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D Kitamura

Publications and source records attributed to D Kitamura.

54 records · Page 3Linked to original sources
PubMed1993

Immunoglobulin heavy and light chain genes rearrange independently at early stages of B cell development.

The compartment of mouse B cell progenitors can be resolved into five developmentally related fractions by multicolor flow cytometry. Using this system and employing mutant mice in which the membrane exon of the mu chain, the lambda 5 gene, or the JH locus was inactivated by gene targeting, we found that expression of the pre-B cell receptor complex is necessary for the transition from the large CD43+ to the small CD43- pre-B cell stage. We report the occurrence of immunoglobulin heavy and light chain gene rearrangement at the stage of large B cell precursors. We show that neither the pre-B cell receptor complex nor any gene rearrangement in the heavy chain locus is required for the induction of kappa light chain gene rearrangement in early B cell progenitors.

Animals↗
PubMed1993

Deletion of the immunoglobulin kappa chain intron enhancer abolishes kappa chain gene rearrangement in cis but not lambda chain gene rearrangement in trans.

Immunoglobulins (Ig) secreted from a plasma cell contain either kappa or lambda light chains, but not both. This phenomenon is termed isotypic kappa-lambda exclusion. While kappa-producing cells have their lambda chain genes in germline configuration, in most lambda-producing cells the kappa chain genes are either non-productively rearranged or deleted. To investigate the molecular mechanism for isotypic kappa-lambda exclusion, in particular the role of the Ig kappa intron enhancer, we replaced this enhancer by a neomycin resistance (neoR) gene in embryonic stem (ES) cells. B cells heterozygous for the mutation undergo V kappa-J kappa recombination exclusively in the intact Ig kappa locus but not in the mutated Ig kappa locus. Homozygous mutant mice exhibited no rearrangements in their Ig kappa loci. However, splenic B cell numbers were only slightly reduced as compared with the wild-type, and all B cells expressed lambda chain bearing surface Ig. These findings demonstrate that rearrangement in the Ig kappa locus is not essential for lambda gene rearrangement. We also generated homozygous mutant mice in which the neoR gene was inserted at the 3' end of the Ig kappa intron enhancer. Unexpectedly, mere insertion of the neoR gene showed some suppressive effect on V kappa-J kappa recombination. However, the much more pronounced inhibition of V kappa-J kappa recombination by the replacement of the Ig kappa intron enhancer suggests that this enhancer is essential for V kappa-J kappa recombination.

Animals↗
PubMed1993

Developmental and functional impairment of T cells in mice lacking CD3 zeta chains.

CD3 zeta is a component of the T cell antigen receptor (TCR) complex and is important for signal transduction. We have established mice selectively lacking CD3 zeta but able to express CD3 eta, a polypeptide produced from the same locus through alternative splicing, using the method of gene targeting in embryonic stem cells. In homozygous mutant mice, the numbers of thymocytes and peripheral T cells were greatly reduced and the expression levels of TCR on these cells were 5-fold lower than those on wild-type cells. By contrast, TCR gamma delta+ intestinal intraepithelial lymphocytes were not obviously affected by the mutation. T cells from homozygous mutants exhibited an impaired proliferative response. These results imply that CD3 zeta has a critical role in the development and signal transduction of T cells in vivo.

Animals↗
PubMed1992

A critical role of lambda 5 protein in B cell development.

The lambda 5 gene is a homolog of immunoglobulin J lambda-C lambda genes, expressed specifically in immature B-lineage cells. Lambda 5-encoded molecules form membrane complexes with mu or D mu proteins in association with an additional protein specifically expressed in immature B cells that is encoded by the Vpre-B gene. We have generated mice in which the lambda 5 gene is inactivated by targeted gene disruption in embryonic stem cells. In these mice, B cell development in the bone marrow is blocked at the pre-B cell stage. However, the blockade is leaky, allowing B cells to populate the peripheral immune system at a low rate. These cells are allelically excluded and able to respond to antigen.

Animals↗
PubMed1992

Targeted disruption of mu chain membrane exon causes loss of heavy-chain allelic exclusion.

Burnet's clonal selection theory suggests that each B lymphocyte is committed to a single antibody specificity. This is achieved by a programme of somatic rearrangements of the gene segments encoding antibody variable (V) regions, in the course of B-cell development. Evidence from immunoglobulin-transgenic mice and immunoglobulin-gene-transfected transformed pre-B cells suggest that the membrane form of the immunoglobulin heavy (H) chain of class mu (microns), expressed from a rearranged H-chain (IgH) locus, may signal allelic exclusion of the homologous IgH locus in the cell and initiation of light (L)-chain gene rearrangement in the Ig kappa loci. We report here that targeted disruption of the membrane exon of the mu chain indeed results in the loss of H-chain allelic exclusion. But, some kappa chain gene rearrangement is still observed in the absence of micron expression.

Alleles↗
PubMed1992

[Studies of physiological functions of cytokines by generating mutant mice through the transgenic and the gene-targeting technology].

Molecular cloning of gene coding for cytokines, growth factors and their receptors have facilitated an accumulation of findings regarding the function of their products. So far, many kinds of functions of such molecules in vitro have been identified. To clarify the physiological roles of such molecules in vivo a variety of cytokine-transgenic mice and a few cytokine-gene targeted mice have been generated. Many of the phenotypes expressed by these animals are as expected from the in vitro assay data, but some are unexpected. In this short article, some of the phenotypes reported are introduced and their significance is discussed.

Animals↗
PubMed1991

B cell development regulated by gene rearrangement: arrest of maturation by membrane-bound D mu protein and selection of DH element reading frames.

In productively rearranged murine VH-DH-JH genes (encoding immunoglobulin heavy chain variable regions), the DH elements are preferentially used in one particular reading frame (RF1), although the recombination breakpoints at the DH-JH border vary. Despite this variability, the bias of RF usage is not due to cellular selection by antigen but is quantitatively established at the stage of DH-JH rearrangement: RF3 is counterselected on the basis of stop codons. RF2 allows the expression of a truncated mu chain (D mu protein) from most DH-JH joints. Using B cells in which the membrane exon of the mu chain is disrupted by homologous recombination on one of the two homologous chromosomes, we obtain evidence that membrane-bound D mu signals arrest of differentiation, presumably by preventing VH-DHJH joining. In addition to RF3 and RF2 counterselection, promotion of DH-JH joining in areas of sequence homology further enforces RF1 usage.

Amino Acid Sequence↗
PubMed1991

A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene.

Of the various classes of antibodies that B lymphocytes can produce, class M (IgM) is the first to be expressed on the membrane of the developing cells. Pre-B cells, the precursors of B-lymphocytes, produce the heavy chain of IgM (mu chain), but not light chains. Recent data suggest that pre-B cells express mu chains on the membrane together with the 'surrogate' light chains lambda 5 and V pre B (refs 2-7). This complex could control pre-B-cell differentiation, in particular the rearrangement of the light-chain genes. We have now assessed the importance of the membrane form of the mu chain in B-cell development by generating mice lacking this chain. We disrupted one of the membrane exons of the gene encoding the mu-chain constant region by gene targeting in mouse embryonic stem cells. From these cells we derived mice heterozygous or homozygous for the mutation. B-cell development in the heterozygous mice seemed to be normal, but in homozygous animals B cells were absent, their development already being arrested at the stage of pre-B-cell maturation.

Animals↗
PubMed1989

Isolation and characterization of a novel human gene expressed specifically in the cells of hematopoietic lineage.

A novel cDNA clone designated as HS1, which show an expression pattern limited to human hematopoietic cells, was isolated. About 2kb mRNA of the clone was accumulated in all the mature and immature lymphoid and myeloid cell lines tested, and two of three erythroblastoid cell lines, but not in any cell lines of non-hematopoietic tissues. The same mRNA was also detected in normal lymphoid and myeloid tissues and peripheral blood lymphocytes, granulocytes and macrophages, but again not in non-hematopoietic tissues. Nucleotide sequence of the HS1 predicts a protein of 486 amino acids (Mr 53,931). N-terminal half of the protein retains unique repeating motifs, each of which shows a significant homology with the helix-turn-helix DNA-binding motif of several proteins reported previously. C-terminal half of the protein retains a region conserved between non-receptor tyrosine kinases (src family), phospholipase C(PLC)-148 and the crk oncogene product. A unique feature of HS1 suggests that the protein may be involved in signal transduction and regulation of gene expression.

Adaptor Proteins, Signal Transducing↗
PubMed1988

Purification of a nuclear trans-acting factor involved in the regulated transcription of a human immunoglobulin heavy chain gene.

The expression of the rearranged human immunoglobulin gamma 1 heavy chain gene (HIG1) was shown to be induced through its enhancer by the positive regulatory trans-acting factor(s) that was contained only in cells of B lineage. The trans-acting factors were purified from mouse myeloma NS1 cells, and HIG1-inducing activity was found mainly in fractions of molecular weight 53-127 kd and in a fraction eluted from a heparin-Sepharose column with 0.5 M KCI. This semipurified fraction contained proteins binding to the conserved octamer sequence, ATGCAAAT, in the promoter region, as well as to sequences in the enhancer region. The 0.5 M KCI eluates from a heparin-Sepharose column were applied to a DNA affinity column of synthetic oligonucleotides of the octamer sequence and the sequence TATTTTAGGAAGCAAA in the HpaII-BgIII region of the HIG1 gene enhancer. The protein eluted from the enhancer sequence-specific DNA affinity column showed a strong inducing activity for the HIG1 gene, and the molecular weight of a predominant protein was 96 kd.

Animals↗
PubMed1987

Purification of an octamer sequence (ATGCAAAT)-binding protein from human B cells.

The highly conserved octamer sequence ATGCAAAT or its inverse complement found in all human and murine immunoglobulin gene promoters has been demonstrated to be necessary in the lymphoid-specific transcription by deletion analysis. Trans-acting factors that interact with the octamer motif are thought to be involved in this tissue-specific expression. Using a gel mobility shift assay, we have identified both lymphoid-specific and ubiquitous nuclear factors that interact with a human gamma 1 heavy chain gene promoter region containing the octamer motif, consistent with the results obtained with murine heavy or light chain promoter regions. We have purified an octamer binding protein from human B cells by sequence-specific DNA affinity chromatography. Renaturation of gel-purified protein allowed the identification of a polypeptide with a molecular weight of 74 kilodaltons (kD) that is capable of recognizing and binding to the octamer motif. This 74 kD protein seems to be also present in T-cells and non-lymphoid cells. The possible function of the factor is discussed.

B-Lymphocytes↗
PubMed1987

Nuclear factors binding to the human immunoglobulin heavy-chain gene enhancer.

The human immunoglobulin heavy chain (IgH) gene contains at least two tissue-specific regulatory regions, which are similar to the mouse IgH gene. One is the J-C enhancer and another is located in the 5' promoter region. Using an electrophoretic mobility shift assay and DNase I footprint, we have examined the interaction of factors in B cell nuclear extracts with the two regulatory regions of the human IgH gene. We have identified a nuclear factor in mouse B cell nuclear extracts which bound to specific sequence in the human IgH enhancer. This factor is apparently not present in mouse fibroblast nuclear extracts. We also found factor(s) which bound to the highly conserved octanucleotide sequence within the human IgH enhancer and 5' promoter regions.

Animals↗
PubMed1987

Regulation of immunoglobulin gene transcription by labile repressor factor(s).

The cloned human gamma 1 heavy chain gene (HIG1) was scarcely expressed in the stable transformants of a mouse fibroblast line, L cell or a T cell line, EL4, and no gamma 1 heavy chain was produced in these cells. Upon treatment with cycloheximide or other kinds of protein synthesis inhibitors, the transcription of HIG1 gene was induced in L cell transformants as well as in T cell transformants. Transcription rate of bacterial gpt gene, which was derived from the plasmid vector used for transfection of HIG1 gene and located just upstream of HIG1 in the transformants, was also greatly enhanced after cycloheximide treatment. But the expression of several endogenous genes in the L cells tested was not affected by the cycloheximide treatment. Nuclear transcription assay indicated that the appearance of HIG1 gene transcripts after treatment with cycloheximide was mainly due to the induction of the transcription. Deletion of an enhancer element from HIG1 gene lowered the inducing activity of cycloheximide in the L cell transformants, but a low level of HIG1 gene expression was still observed. These results suggested that trans-acting factors similar to those present in B lymphoid cells are also functioning in non-B lymphoid cells but their activity is inhibited by short-lived repressor protein(s). Such repressor protein(s) appear to act on regulatory element(s) of the immunoglobulin heavy chain gene including enhancer region as well as 5' flanking region.

Animals↗
PubMed1987

The cis-acting regulatory elements of immunoglobulin heavy chain gene involved in enhanced immunoglobulin production after lipopolysaccharide (LPS) stimulation.

In the present study, the transient expression of the human immunoglobulin heavy chain gene (HIG1) was analyzed in a mouse pre-B-cell line, 70Z/3, after LPS stimulation. HIG1 gene and its recombinant plasmids were transfected by the calcium phosphate method into 70Z/3 cells and the cells were stimulated with lipopolysaccharide (LPS) 48 hr after DNA transfection. The amounts of human heavy chain gene products greatly increased in 70Z/3 cells after LPS stimulation, but the increment was diminished by deletion of the heavy chain gene enhancer element (MluI-HpaI fragment) in the J-C intron from the HIG1 gene. The gene, delta 3, which contained the 5' promoter region and the rearranged VDJ region of HIG1 but lacked the enhancer element, was weakly transcribed in 70Z/3 cells after LPS stimulation. Insertion of the enhancer element into the delta 3 gene greatly enhanced the transcription of the VDJ gene. The highest enhancement of the VH gene transcription rate was obtained when the 3' half of the enhancer element was ligated to the delta 3 gene. The present data suggest that the 3' half of the enhancer element of the heavy chain gene may play an important role in the enhanced production of immunoglobulin which is induced with LPS stimulation.

Animals↗
PubMed1986

Trans-acting nuclear protein responsible for induction of rearranged human immunoglobulin heavy chain gene.

A complete human gamma 1 heavy chain gene (HIG1) was transferred into mouse cells by protoplast fusion. The HIG1 gene was strongly expressed in mouse myeloma cells but not in mouse fibroblasts (L cells). Nuclear extracts from myeloma cells were injected into L cell transformants containing one copy of the HIG1 gene; this triggered accurate transcription of the HIG1 gene in the transformants. The induction of HIG1 gene expression by a myeloma nuclear factor (or factors) appeared to depend on the enhancers in the heavy chain gene. Nuclear proteins prepared from cells of the B lineage could induce the transcription of HIG1 gene in the L cell transformants, while those from the cells of non-B-lineage could not. The present study shows that positive regulatory trans-acting factors are involved in the activation of the immunoglobulin heavy chain gene through its enhancers and are contained only in cells of the B lineage.

B-Lymphocytes↗
PubMedDate not supplied

A dual role for B cells in Plasmodium chabaudi chabaudi (AS) infection?

CD4+ T cells are necessary for a protective immune response against the erythrocytic stages of the malaria parasite Plasmodium chabaudi chabaudi AS. B cells are not required for control of early acute parasitaemias, but appear to be important for final clearance of the infection, most probably by producing specific antibodies against the parasite. However, immune sera and immune IgG are unable to replace the protective capacity of B cells in adoptive transfer of immunity to P. chabaudi AS. It is therefore conceivable that B cells are required to achieve protective immunity, not only as effector plasma cells, but because they may also play a second important role. We have recently suggested that B cells may regulate the Th response to P. chabaudi AS during a primary infection. We discuss here the possibility of a dual requirement of B cells in achieving protective immunity to P. chabaudi AS.

Animals↗