N-region Diversity Research Articles

Genetic basis of the neonatal antibody repertoire: germline V-gene expression and limited N-region diversity

We report here on the molecular characterization of heavy and light chain V-regions of antibodies isolated from the highly connected idiotypic network of the newborn BALB/c mouse. Nucleotide sequence analysis of eight hybridomas confirmed their germline origin. Furthermore, in contrast to most hybridomas and myelomas derived from adult mice, the majority of these clones were found to lack N-region sequences. These data show that somatic processes amplifying junctional diversity are relatively inactive early in ontogeny, and that germline gene expression alone ensures idiotypic complementarities in the developing immune system.

Allelic T-cell receptor α complexes have little or no influence on susceptibility to type 1 diabetes

We performed a multiple-affected-sib study to determine if T-cell receptor α-chain alleles affect susceptibility to insulin-dependent diabetes mellitus. Restriction fragment length polymorphisms were used to follow the segregation of allelic T-cell receptor α complexes within the families. The segregation of T-cell receptor α alleles in 29 multiplex families revealed no significant tendency for affected sibs to share T-cell receptor α-chain alleles more often than would be expected by chance alone ( p > 0.2). In contrast, the same type of analysis for HLA alleles easily detected the well-known linkage of insulin-dependent diabetes mellitus susceptibility to the HLA complex ( p = 0.003). We suggest that the importance of HLA alleles in insulin-dependent diabetes mellitus susceptibility and the lack of importance of T-cell receptor α alleles result from the different strategies by which HLA and T-cell receptor molecules achieve antigen-binding diversity: multiple loci and allelic diversity in the case of HLA; combinatorial, junctional, and N-region diversity in the case of the T-cell receptor. In this paper we also describe three new restriction fragment length polymorphisms of the T-cell receptor α complex and a new method for testing the significance of linkage in multiple-affected-sib studies.

Base substitution mutagenesis by terminal transferase: its role in somatic mutagenesis

We have addressed the possibility of terminal transferase involvement in somatic mutagenesis and the creation of N-region diversity, by measuring the ability of TdT to enhance single-base substitution mutagenesis during in vitro DNA synthesis. Using 3 independent assays we find that terminal transferase produces only a small increase in base-substitution mutagenesis when assayed in the presence of DNA polymerase-β. In the presence of either polymerase-α or E. coli polymerase-I, however, no detectable increase in TdT-induced mutagenesis is seen. Furthermore, in an assay capable of detecting a variety of mutational events, terminal transferase primarily produces complex addition/deletion mutations, as well as a few multiple, tightly-clustered, single-base mutations. We conclude that the majority of the scattered single-base changes that occur during antibody gene differentiation are not catalyzed by terminal transferase, but instead result from another error-prone DNA synthetic process (possibly utilizing DNA polymerase-β).

Mammalian T-lymphocyte antigen receptor genes: genetic and nongenetic potential to generate variability.

T lymphocytes of higher vertebrates are able to specifically recognize a seemingly unlimited number of foreign antigens via their receptors, the T cell antigen receptors (TCRs). T lymphocytes mature by passing through the thymus and acquire antigen specificity by expressing the TCR molecules on their cell surface. Genetic and somatic diversification mechanisms give rise to the enormous degree of TCR variability observed in mature T cells: germline and combinatorial diversity as well as junctional and the so-called N-region diversity. In contrast to the situation in immunoglobulin genes somatic hypermutation does not seem to play a significant role in TCR diversification. It is argued here that the enzyme terminal nucleotidyl-transferase is potentially a major factor in generating the immense diversity. We propose furthermore that this enzyme ensures the flexibility of T cell responses to novel antigens by random insertion of so-called N-region nucleotides. Apart from the physiological functions of TCR genes any involvement in the etiology of T cell neoplasia remains to be proven.

Preferential expression of a defined T-cell receptor beta-chain gene in hapten-specific cytotoxic T-cell clones.

A multitude of different antigens can be recognized by T cells through specific receptors. Both the alpha- and beta-chains of the T-cell receptor contribute to the antigen recognition portion. The repertoire of beta-chain variable region (V beta) gene segments is limited to some 20 elements which seem to be used randomly in different T cells. Diversity at the beta-chain level can be created in several ways: a multiplicity of germline gene segments; combinatorial diversity by rearranging different V, diversity (D), joining (J) and constant (C) region elements; junctional diversity by joining gene segments at different sites; N-region diversity, that is, insertion of random nucleotides at junctional sites; and somatic mutation. However, the major sources and the extent of diversity of the T-cell receptor are unclear. To address this issue, 42 H-2Kb-restricted, 2,4,6-trinitrophenyl (TNP)-specific cytotoxic T-cell (Tc) clones from C57BL/6 mice were characterized with respect to expression of different beta-chain gene segments in messenger RNA using specific oligonucleotide probes. We report here that nearly half of the Tc clones use identical elements for productive beta-chain gene rearrangement. Thus, there is a restriction in the use of beta-chain gene segments in this panel of Tc clones which favours a particular V beta--D beta--J beta--C beta combination with a defined D beta element.

Diversity and rearrangement of the human T cell rearranging γ genes: Nine germ-line variable genes belonging to two subgroups

We describe nine T cell γ variable (V) gene segments isolated from human DNA. These genes, which fall into two subgroups, are mapped in two DNA regions covering 54 kb and probably represent the majority of human V γ genes. One subgroup (V γI) contains eight genes, consisting of four active genes and four pseudogenes. The single V γII gene is potentially active. Sequence analysis of the V γI genes shows variation clustered in hypervariable regions, but somatic variability is restricted to N-region diversity. Studies on rearrangement in T cell lines and in thymic DNA show that major rearrangements can be observed that are attributable to the five active V γ genes. In addition, human cells with the phenotype of helper T cells can undergo productive V γ-J γ joining.