The somatic diversity immunglobulin and T-cell receptor diversity is largely provided by the junctional variation created during site-specific rearrangement of separately encoded gene segments. Using a transient transfection assay, we demonstrate that the recombination activating genes Ragl and Rag2 direct site-specific rearrangement on an artificial substrate in poorly differentiated as well as in differentiated nonlymphoid cell lines. In addition to a high frequency of precise recombination events, coding joints show deletions and more rarely P-nucleotide insertions, reminiscent of immunoglobulin and T-cell receptor junctions found in fetal tissues. N-region insertions, which are characteristic of adult junctional diversity, are obtained at high frequency upon transfection of a terminal deoxynucleotidyltransferase expression vector together with Ragl and Rag2. These results show that only three lymphoid-specific factors are needed to generate all types ofjunctional diversity observed during lymphoid development.Immunoglobulin (Ig) and T-cell receptor (TCR) gene assembly is achieved through site-specific recombination events, from separately encoded variable (V), in some cases diversity (D), and junction (J) gene segments. Much of the immunoglobulin and TCR diversity is generated by the combinatorial rearrangement of a large number of V, D, and J gene segments (for a review see refs. 1 and 2).Recombination signal sequences (RSSs) situated adjacent to each gene segment provide the targets for recombination. RSSs are composed of a palindromic heptamer and an (A+T)-rich nonamer separated by a spacer of 12 or 23 base pairs (bp) (3). Rearrangement only occurs between RSSs with spacers of different length. RSSs are sufficient to target rearrangement of artificial substrates containing no other antigen receptor sequences (4-7).Two main types of joints are formed during the recombination process: coding joints created by the juxtaposition of the gene segments and reciprocal joints by contiguous RSSs (8). Whereas the heptamers in the reciprocal joints are generallyjoined back to back without nucleotide insertions or deletions, the coding joints are subjected to extensive processing (8). The junctions formed during rearrangement constitute another source of diversity. Several nucleotides can be deleted and two types of insertions can be found. Random nucleotide additions, resulting in N-region insertions, are thought to be introduced by terminal deoxynucleotidyltransferase (TdT) (9-11). P-nucleotide insertions represent the inverted repeat of the adjacent coding sequence. Their addition has been proposed to be a compulsory step of the recombination mechanism (12).Genomic DNA transfection experiments have allowed the isolation of two recombination activating genes, Ragl and Rag2 (13-15). Together these genes are able to confer sitespecific recombination activity to NIH 3T3 fibroblasts. Although it is not formally excluded that they activate other genes that would be responsible for the recombination activity, Ragl...

No abstract

Protocadherins gamma (Pcdhgamma) are a family of transmembrane proteins in which variable extracellular domains are associated with an invariant cytoplasmic domain, potentially allowing these proteins to trigger common cellular responses through diverse extracellular signals. We studied the expression of the family by in situ hybridisation and immunohistochemistry for the conserved portion of the mRNA or protein. During mouse development, Pcdhgamma expression is highest in neural tissues, but is also present in some nonneural tissues. In the adult, Pcdhgamma expression is maintained at high levels in brain, in particular in hippocampus and in the Purkinje cells of the cerebellum, whereas it is downregulated in spinal cord. Using antibodies against the conserved cytoplasmic domain, we show that in cultured embryonic spinal cord neurons, Pcdhgamma protein is present initially in both axonal and dendritic growth cones. At later stages of differentiation in vitro, Pcdhgamma distribution becomes polarised to the somatodendritic compartment. We propose that members of the Pcdhgamma family may play roles in neuronal growth and maturation.