Taq I, Alu I, Rsa I, or Hae III. DNA fragments were size-selected by electrophoresis through 1% low melting agarose and subcloned into appropriate M13 vectors.