On-line version ISSN 1678-4502
Braz. J. Genet. vol. 20 no. 1 Ribeirão Preto Mar. 1997
The genomic organization and complete sequencing of the human T-cell receptor b locus
Geraldo A.S. Passos Jr.
Departamento de Morfologia, Disciplina de Genética, Faculdade de Odontologia de Ribeirão Preto,
Universidade de São Paulo, 14049-904 Ribeirão Preto, SP, Brasil. Fax: (016) 633-0999, E-mail: email@example.com
c/c to firstname.lastname@example.org and Grupo de Imunogenética Molecular, Departamento de Genética,
Faculdade de Medicina de Ribeirão Preto, USP, 14040-900 Ribeirão Preto, SP, Brasil.
In 1996, human genome research was marked by a milestone from Leroy Hoods group who published the largest contiguous human DNA sequenced to date, i.e., the 685 kb of DNA of the T-cell receptor (TCR) b locus (Rowen et al., 1996). This achievement resulted from a combination of physical mapping and automated DNA sequencing methodology and the correct previous choice of DNA region to be analyzed. The human TCR b locus spans about 0.7 Mb, the appropriate size for current large-scale DNA sequencing. Moreover, this locus plays a vital role in immunity.
T-cell receptors are heterodimeric polypeptides expressed on the surface of T-cells held together by covalent disulfide bonds. TCR a/b is present in both T-helper cells (CD4+) and T-cytotoxic (CD8+) and TCR g/d is expressed in a minority of lymphocytes (10% of circulating lymphocytes) that recognize Mycobacterium, tumor cells and stressed cells (Raulet, 1989; Kabelitz, 1992). Similar to an immunoglobulin molecule, the TCR chains contains a variable region that interacts with the antigen (amino terminal) and a constant region (carboxy terminal) attached to the cell membrane. The recognition of a non-self antigen by TCR a/b is made in association with the self major histocompatibility complex (MHC) molecules expressed by antigen-presenting cells (APC) such as macrophages, monocytes, B-cells and dendritic cells. T-cells do not recognize a soluble antigen directly but only those present on the surface of APC or target tumor cells (Kersh and Allen, 1996). The essential role of the immune system is the discrimination between the self and non-self antigens. So, TCR a/b represents a "conceptual molecule" of current immunology, since it recognizes simultaneously the self (MHC) and the non-self (antigen). Each individual T-cell expresses a clonally distributed TCR. The constant region of a TCR chain is coded by a C gene and the variable region results from the junction of non-contiguous DNA segments (V gene and J segment for a and g chains; V gene, D-J segments for b and d chains). The maturation of a T-cell depends on the somatic recombination V-J and V-D-J DNA segments assembling an active TCR gene. The V(D)J recombination is mediated by the recombinases RAG-1 and RAG-2 (Oettinger et al., 1990; Mombaerts et al., 1992). The generation of the diversity in TCR is based on i) the multiple germline V, D and J elements, ii) combinatorial joining of these elements during T-cell maturation, iii) N diversity, i-e, the addition of non-germline nucleotides to the gene segments by terminal deoxynucleotidyl transferase, and iv) the combinatorial heterodimeric associations between a and b chains.
Organization of the TCR b locus
The human TCR b locus maps to chromosome 7q35 (Isobe et al., 1985) and comprises about 65 Vb genes. Until recently, the data contributing to the partial organization of the TCR b family were derived from the cDNA analysis of ~270 different b transcripts (Concannon et al., 1986; Tillinghast et al., 1986; Kimura et al., 1987) and from a few cloned germline DNAs of the b locus (Li et al., 1991; Slightom et al., 1994; Zhao et al., 1994). The Vb gene segments have been divided by cDNA analysis into 26 subfamilies, whose members exibit 75% DNA sequence homology. cDNA analysis has provided only a partial idea of the genomic organization because it analyzes only the active genes. Thus, a limited picture for the TCR b locus emerged as follows; 5-(unknown number and order of Vb elements)- (Db1-Jb1.1-1.6-Cb1-Db2-Jb2.1-2.7-Cb2-Vb20). Today, with the complete sequence of the 685-kb TCR b locus (Rowen et al., 1996) we can have insights into the organization, evolution and diversification of this gene cluster. Eighty-one TCR elements and two other non- TCR genes lie in this sequence. One is the dopamine-b-hydroxylase-like gene at the 5 end of this sequence and eight trypsinogen genes divided into two clusters, three immediately 3 to the dopamine-b-hydroxylase-like gene and five immediately 5 to the Db1 gene segment. Dot matrix sequence comparisons in relation to TCR cDNA sequences identified 65 Vb genes, six of which were new. With one exception all of these genes are located between the dopamine-b-hydroxylase-like gene and the Db1 gene segment. Each of the duplicated DJC clusters, separated by 2.5 kb, contains one Db and six or seven Jb gene segments and a Cb gene. An enhancer element is located at the 3 extremity of the Cb2 gene and the 65th Vb gene has an inverted translation reading frame compared to the other TCR elements. Of the 685 kb of the locus 4.6% are coding regions. A new nomenclature was proposed based on the complete sequence. The Vb subfamilies were assigned consecutively, numbered starting at the 5 end of the locus and the individual subfamily members were then numbered sequentially after the subfamily designation. A b locus translocation from chromosome 7 to chromosome 9 was also observed. The segment represents a duplication and translocation of a DNA segment from the 3 end of the b locus that includes at least seven Vb elements and a functional trypsinogen gene denoted T9. The physical map of the human TCR b locus is also available via Internet: http://imgt.cnusc.fr:8104 or http://www.ebi.ac.uk/imgt.
Characterization of the Vb gene segments
A Vb gene contains five regions: i) a promoter, ii) a first exon coding for a signal peptide, iii) an intron with 5 and 3 RNA splicing signals, iv) a second exon encoding the V element and v) a DNA rearrangement signal sequence. A clustering of the members of individual b subfamilies was revealed by phylogenetic analysis using > 75% sequence homology as a criterion. The 65 Vb gene segments are divided into 46 functional genes, 19 pseudogenes, and 22 other additional sequences with limited similarity to Vb gene segments were identified, each bearing several major lesions other than pseudogenes. Although these sequences provide no functional information, they contribute to a dynamic view of the evolutionary changes at this locus. The Vb gene segments contain conventional RNA splicing signals 5 GT and 3 AG for the introns. The TCR b DNA rearrangement signals are like the other gene segment counterparts (Hesse et al., 1989; Koop et al., 1994), such as human and mouse immunoglobulin, with the classical heptamer-spacernonamer structure. The expression of the Vb genes shows a heterogeneous pattern, with some Vb and Jb gene segments being expressed more frequently than others. These differences could arise from clonal selection, different promoter strengths, half-life of the mRNAs, or specific DNA rearrangement probabilities. The expression insights were obtained by comparison of TCR germline and cDNA sequences. Moreover, the cDNA analysis was useful for establishing exon-intron boundaries and for identifying non-TCR genes within the TCR locus, such as the dopamine-b-hydroxylase- like and trypsinogen genes.
Evolution of the locus itself
The b locus presents a high degree of complexity, 47% of its sequences being composed of locus-specific repeats (homology units) that have been duplicated 2 to 10 times, with eight major locus-specific repeats across the multigene family, as deduced from dot-matrix analysis.
Variations in the sequence
The polymorphism data at the TCR b locus (as RFLPs) have been obtained from limited cDNA and germline DNA analyses. Overlapping cosmid clones originated from different haplotypes (chromosomes) showed discrepant rates of sequence variation. Two large insertion-deletion polymorphisms affecting three Vb [Vb13S2 (6-2), Vb7S2 (4-3) and Vb9S2 (3-2)] and two trypsinogen genes (T6 and T7), respectively, exhibited allele frequencies of 0.37 (insertion) and 0.61 (deletion) for Vb polymorphism and 0.54 (insertion) and 0.46 (deletion) for the trypsinogen polymorphisms. The Vb deletion can be associated with loss of autoimmune tendencies, but it is not clear why the loss of the functional trypsinogen T6 gene would confer a selective advantage.
Concluding remarks and perspectives
The last year was marked by a historical achievement in the human genome program: the TCR b locus was totally sequenced, its 685 kb being the longest contiguous stretch of DNA analyzed to date in humans. Another cluster of immune response genes, the lambda locus, responsible for the lambda light chain antibodies located on chromosome 22q11, has been recently mapped using contigs of YACs and cosmids (Frippiat et al., 1995; Kawasaki et al., 1995) and is the next candidate for total sequencing. This knowledge opens new perspectives to explore the normal and pathological function and evolution of the human immune system.
Publication supported by FAPESP.
Concannon, P., Pickering, L.A., Kung, P. and Hood, L. (1986). Diversity and structure of human T-cell receptor beta-chain variable region genes. Proc. Natl. Acad. Sci. USA 83: 6598-6602. [ Links ]
Frippiat, J.-P., Williams, S.C., Tomlinson, I.M., Cook, G.P., Cherif, D., Le Paslier, D., Collins, J.E., Dunham, I., Winter, G. and Lefranc, M.P. (1995). Organization of the human immunoglobulin lambda light-chain locus on chromosome 22q11.2. Hum. Mol. Genet. 4: 983-991. [ Links ]
Hesse, J.E., Lieber, M.R., Mizuuchi, K. and Gellert, M. (1989). V(D)J recombination: a functional definition of the joining signals. Genes Dev. 3: 1053-1061. [ Links ]
Isobe, M., Erikson, J., Emmanuel, B.S., Nowell, P.C. and Croce, C.M. (1985). Location of the gene for b subunit of human T-cell receptor at band 7q35, a region prone to rearrangements in T-cells. Science 228: 580-582.
Kabelitz, D. (1992). Function and specificity of human g/d-positive T-cells. Crit. Rev. Immunol. 11: 281-303.
Kawasaki, K., Minoshima, S., Schooler, K., Kudoh, J., Asakawa, S., de Jong, P.J. and Shimizu, N. (1995). The organization of the human immunoglobulin l gene locus. Genome Res. 5: 125-135. [ Links ]
Kersh, G.J. and Allen, P.M. (1996). Essential flexibility in the T-cell recognition of antigen. Nature 380: 495-498. [ Links ]
Kimura, N., Toyonaga, B., Yoshikai, Y., Du, R.P. and Mak, T.W. (1987). Sequences and repertoire of the human T-cell receptor alpha and beta chain variable region genes in thymocytes. Eur. J. Immunol. 17: 375-383. [ Links ]
Koop, B.F., Rowen, L., Wang, K., Kuo, C.L., Seto, D., Lenstra, J.A., Howard, S., Shan, W., Deshpande, P. and Hood, L. (1994). The human T-cell receptor TCRAC/TCRDC (C alpha/C delta) region: organization, sequence, and evolution of 97.6 kb of DNA. Genomics 19: 478-493. [ Links ]
Li, Y., Szabo, P. and Posnett, D.N. (1991). The genomic structure of human V beta 6 T-cell antigen receptor genes. J. Exp. Med. 174: 1537-1547. (Published erratum appears in J. Exp. Med. 1992 Vol. 175: 617). [ Links ]
Mombaerts, P., Jacomini, J., Johnson, R.S., Herrup, K., Tonegawa, S. and Papaioannou, V.E.. (1992). RAG-2 deficient mice have no mature B and T lymphocytes. Cell 68: 869-877. [ Links ]
Oettinger, M.A., Schatz, D.G., Gorka, C. and Baltimore, D. (1990). RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science 248: 1517-1522. [ Links ]
Raulet, D.H. (1989). Antigens for g/d T-cells. Nature 339: 342-343.
Rowen, L., Koop, B.F. and Hood, L. (1996). The complete 685-kilobase DNA sequence of the human b T-cell receptor locus. Science 272: 1755-1762.
Slightom, J.L., Siemieniak, D.R., Sieu, L.C., Koop, B.F. and Hood, L. (1994). Nucleotide sequence analysis of 77.7 kb of the human V beta T-cell receptor gene locus: direct primer-walking using cosmid template DNAs. Genomics 20: 149-168. [ Links ]
Tillinghast, J.P., Behlke, M.A. and Loh, D.Y. (1986). Structure and diversity of the human T-cell receptor beta chain. Science 233: 879-883. [ Links ]
Zhao, T.M., Whitaker, S.E. and Robinson, M.A. (1994). A genetically determinated insertion/deletion related polymorphism in human T-cell receptor beta chain (TCRB) includes functional variable gene segments. J. Exp. Med. 180: 1405-1414. [ Links ]