Hideo Yamagishi

Excision products of the T cell receptor gene support a progressive rearrangement model of the alpha/delta locus.

We have cloned extrachromosomal circular DNAs containing T cell receptor (TCR) delta gene segments in adult mouse thymocytes and splenocytes. We find that the frequency of circular DNA clones carrying germline delta sequences is lower than that of J alpha probe‐positive clones, possibly related to increasing 5′ distance from the most upstream J alpha segment. This suggests that the TCR alpha/delta locus is successively rearranged from within and that the delta‐containing excision products are progressively diluted out by the subsequent cell division which includes further alpha gene rearrangements. In addition, examination of delta gene excision products revealed newly identified V delta subfamilies, the reciprocal joining of two D delta elements, J delta 2 usage in thymocytes and novel sequences homologous to the human delta‐gene deleting elements.

Immunoglobulin gene hyperconversion ongoing in chicken splenic germinal centers.

It has been believed that the peripheral lymphocytes in chickens proliferate by self‐renewing amplification of the preimmune repertoire generated in bursa. We amplified rearranged immunoglobulin variable (V) region genes from the single germinal centers induced by immunization. The sequence analysis of these genes revealed that most were derived from distinct B‐cell clones which expanded locally, generating somatic antibody mutants at a high rate. Somatic hypermutations included unlinked base changes and the linked base modifications interpreted as unidirectional transfer of sequences from V region pseudogenes. This finding demonstrates the ongoing post‐bursal diversification of B‐cells in splenic germinal centers by templated gene conversion as well as untemplated point mutations.

Appearance of extrachromosomal circular DNAs during in vivo and in vitro ageing of mammalian cells

Appearance of extrachromosomal circular DNAs with in vivo and in vitro cellular ageing was examined by using a new technique of mica-press-adsorption for electron microscopy. The size distribution and the copy number of circular DNA complexes varied, depending on the cellular age. Extrachromosomal circular DNA complexes of variable length of more than 0.5 microns or 1.5 kilobase (kb) appeared during in vivo ageing of rat lymphocytes and in vitro ageing of cultured human lung fibroblasts. A restricted size class of circular forms of less than 0.5 microns in contour length was amplified in human skin fibroblasts from aged normal or Werners syndrome subjects. These circular DNA molecules are suggested to be products of DNA rearrangements or gene amplification occurring in the chromosome.

Physical mappings of chloroplast DNA from liverwort Marchantia polymorpha L. cell suspension cultures

SummaryPhysical maps of liverwort Marchantia polymorpha chloroplast DNA were constructed with restriction endonucleases, BamHI, SmaI, KpnI, and XhoI. The molecular size, calculated from the sum of the restriction fragments, is 121.0±1.0 kilobase (kb) pairs. This value is in good agreement with that of 118.7±2.0 kb pairs obtained from contour length measurements of the chloroplast DNA electron micrographs. The physical map indicates that the chloroplast DNA contains two copies of inverted repeats. The length of the inverted repeat is at most 11.7 kb pairs. Southern hybridization analysis indicates that two sets of the chloroplast ribosomal RNA genes are located in the inverted repeat regions. The site of the ribulose bisphosphate (RuBP) carboxylase (large subunit) gene was also determined on a BamHI fragment, Ba5, by using 32P-labeled DNA fragments containing the tobacco chloroplast RuBP carboxylase gene as a probe.

Exacerbating Role of γδ T Cells in Chronic Colitis of T-Cell Receptor α Mutant Mice

BACKGROUND & AIMS T-cell receptor (TCR) gammadelta T cells are an important component of the mucosal immune system and regulate intestinal epithelial homeostasis. Interestingly, there is a significant increase in gammadelta T cells in the inflamed mucosa of patients with ulcerative colitis (UC). However, the role of gammadelta T cells in chronic colitis has not been fully identified. METHODS TCRalpha-deficient mice, which spontaneously develop chronic colitis with many features of human UC including an increase in gammadelta T-cell population, represent an excellent model to investigate the role of gammadelta T cells in UC-like colitis. To identify the role of gammadelta T cells in this colitis, we herein have generated TCRgamma-deficient mice through deletion of all TCR Cgamma genes (Cgamma1, Cgamma2, Cgamma3, and Cgamma4) using the Cre/loxP site-specific recombination system and subsequently crossing these mice with TCRalpha-deficient mice. RESULTS An increase in colonic gammadelta T cells was associated with the development of human UC as well as UC-like disease seen in TCRalpha-deficient mice. Interestingly, the newly established TCRalpha(-/-) x TCRgamma(-/-) double mutant mice developed significantly less severe colitis as compared with TCRalpha-deficient mice. The suppression of colitis in TCRalpha(-/-) x TCRgamma(-/-) double mutant mice was associated with a significant reduction of proinflammatory cytokine and chemokine productions and a decrease in neutrophil infiltration. CONCLUSIONS gammadelta T cells are involved in the exacerbation of UC-like chronic disease. Therefore, gammadelta T cells may represent a promising therapeutic target for the treatment of human UC.

Isolation and characterization of transducing coliphage fd carrying a kanamycin resistance gene.

The DNA segment (Tn903) with a size of 3100 nucleotide pairs which carries a gene specifying kanamycin resistance derived from a chimeric plasmid pML21 (Hershfield et al., 1976) was transposed to various sites on the filamentous phage fd DNA. Wild type fd can be restored by excision of Tn903 from the resulting hybrid DNA molecule. The fd DNA carrying Tn903 when converted to the mature phage particle, was capable of transducing the kanamycin marker, and its replicative form DNA could be maintained in a bacterial cell like a plasmid.

Small circular DNA complexes in eucaryotic cells

Abstract A small number of eucaryotic cells (100 to 1000 cells) were pressed by mica sheet; then the extruded contents were adsorbed on mica and processed for electron microscopy. In the absence of divalent cation, small polydisperse circular DNA molecules bound to proteins or membrane material were preferentially adsorbed. The small circular DNA complexes have been found in every eucaryotic cell, primary lymphoid tissue cells of bursa and thymus, primary cell lines of retina and liver, and established cultured cell lines of embryonal teratocarcinoma, F9 and PCC3, HeLa and 3T6. Size distribution of these DNA complexes varies, depending on the cell source. The circles less than 1 μm in contour length predominate in cultured cell lines and the larger ones in primary cell lines and cells in situ . Polydisperse covalently closed circular DNAs were recovered from thymus lymphocytes by the conventional dye-CsCl buoyant density method. Their size distribution was similar to that of the small circular DNA complexes detected by the mica-press-adsorption method. They are present in several tens to hundreds of copies per cell representing, at a maximum, 0.02% of the total cellular DNA. The possibility that small circular DNA complexes may result from gene rearrangement as well as from replicon “misfiring” ( A. Varshavsky, 1981 , Proc. Nat. Acad. Sci. USA 78 , 3673–3677) are discussed.

Structure of extrachromosomal circular DNAs excised from T-cell antigen receptor alpha and delta-chain loci

Small polydisperse circular (spc) DNA was isolated from mouse thymocytes, fragmented by HindIII digestion and cloned into the vector. Sixty DNA clones were randomly selected from the 10,400 phage library. The average size of insert was one-fifth of the original circular molecule. Twenty spc-DNA clones were homologous to DNA probes derived from T-cell antigen receptor (TCR) alpha-chain loci. We have characterized nine clones by DNA sequencing; they contain new germline sequences of the TCR alpha-chain variable (V alpha) and joining (J alpha) gene segments and the products out of the recombination of a V alpha with a J alpha gene segment. An additional four spc-DNA clones carried a new rearranging gene of the TCR delta-chain that is located between V alpha and J alpha genes. At least nine of 60 DNA clones carried the recombination junction of a heptamer-heptamer head-to-head structure expected from an excised product of V-J joining. This shows that most extrachromosomal circular DNAs in the thymus are formed by a sequence-dependent recombination mechanism. We suggest that a functional T-cell receptor V alpha gene can be constructed by somatic random rearrangements through successive looping-out, excision and deletion.

Effect of Environmental Antigens on the Ig Diversification and the Selection of Productive V-J Joints in the Bursa

In chickens, a single set of unique functional segments of both Ig H and L chain genes is rearranged during early embryogenesis to generate a pool of B cell progenitors that will be diversified in the bursa by gene conversion, forming the preimmune repertoire. After hatching, bursal cells are exposed to environmental Ags in the bursal lumen. We prepared B cells from each single bursal follicle and used PCR-directed Ig L chain gene analysis to study the differentiation of B cells and the effect of antigenic stimulation from the bursal lumen on the neonatal chicken B cell repertoire formation. Selective amplification of B cell clones with a productive V-J joint was observed during the late embryonic stage, possibly by the interaction with ligands expressed on the bursal stroma and further accelerated in the neonatal chicken. Administration of the artificial Ags into the bursal lumen before the isolation of bursa by bursal duct ligation in the embryo caused a significant increase in lymphocytes with a productive V-J joint in the neonatal chicken bursa compared with the isolated bursa. Intra- and interclonal diversity of a complementarity-determining region measured by an evolutionary distance increased during bursal development. Clonal diversification did not require stimulation by artificial Ags from the bursal lumen. Thus, the preimmune repertoire in the bursa is generated by gene conversion during Ag-independent B cell proliferation, and antigenic stimulation from the bursal epithelium to bursal B cells plays roles in the selection of clones with a productive V-J joint.

Characterization of the Japanese pufferfish (Takifugu rubripes) T-cell receptor α locus reveals a unique genomic organization

Abstract. Polymerase chain reactions with degenerate V gene segment primers were used to isolate the putative T-cell receptor α-chain gene (TCRA) from Japanese pufferfish (Takifugu rubripes). The putative TCRA chain cDNA is composed of an N-terminus leader peptide followed by the variable region and the constant region. The variable portion of the TCRA gene is encoded by V and J gene segments separated in the germline. As in mammals, the V-J junction sequences are GC rich and highly diversified. Amino acid residues that are required to maintain the function and structural integrity of the TCRA polypeptide, including the conserved Trp-Tyr-Lys and Tyr-Tyr-Cys motifs in the V gene segments, the Lys-Leu-X-Phe-Gly-X-Gly-Thr-X-Leu motif in the J gene segment, the three cysteine residues in the constant region and the charged residues in the transmembrane region are all preserved in the pufferfish. These conserved features suggest that the TCRA gene families in fish and mammals have evolved from a common ancestor.