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T Iwasato

Publications and source records attributed to T Iwasato.

10 recordsLinked to original sources

Cortex-restricted disruption of NMDAR1 impairs neuronal patterns in the barrel cortex.

In the rodent primary somatosensory cortex, the configuration of whiskers and sinus hairs on the snout and of receptor-dense zones on the paws is topographically represented as discrete modules of layer IV granule cells (barrels) and thalamocortical afferent terminals. The role of neural activity, particularly activity mediated by NMDARs (N-methyl-D-aspartate receptors), in patterning of the somatosensory cortex has been a subject of debate. We have generated mice in which deletion of the NMDAR1 (NR1) gene is restricted to excitatory cortical neurons, and here we show that sensory periphery-related patterns develop normally in the brainstem and thalamic somatosensory relay stations of these mice. In the somatosensory cortex, thalamocortical afferents corresponding to large whiskers form patterns and display critical period plasticity, but their patterning is not as distinct as that seen in the cortex of normal mice. Other thalamocortical patterns corresponding to sinus hairs and digits are mostly absent. The cellular aggregates known as barrels and barrel boundaries do not develop even at sites where thalamocortical afferents cluster. Our findings indicate that cortical NMDARs are essential for the aggregation of layer IV cells into barrels and for development of the full complement of thalamocortical patterns.

Animals↗

NMDA receptor-dependent refinement of somatotopic maps.

We have examined the role of NMDA receptor-mediated neural activity in the formation of periphery-related somatosensory patterns, using genetically engineered mice. We demonstrate that ectopic expression of a transgene of an NMDAR1 splice variant rescues neonatally fatal NMDAR1 knockout (KO) mice, although the average life span varies depending on the level of the transgene expression. In NMDAR1 KO mice with "high" levels of the transgene expression, sensory periphery-related patterns were normal along both the trigeminal and dorsal column pathways. In the KO mice with "low" levels of the transgene expression, the patterns were absent in the trigeminal pathway. Our results indicate that NMDA receptor-mediated neural activity plays a critical role in pattern formation along the ascending somatosensory pathways.

Alternative Splicing↗

Transforming growth factor-beta induced class switch recombination may be modified during the cell proliferation restored by IL-2 stimulation.

Mouse spleen cells were stimulated, first by transforming growth factor beta (TGF-beta) and then by interleukin-2 (IL-2) in the presence of bacterial lipopolysaccharide (LPS). LPS plus IL-2 stimulation restored the cell proliferation suppressed by TGF-beta and increased the number of B cells. Both TGF-beta alone or TGF-beta plus IL-2 enhanced production of surface IgA-positive (sIgA+) cells in LPS-stimulated B-cell cultures. We characterized extrachromosomal circular DNAs generated in TGF-beta-primed and IL-2-stimulated B cells and identified the breakpoints of S mu/S gamma 3 recombinants which were the major intermediates of S mu and S alpha switch recombination. All switch recombination sites were dispersed evenly within both S mu and S gamma 3 regions. This even distribution of S mu/S gamma 3 breakpoints is in contrast to the site preference of class switch breakpoints induced by TGF-beta alone. These results suggest that IL-2-stimulated cell proliferation may modify class switch recombination primed by TGF-beta.

Amino Acid Sequence↗

The complete murine immunoglobulin class switch region of the alpha heavy chain gene-hierarchic repetitive structure and recombination breakpoints.

A 7255-base pair (bp) sequence, including the previously sequenced murine segments of I alpha, S alpha, and C alpha, has been completed. Homology matrix comparison revealed a switch repetitive region of 4.2 kilobases (kb) composed of 20-80-bp homology runs, including the previously assigned S alpha region. We distinguished several stretches of duplication, i.e. the central 0.8-kb repetitive region, with some 80-bp staggered consensus repeats containing 20-30-bp subsets, made up of the primordial pentamers CTG(A/G)G. All the break-points of the S alpha switch recombination, including those generated by the translocation of the c-myc protooncogene and those catalyzed by bacterial extracts, are located within the consensus sequence subsets of the 4.2-kb repetitive region.

Animals↗

Molecular characterization of extrachromosomal circular DNAs from an embryonal carcinoma cell line induced to differentiate into neuron-like cells in vitro.

Extrachromosomal circular DNAs isolated from a P19 embryonal carcinoma cell line were induced to differentiate into neuron-like cells by retinoic acid and cloned into an EcoRI site of a phage vector. Of the 26 DNA inserts (2.1 kb in average length) analyzed, 16 contained repetitive sequences. Out of 10 DNA inserts with unique sequence, 6 carried linear chromosomal sequences and 4 showed chromosomal rearrangements in Southern blots. Two unique fragments with germline configuration were enriched in circular DNA clone libraries. We assigned the breakpoints of 3 circular DNA fragments to positions in the germline sequence. Patchy short inverted repeats were found in the vicinity of breakpoints. An intrastrand loop structure between such inverted short homology region may be required for the circularization of excised DNA.

Animals↗

Biased distribution of recombination sites within S regions upon immunoglobulin class switch recombination induced by transforming growth factor beta and lipopolysaccharide.

We have characterized extrachromosomal circular DNAs from adult mouse spleen cells that were induced to switch to immunoglobulin A (IgA) with bacterial lipopolysaccharide (LPS) and transforming growth factor beta (TGF-beta), and identified breakpoints of S mu/S gamma 3, S mu/S gamma 2, S mu/S alpha, S gamma 3/S alpha, and S gamma 2/S alpha recombinants. The S mu recombination donor sites clustered in the 3' half of the S mu region, while the S alpha recombination acceptor sites clustered in the 5' half of the S alpha region. In addition, donor and acceptor sites of S gamma regions also clustered in the 3' and 5' parts, respectively. These site preferences are in sharp contrast to the dispersed distribution of S mu/S gamma 1 breakpoints within both S mu and S gamma 1 regions upon IgG1 switch induced by LPS and interleukin 4. Our results support the hypotheses that TGF-beta increases the frequency of switch recombination events to IgA and that the switch recombination to IgA often proceeds by successive recombination of S mu/S gamma and S gamma/S alpha.

Animals↗

Novel excision products of T cell receptor gamma gene rearrangements and developmental stage specificity implied by the frequency of nucleotide insertions at signal joints.

We have cloned circular DNA excised by T cell receptor (TcR) gamma 1, gamma 2 and gamma 3 gene rearrangements in fetal and adult mouse thymocytes. Circular DNA contained a signal joint reciprocal to the genomic V-J coding joint. Although signal joints without nucleotide insertions are common in immunoglobulin (Ig) and TcR gene rearrangements, the signal joint of gamma found in adult thymocytes contained non-germ-line element (N) insertions at high frequency, while no insertions were found in fetal thymocytes. Thus developmental stage specificity of TcR gamma gene rearrangements is faithfully reflected on the signal joint of excision products. In addition, examination of gamma gene excision products revealed circular DNA products of TcR gamma-alpha transrearrangements, but no evidence of V gamma gene replacement in a rearranged segment.

Age Factors↗

Deletions of immunoglobulin C kappa region characterized by the circular excision products in mouse splenocytes.

We have identified circular DNAs containing the kappa light chain constant region (C kappa), as well as the excision products of V kappa-J kappa and V lambda-J lambda joining in adult mouse splenocytes. Analysis of C kappa-positive circular DNA clones revealed two recombination sites (intron recombining sequence [IRS]1 and -2) within the germline J kappa-C kappa intron region and the recombining sequence (RS) located downstream of the C kappa exon. While RS contains a conserved heptamer and nonamer separated by a 23-bp spacer on the 5' side, IRS1 sequence is an isolated heptamer without an obvious nonamer, and IRS2 contains a variant heptamer, CACAAAA. Since IRS1 and IRS2 recombined with both RS (23-bp spacer signal) and V kappa (12-bp spacer signal) with significant frequency, intron recombination sites seem to have dual recombination signals. These findings provide direct evidence that C kappa deletion preceding lambda gene rearrangement can occur by looping out and excision. Increased accessibility of inefficient recombinational loci within the intron may enable recombinase to accept wide signal sequence variation.

Animals↗

Circular DNA is excised by immunoglobulin class switch recombination.

We have purified extrachromosomal circular DNAs from adult mouse spleen cells, and cloned into a phage vector the BamHl fragments hybridizing with C mu and S gamma 1 probes. We obtained 52 S mu+S gamma 1+ clones by screening 1.4 million phage clones derived from spleen cells stimulated with bacterial lipopolysaccharide and interleukin 4. We have identified the breakpoints of six clones that contain S gamma 1 and S mu sequences fused in the 5' to 3' orientation. All these switch recombination sites were assigned to the central repetitive sequences of the S mu and S gamma 1 regions. Since the common S mu-S gamma 1 sequences at the recombination sites are at most 2 bases long, typical homologous recombination cannot account for their joining. These findings provide direct evidence that mu-gamma 1 class switching can occur by the looping out and excision of chromosomal DNA, with formation of a circle.

Animals↗

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.

Animals↗