The EMBO Journal vol.9 no.7 pp.2141 -2150, 1990 Genomic organization of the mouse T cell receptor Va family Evelyne Jouvin-Marche, Isabelle Hue', Patrice N. Marche, Carine Liebe-Gris, Jean-Pierre Marolleau, Bernard Malissen', Pierre-Andre Cazenave and Marie Malissen' Unite d'Immunochimie Analytique, Ddpartement d'Immunologie, Institut Pasteur (CNRS LA 359 and Universite Pierre et Marie Curie), 25, rue du Dr Roux 75724 Paris Cedex 15, France and 'Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France Communicated by P.A.Cazenave Based on the analysis of Va gene segment deletions in a panel of T lymphomas, we have constructed a map of the mouse T cell receptor a/6 region and assigned the relative position of 72 distinct V gene segments. Three major observations have emerged from such studies. First, members of a given Va subfamily are not organized in discrete units along the chromosome but largely interspersed with members of other Va subfamilies. Second, analysis of the deletion map suggests the existence of repetitive patterns (Va clusters) in the chromosomal distribution of the Va gene segments. Third, the present- day organization of the Vca/6 region may be readily explained by a series of sequential duplications involving three ancestral Va clusters. Direct evidence for the existence of these unique structural features has been gained by cloning - 370 kb of DNA and positioning 26 distinct Va gene segments belonging to six different subfamilies. Finally, the relationships existing between the Va/6 gene segment organization and usage are dis- cussed in terms of position-dependent models. Key words: gene rearrangement/mouse T cell receptor/ repertoire/Va gene segment organization Introduction Mouse T cells can be divided into two subsets based on the structure of their specific receptors for antigen (T cell receptor, TCR). In the adult, most peripheral T cells express a TCR heterodimer consisting of a and chains, whereas a small percentage (1-10%) of peripheral T cells and a majority of intraepithelial T lymphocytes express an alternative TCR form made of -y and 6 chains (reviewed in Davis and Bjorkman, 1988). Each of these four TCR chains includes one variable (V) and one constant (C) region. As previously observed for the immunoglobulin heavy and light chain genes, functional TCR V genes are formed by the somatic rearrangement and juxtaposition of variable (V), diversity (D, in the case of TCR and TCR 6) and joining (J) gene segments. The organization of the a and 6 gene elements displays unique structural features among antigen receptor genes. First, a and 6 genes belong to the same genetic complex encoded on chromosome 14 (Chien et al., Oxford University Press 1987a). Second, the Db and J6 gene segments and the CA gene are located between the Va and Ja gene segments (Chien et al., 1987b). Most of the Vc -JJa rearrangements have been shown to result in the excision of the Db-J6 - C cluster (Chien et al., 1987a: Fujimoto and Yamagishi, 1987; Lindsten et al., 1987; Okazaki and Sakano, 1988). As a con- sequence, the majority of the Va gene segments have therefore been postulated to map at an undetermined distance 5' to the Db1 gene segment and to rearrange via deletional mechanisms. Based on the analysis of V segment usage in cloned T cells, the mouse Vca family has been estimated to compn'se approximately 75-100 members (Arden et al., 1985; Becker et al., 1985). These V gene segments can be grouped into distinct subfamilies based upon sequence similarity. V gene segments that show > 75 % similarity at the nucleotide level are considered members of the same subfamily (Brodeur and Riblet, 1984). Operationally, members of a given subfamily can be identified on Southern blots by hybridization to a prototypic V gene segment probe. Southern blot analyses have indicated that the Va subfamilies identified contain from one to ten members (Arden et al., 1985). Furthermore, analyses of restriction fragment length polymorphism of TCR Va gene segments among different inbred mouse strains have suggested that the members of a given subfamily are not organized in discrete clusters but instead are probably interspersed with members of other sub- families (Singer et al., 1988; Jouvin-Marche et al., 1989; Klotz et al., 1989). The unique organization of the a and 6 gene elements raises the possibility that a shared pool of V gene segments is used to produce both a and 6 chain genes. There is indeed some overlap in V segment usage: seven of the 11 identified VA gene segment subfamilies (Elliot et al., 1988; Raulet, 1989; Takeshita et al., 1989) overlap with or are identical to known Va subfamilies. However, four of the VA subfamilies have not been observed in surveys of large numbers of a genes and are possibly exclusively used to produce 6 chain genes (see Discussion). To study the organization of the mouse Va/6 gene segment families, we have used two complementary approaches. First we have exploited the fact that most Va -Ja rearrangements occur via deletion (see above). On a chromosome that has experienced a Va -Ja deletional rearrangement, the Va gene segments located between the rearranging Va and Ja gene segments are lost. In a single T cell, if both copies of chromosome 14 have undergone a Vca-Ja deletional rearrangement, the identity of the Va gene segments prox- imal and distal to the 3' most rearranged Va gene segment can be determined by Southern blot analysis of the T cell genomic DNA with various Vai-specific probes. Accordingly, we have analysed a panel of seven independently derived T lymphomas which have each rearranged different Voa gene segments on both homologous chromosomes, and constructed a deletion map indicating the relative order of Vax gene segments along the chromosome. 2141