The sex-linked fidget mutation abolishes Brn4/Pou3f4 gene expression in the embryonic inner ear.
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We have demonstrated that the phenotype of the mouse mutant sex-linked fidget ( slf ) is caused by developmental malformations of the inner ear that result in hearing loss and vestibular dysfunction. Recently, pilot mapping experiments suggested that the mouse Brn4 / Pou3f4 gene co-segregated with the slf locus on the mouse X chromosome. These mapping data, in conjunction with the observation that the vertical head-shaking phenotype of slf mutants is identical to that observed in mice with a targeted deletion of the Brn4 gene, suggested that slf is a mutant allele of the Brn4 gene. In this paper, we have identified the nature of the slf mutation, and demonstrated that it is an X chromosomal inversion with one breakpoint close to Brn4. This inversion selectively eliminates the expression of the Brn4 gene in the developing inner ear, but not the neural tube. Finally, these results demonstrate that the slf mutation is a good mouse model for the most prevalent form of X-linked congenital deafness in man, which is associated with mutations in the human Brn4 ortholog, POU3F4. (+info)
DAR, a new RhD variant involving exons 4, 5, and 7, often in linkage with ceAR, a new Rhce variant frequently found in African blacks.
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The highly polymorphic Rh system is encoded by 2 homologous genes RHD and RHCE. Gene rearrangements, deletions, or point mutations may cause partial D and CE antigens. In this study, a new RHD variant, DAR, and a new RHCE variant, ceAR, are described in 4 Dutch African Blacks. Serologically, DAR showed weaker reactions with a monoclonal antibody and polyclonal antiserum against D. The DAR phenotype was characterized by complete loss of at least 9 of 37 Rh D epitopes. Erythrocytes expressing ceAR were all typed as VS(-), V(+). DNA analysis showed a partial D allele with only 3 mutations: C602G (exon 4), T667G (exon 5), and T1025C (exon 7). The ceAR allele carried G48C (exon 1), a hybrid exon 5 (A712G, C733G, A787G, and T800A), and A916G (exon 6). To study the frequency of these variants, 326 South-African Blacks was screened genomically. Of the 326 donors, 16 (4.9%) carried the DAR allele, 20 (6.1%) the ceAR allele, and 14 (4.3%) both mutated alleles. Five of these donors (1.5%) had the DAR phenotype, indicating that they carried the DAR allele homozygously or next to a D-negative allele. Immunogenicity of the D antigen for individuals with the DAR phenotype was proven, because 1 of the 4 Dutch individuals produced allo-antibodies against D after multiple transfusions with D-positive blood. In a multiethnic society, the prevalence of this D phenotype will increase and is therefore relevant in transfusion practice and in prevention of hemolytic disease of the newborn. (+info)
Primary central nervous system lymphomas are derived from germinal-center B cells and show a preferential usage of the V4-34 gene segment.
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Primary central nervous system lymphomas (PCNSLs) have recently received considerable clinical attention due to their increasing incidence. To clarify the histogenetic origin of these intriguing neoplasms, PCNSLs from 10 HIV-negative patients were analyzed for immunoglobulin (Ig) gene rearrangements. All tumors exhibited clonal IgH gene rearrangements. Of the 10 cases, 5 used the V4-34 gene segment, and all of these lymphomas shared an amino acid exchange from glycine to aspartate due to a mutation in the first codon of the complementarity-determining region 1. No preferential usage of D(H), J(H), V(kappa), J(kappa), V(lambda), or J(lambda) gene segments was observed. All potentially functional rearrangements exhibited somatic mutations. The pattern of somatic mutations indicated selection of the tumor cells (or their precursors) for expression of a functional antibody. Mean mutation frequencies of 13. 2% and 8.3% were detected for the heavy and light chains, respectively, thereby exceeding other lymphoma entities. Cloning experiments of three tumors showed ongoing mutation in at least one case. These data suggest that PCNSLs are derived from highly mutated germinal-center B cells. The frequent usage of the V4-34 gene and the presence of a shared replacement mutation may indicate that the tumor precursors recognized a shared (super) antigen. (+info)
Mutational analysis of RAG1 and RAG2 identifies three catalytic amino acids in RAG1 critical for both cleavage steps of V(D)J recombination.
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RAG1 and RAG2 initiate V(D)J recombination, the process of rearranging the antigen-binding domain of immunoglobulins and T-cell receptors, by introducing site-specific double-strand breaks (DSB) in chromosomal DNA during lymphocyte development. These breaks are generated in two steps, nicking of one strand (hydrolysis), followed by hairpin formation (transesterification). The nature and location of the RAG active site(s) have remained unknown. Because acidic amino acids have a critical role in catalyzing DNA cleavage by nucleases and recombinases that require divalent metal ions as cofactors, we hypothesized that acidic active site residues are likewise essential for RAG-mediated DNA cleavage. We altered each conserved acidic amino acid in RAG1 and RAG2 by site-directed mutagenesis, and examined >100 mutants using a combination of in vivo and in vitro analyses. No conserved acidic amino acids in RAG2 were critical for catalysis; three RAG1 mutants retained normal DNA binding, but were catalytically inactive for both nicking and hairpin formation. These data argue that one active site in RAG1 performs both steps of the cleavage reaction. Amino acid substitution experiments that changed the metal ion specificity suggest that at least one of these three residues contacts the metal ion(s) directly. These data suggest that RAG-mediated DNA cleavage involves coordination of divalent metal ion(s) by RAG1. (+info)
Mutations of acidic residues in RAG1 define the active site of the V(D)J recombinase.
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The RAG1 and RAG2 proteins collaborate to initiate V(D)J recombination by binding recombination signal sequences (RSSs) and making a double-strand break between the RSS and adjacent coding DNA. Like the reactions of their biochemical cousins, the bacterial transposases and retroviral integrases, cleavage by the RAG proteins requires a divalent metal ion but does not involve a covalent protein/DNA intermediate. In the transposase/integrase family, a triplet of acidic residues, commonly called a DDE motif, is often found to coordinate the metal ion used for catalysis. We show here that mutations in each of three acidic residues in RAG1 result in mutant derivatives that can bind the RSS but whose ability to catalyze either of the two chemical steps of V(D)J cleavage (nicking and hairpin formation) is severely impaired. Because both chemical steps are affected by the same mutations, a single active site appears responsible for both reactions. Two independent lines of evidence demonstrate that at least two of these acidic residues are directly involved in coordinating a divalent metal ion: The substitution of Cys for Asp allows rescue of some catalytic function, whereas an alanine substitution is no longer subject to iron-induced hydroxyl radical cleavage. Our results support a model in which the RAG1 protein contains the active site of the V(D)J recombinase and are interpreted in light of predictions about the structure of RAG1. (+info)
Hairpin formation in Tn5 transposition.
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The initial chemical steps in Tn5 transposition result in blunt end cleavage of the transposon from the donor DNA. We demonstrate that this cleavage occurs via a hairpin intermediate. The first step is a 3' hydrolytic nick by transposase. The free 3'OH then attacks the phosphodiester bond on the opposite strand, forming a hairpin at the transposon end. In addition to forming precise hairpins, Tn5 transposase can form imprecise hairpins. This is the first example of imprecise hairpin formation on transposon end DNA. To undergo strand transfer, the hairpin must to be resolved by a transposase-catalyzed hydrolytic cleavage. We show that both precise and imprecise hairpins are opened by transposase. A transposition mechanism utilizing a hairpin intermediate allows a single transposase active site to cleave both 3' and 5' strands without massive protein/DNA rearrangements. (+info)
Recombinant balanced and unbalanced translocations as a consequence of a balanced complex chromosomal rearrangement involving eight breakpoints in four chromosomes.
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We report on a family with a balanced complex chromosomal rearrangement (CCR) involving eight breakpoints between chromosomes 6, 7, 18, and 21 in the father. All three sons inherited one derivative chromosome from the father and in addition each inherited a different recombinant chromosome resulting in a partial trisomy 6q in the first, an apparently balanced karyotype in the second, and a partial trisomy 7q in the third son. Fluorescence in situ hybridisation (FISH) and microsatellite analysis were essential for the identification of the breakpoints. In addition, the results were confirmed by a 24-colour FISH experiment using the spectral karyotyping (SKYtrade mark) system. Paternal origin of the de novo CCR in the father was demonstrated for the first time by haplotype analysis. This is the second report of a CCR leading to simpler but unbalanced translocations in offspring as a consequence of recombination during gametogenesis, and the first report of a family case of CCR exhibiting as many as eight breakpoints in the transmitting carrier. The initial prediction that viable offspring would be quite unlikely had to be revised after the birth of three children. Genetic counselling of carriers of balanced complex rearrangements has to consider a higher probability for unbalanced recombinations than has been so far commonly assumed. (+info)
Aberrant rel/nfkb genes and activity in human cancer.
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Rel/NF-kappaB transcription factors are key regulators of immune, inflammatory and acute phase responses and are also implicated in the control of cell proliferation and apoptosis. Remarkable progress has been made in understanding the signal transduction pathways that lead to the activation of Rel/NF-kappaB factors and the consequent induction of gene expression. Evidence linking deregulated Rel/NF-kappaB activity to oncogenesis in mammalian systems has emerged in recent years, consistent with the acute oncogenicity of the viral oncoprotein v-Rel in animal models. Chromosomal amplification, overexpression and rearrangement of genes coding for Rel/NF-kappaB factors have been noted in many human hematopoietic and solid tumors. Persistent nuclear NF-kappaB activity was also described in several human cancer cell types, as a result of constitutive activation of upstream signaling kinases or mutations inactivating inhibitory IkappaB subunits. Studies point to a correlation between the activation of cellular gene expression by Rel/NF-kappaB factors and their participation in the malignant process. Experiments implicating NF-kappaB in the control of the apoptotic response also support a role in oncogenesis and in the resistance of tumor cells to chemotherapy. This review focuses on the status of the rel, nfkb and ikb genes and their activity in human tumors and their association with the onset or progression of malignancies. (+info)