We have developed a simple PCR strategy, termed vector-hexamer PCR, that is unique in its ability to easily recover every insert end from large insert clones in YAC and BAC vectors. We used this method to amplify and isolate all insert ends from a YAC contig covering the mouse Igh locus. Seventy-seven ends were amplified and sequenced from 36 YAC clones from four libraries in the pYAC4 vector. Unexpectedly, 40% of the insert ends of these YACs were LINE1 repeats. Nonrepetitive ends were suitable for use as probes on Southern blots of digested YACs to identify overlaps and construct a contig. The same strategy was used successfully to amplify insert ends from YACs in the pRML vector from the Whitehead Institute/MIT-820 mouse YAC library and from BACs in pBeloBAC11. The simplicity of this technique and its ability to isolate every end from large insert clones are of great utility in genomic investigation.[The nucleotide sequence data reported in this paper are accessible in GenBank under accession nos. B07512-B07598.]
The mouse H4 minor histocompatibility antigen (HA) includes a single H2Kb-bound peptide that stimulates rejection of skin allografts and generation of cytolytic T lymphocytes (CTL). We evaluated the diversity of the CTL response to this single minor HA peptide by sequencing alpha and beta chains of T-cell receptors (Tcr) from H4-specific CTLs as a first step toward understanding the diversity of Tcrs specific for single minor HA. We selected H4-specific CTL clones from short-term lines that were restricted by Kb (19 clones), Kbm5 (7 clones), and Kbm11 (10 clones). Whereas multiple V alpha and V beta family members were identified in the panel of Kb-restricted CTLs, five VB genes and one VA subfamily were predominant in CTLs derived from multiple individuals. Similar distributions were observed with Kbm5- and Kbm11-restricted CTLs, suggesting that these two mutants did not alter Tcr gene usage observable at the VA and/or VB gene levels. Negatively charged residues were present in the CDR3 regions of 12/13 unique Kb-restricted beta chains. A comparable observation was made with Kbm5-restricted CTLs, and the distributions of these residues among CDR3 positions were similar in the two CTL panels. However, the Kbm11 mutation dramatically altered the distribution of these residues resulting in their presence in positions 10-12 of CDR3 regions in all CTLs. These results indicate that the Tcrs expressed by CTLs specific for this single minor HA peptide are oligoclonal and characterized by the presence of negatively charged residues in beta CDR3 regions, the distribution of which is profoundly altered by the Kbm11 mutation.
Previous studies illustrated the influence of T cell subsets on susceptibility or resistance to demyelination in the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. Genetic segregation analysis showed a correlation with disease phenotype in this model with particular V(beta) genes. In this study we investigated the contribution of specific V(beta) TCR to the pathogenesis of virus-induced demyelinating disease. Spectratype analysis of cells infiltrating the CNS early in infection demonstrated an over-representation of V(beta)8(+) T cells in mice expressing a susceptible H-2 haplotype. We infected transgenic mice expressing the V(beta)8.2 TCR directed against a non-TMEV antigen and found an increase in demyelinating disease in mice of either susceptible or resistant background compared with littermate controls. In addition, depletion studies with an anti-V(beta)8-specific antibody in both susceptible (B10.Q) and resistant (C57BL/6) mice resulted in increased demyelination. TCR analysis of VP2-specific cytotoxic T cell clones from mice with a resistant genotype identified only the V(beta)8.1 TCR, suggesting that limited T cell diversity is critical to TMEV clearance. Together, these results support a protective role for V(beta)8(+) T cells in virus-induced demyelinating disease.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.