RESUMO
Haematopoietic stem cell transplantation (HSCT) is an immunological treatment that has been used for more than 40 years to cure a variety of diseases. The procedure is associated with serious side effects, due to the severe impairment of the immune system induced by the treatment. After a conditioning regimen with high-dose chemotherapy, sometimes in combination with total body irradiation, haematopoietic stem cells are transferred from a donor, allowing a donor-derived blood system to form. Here, we discuss the current knowledge of humoral problems and B cell development after HSCT, and relate these to the current understanding of human peripheral B cell development. We describe how these studies have aided the identification of subsets of transitional B cells and also a robust memory B cell phenotype.
Assuntos
Subpopulações de Linfócitos B/citologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Linfopoese/fisiologia , Animais , Formação de Anticorpos , Antígenos de Diferenciação de Linfócitos B/análise , Linhagem da Célula , Sobrevivência de Enxerto , Humanos , Hospedeiro Imunocomprometido , Memória Imunológica , Imunofenotipagem , Infecções/etiologia , Antígenos Comuns de Leucócito/biossíntese , Linfopenia/etiologia , Camundongos , Condicionamento Pré-Transplante/efeitos adversosRESUMO
Somatic hypermutation and isotype switch recombination occur in germinal center B cells, are linked to transcription, and are similarly affected by deficiency in MutS homologue (MSH)2. Class-switch recombination is abrogated by disruption of genes encoding components of the catalytic subunit of DNA-dependent protein kinase (DNA-PK(cs))/Ku complex and likely involves nonhomologous end joining (NHEJ). That somatic hypermutation might also be associated with end joining is suggested by its association with the creation of deletions, duplications, and sites accessible to terminal transferase. However, a requirement for NHEJ in the mutation process has not been demonstrated. Here we show that somatic mutation in mice deficient in NHEJ can be tested by introduction of rearranged immunoglobulin and T cell receptor transgenes: the transgene combination not only permits reconstitution of peripheral lymphoid compartments but also allows formation of germinal centers, despite the wholly monoclonal nature of the lymphocyte antigen receptors in these animals. Using this strategy, we confirm that somatic hypermutation like class-switching can occur in the absence of recombination-activating gene (RAG)1 but show that the two processes differ in that hypermutation can proceed essentially unaffected by deficiency in DNA-PK(cs) activity.
Assuntos
Proteínas de Ligação a DNA , Genes RAG-1 , Mutagênese , Proteínas Serina-Treonina Quinases/genética , Recombinação Genética , Animais , Sequência de Bases , Domínio Catalítico , Proteína Quinase Ativada por DNA , Rearranjo Gênico , Centro Germinativo , Proteínas de Homeodomínio , Imunoglobulinas/genética , Camundongos , Camundongos SCID , Dados de Sequência Molecular , Muramidase/imunologia , Subunidades Proteicas , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos T/genética , Transposases/genéticaRESUMO
Matrix protein 2 ectodomain (M2e) is considered an attractive component of a broadly protective, universal influenza A vaccine. Here we challenge the canonical view that antibodies against M2e are the prime effectors of protection. Intranasal immunizations of Balb/c mice with CTA1-3M2e-DD-generated M2e-specific memory CD4 T cells that were I-Ad restricted and critically protected against infection, even in the complete absence of antibodies, as observed in JhD mice. Whereas some M2e-tetramer-specific memory CD4 T cells resided in spleen and lymph nodes, the majority were lung-resident Th17 cells, that rapidly expanded upon a viral challenge infection. Indeed, immunized IL-17A-/- mice were significantly less well protected compared with wild-type mice despite exhibiting comparable antibody levels. Similarly, poor protection was also observed in congenic Balb/B (H-2b) mice, which failed to develop M2e-specific CD4 T cells, but exhibited comparable antibody levels. Lung-resident CD69+ CD103low M2e-specific memory CD4 T cells were αß TCR+ and 50% were Th17 cells that were associated with an early influx of neutrophils after virus challenge. Adoptively transferred M2e memory CD4 T cells were strong helper T cells, which accelerated M2e- but more importantly also hemagglutinin-specific IgG production. Thus, for the first time we demonstrate that M2e-specific memory CD4 T cells are broadly protective.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Vírus da Influenza A/imunologia , Infecções por Orthomyxoviridae/imunologia , Proteínas da Matriz Viral/imunologia , Animais , Anticorpos Antivirais/sangue , Antígenos de Histocompatibilidade Classe II/metabolismo , Hibridomas , Memória Imunológica , Interleucina-17/genética , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Ligação Proteica , Domínios Proteicos/genética , Vacinação , Proteínas da Matriz Viral/genéticaRESUMO
Multiple DNA polymerases exist in eukaryotes. Polymerases alpha, delta and epsilon are mainly responsible for chromosomal DNA replication in the nucleus and are required for proliferation. In contrast, the repair polymerases beta and eta are not essential for cellular proliferation in yeast or mice, but a lack of either polymerase can lead, respectively, to defects in base excision repair or the ability to replicate past lesions induced by ultraviolet (UV) radiation [1-3]. Here, we have focused on polymerase zeta. This was first described as a non-essential product of the yeast REV3/REV7 genes involved in UV-induced mutagenesis, and was later implicated in trans-lesion synthesis [4,5]. Unlike in yeast, the mouse homologue (mRev3) was found to be essential for life. Homozygous mutant mice died in utero. Mutant embryos were considerably reduced in size at day 10.5 of development and usually aborted around day 12.5. It is likely that this block reflects a need for mRev3 in proliferative clonal expansion (rather than in the production of a particular cell type) as mutant blastocysts showed greatly diminished expansion of the inner cell mass in culture. Thus, mRev3 could be required to repair a form of externally induced DNA damage that otherwise accumulates during clonal expansion or, consistent with the high homology shared between its Rev7 partner and the mitotic checkpoint gene product Mad2 [6], mRev3 might play a role in cell proliferation and genomic stability even in the absence of environmentally induced damage.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Desenvolvimento Embrionário e Fetal/genética , Proteínas Fúngicas/genética , Genes Letais , Proteínas de Saccharomyces cerevisiae , Animais , Sequência de Bases , Primers do DNA , Camundongos , Camundongos MutantesRESUMO
The SP6 kappa-promoter pentadecamer (pd) element was found to be unable to stimulate transcription when present in one copy as the only promoter element in a minimal promoter but showed weak stimulatory activity when present as a multimer (four copies). One copy of the pd element acted synergistically with an octamer element, but not with a SP1 site, to stimulate transcription. The effect was orientation dependent with regard to the pd element. Gel shift analysis showed that pd-binding proteins were expressed in transformed as well as nontransformed B lymphocytes, irregardless of their differentiation stage, and in HeLa cells. Two major complexes, binding to different sites within the pd element, were observed in gel shifts. A low-molecular-weight form dominated in resting cells, while a higher-molecular-weight form appeared after mitogenic stimulation. Southwestern analysis showed that the low-molecular-weight pd-binding protein had a molecular mass of 35 kDa, which was confirmed by fractionation by denaturating polyacrylamide gel electrophoresis and molecular sieving. The higher-molecular-weight complex was sensitive to detergent treatment, while the low-molecular-weight complex was not. Mutation analysis showed that the two pd-binding complexes interacted with distinct sites within the element and that dual occupancy was required for functional activity. The functional synergy between the pd element and the octamer was more pronounced in plasmacytomas than in B-cell lymphomas.
Assuntos
Linfócitos B/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas , Animais , Linfócitos B/imunologia , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Cloranfenicol O-Acetiltransferase/biossíntese , Cloranfenicol O-Acetiltransferase/metabolismo , Células HeLa , Humanos , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Peso Molecular , Oligodesoxirribonucleotídeos , Plasmídeos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Baço/efeitos dos fármacos , Baço/imunologia , TATA Box , TransfecçãoRESUMO
The majority of activated B cells differentiate into IgA plasma cells, with the gut being the largest producer of immunoglobulin in the body. Secretory IgA antibodies have numerous critical functions of which protection against infections and the role for establishing a healthy microbiota appear most important. Expanding our knowledge of the regulation of IgA B-cell responses and how effective mucosal vaccines can be designed are of critical importance. Here we discuss recent developments in the field that shed light on the uniqueness and complexity of mucosal IgA responses and the control of protective IgA responses in the gut, specifically.
Assuntos
Linfócitos B/imunologia , Imunoglobulina A/metabolismo , Mucosa Intestinal/imunologia , Vacinas/imunologia , Animais , Humanos , Imunidade Humoral , Imunidade nas Mucosas , Imunoglobulina A/genética , Memória Imunológica , Ativação LinfocitáriaRESUMO
Burkitt's lymphomas harbour chromosomal translocations bringing c-MYC into the vicinity of one of the immunoglobulin gene loci. Point mutations have been described within c-MYC in several Burkitt's lymphomas and it has been proposed that translocation into the Ig loci might have transformed c-MYC into a substrate for the antibody hypermutation mechanism. Here we test this hypothesis by exploiting a Burkitt's lymphoma line (Ramos) that we have previously shown to hypermutate its immunoglobulin genes constitutively. We find that, during in vitro culture, Ramos mutates the c-MYC allele that is translocated into the IgH locus whilst leaving the untranslocated c-MYC and other control genes essentially unaffected. The mutations are introduced downstream of the c-MYC transcription start with the pattern of substitutions being characteristic of the antibody hypermutation mechanism; the mutation frequency is 2-3-fold lower than for the endogenous functional IgH allele. Thus chromosomal translocations involving the Ig loci may not only contribute to transformation by deregulating oncogene expression but could also act by potentiating subsequent oncogene hypermutation.
Assuntos
Alelos , Linfoma de Burkitt/genética , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Mutação , Proteínas Proto-Oncogênicas c-myc/genética , Translocação Genética , Sequência de Bases , Humanos , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
The physiological mechanism for producing antigen-specific antibodies is based on a two-phase neo-Darwinian process: the first phase consists of diversity generation (formation of the repertoire), and the second phase is antigen-mediated selection. In this article, we consider how the natural immunoglobulin gene-diversification processes can be exploited both in vivo and in vitro in order to allow the generation of novel antibody (and heterologous protein) repertoires.
Assuntos
Diversidade de Anticorpos/genética , Rearranjo Gênico do Linfócito B , Seleção Genética , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Afinidade de Anticorpos/genética , Especificidade de Anticorpos/genética , Linhagem Celular , Genes de Imunoglobulinas , Humanos , Camundongos , Camundongos Transgênicos , MutaçãoRESUMO
Whereas gut IgA responses to the microbiota may be multi-centered and diverse, little is known about IgA responses to T-cell-dependent antigens following oral immunizations. Using a novel approach, gut IgA responses to oral hapten (4-hydroxy-3-nitrophenyl)acetyl-cholera toxin (NP-CT) conjugates were followed at the cellular and molecular level. Surprisingly, these responses were highly synchronized, strongly oligoclonal, and dominated by affinity matured cells. Extensive lineage trees revealed clonal relationships between NP-specific IgA cells in gut inductive and effector sites, suggesting expansion of the same B-cell clone in multiple Peyer's patches (PPs). Adoptive transfer experiments showed that this was achieved through re-utilization of already existing germinal centers (GCs) in multiple PPs by previously activated GC GL7(+) B cells, provided oral NP-CT was given before cell transfer. Taken together, these results explain why repeated oral immunizations are mandatory for an effective oral vaccine.
Assuntos
Afinidade de Anticorpos/imunologia , Trato Gastrointestinal/imunologia , Centro Germinativo/imunologia , Imunoglobulina A/imunologia , Nódulos Linfáticos Agregados/imunologia , Administração Oral , Transferência Adotiva , Animais , Antígenos/administração & dosagem , Antígenos/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Toxina da Cólera/imunologia , Trato Gastrointestinal/metabolismo , Ordem dos Genes , Centro Germinativo/metabolismo , Imunização , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Camundongos , Modelos Imunológicos , Nitrofenóis/imunologia , Nódulos Linfáticos Agregados/metabolismo , Fenilacetatos/imunologia , Plasmócitos/imunologia , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologiaRESUMO
The ultimate goal for vaccination is to stimulate protective immunological memory. Protection against infectious diseases not only relies on the magnitude of the humoral immune response, but more importantly on the quality and longevity of it. Adjuvants are critical components of most non-living vaccines. Although little attention has been given to qualitative aspects of the choice of vaccine adjuvant, emerging data demonstrate that this function may be central to vaccine efficacy. In this review we describe efforts to understand more about how adjuvants influence qualitative aspects of memory development. We describe recent advances in understanding how vaccines induce long-lived plasma and memory B cells, and focus our presentation on the germinal center reaction. As mucosal vaccination requires powerful adjuvants, we have devoted much attention to the adenosine diphosphate (ADP)-ribosylating cholera toxin and the CTA1-DD adjuvants as examples of how mucosal adjuvants can influence induction of long-term memory.
Assuntos
Adjuvantes Imunológicos , Linfócitos B/imunologia , Toxinas Bacterianas/imunologia , Toxina da Cólera/imunologia , Centro Germinativo/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , ADP-Ribose Cíclica/metabolismo , Humanos , Imunidade nas Mucosas , Memória Imunológica , VacinaçãoRESUMO
OBJECTIVE: Serum is usually added to growth media when mammalian cells are cultured in vitro to supply the cells with growth factors, hormones, nutrients and trace elements. Defined proteins and metal ions, such as insulin, growth factors, transferrin and sodium selenite, are sometimes also included and can in some cases substitute serum components. How adaptation to serum free media influences cells has not been studied in detail. MATERIALS AND METHODS: We have adapted the Burkitt's lymphoma line Ramos to a serum-free medium that supports long-term survival and studied gene expression changes that occurred during the adaptation process. RESULTS AND CONCLUSIONS: The adaptation process was characterized by initial cell population growth arrest, and after that extensive cell death, followed by proliferation and long-term survival of clonal cultures. Proliferation and cell cycle progression of the serum-free cultures closely mimicked that of serum-dependent cells. Affymetrix micro-array technology was used to identify gene expression alterations that had occurred during the adaptation. Most changes were subtle, but frequently the genes with altered expression were involved in basal cellular functions such as cell division, cell cycle regulation, apoptosis and cell signalling. Some alterations were restored when the cells were transferred back to serum-containing medium, indicating that expression of these genes was controlled by components in serum. Others were not, and may represent changes that were selected during the adaptation process. Among these were, for example, several genes within the Wnt signalling pathway.
Assuntos
Linfoma de Burkitt/genética , Regulação Neoplásica da Expressão Gênica , Northern Blotting , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Proliferação de Células , Meios de Cultura Livres de Soro , Humanos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Immunoglobulin kappa promoters show sequence divergence but conserved function between different subgroups. Here we show that three separate 5' elements are required for synergistic stimulation of transcription with the decamer in a kappa promoter. These sites are a 5' E-box, a 3' AT-rich region in the pentadecamer (pd) element, and the kappa-Y element. Elf-1 is a novel kappa-Y element ligand induced upon mitogenic stimulation of resting B lymphocytes. Furthermore, the 5' E2A-like E-box in the pd element could be substituted by an upstream stimulatory factor motif with conservation of function. Thus, the synergistic activation requirements of kappa transcription is strictly dependent on the quantitative presence of transcription factor-binding motifs 5' of the decamer, but these differ qualitatively in that they may bind an array of proteins with conserved function.
Assuntos
Cadeias kappa de Imunoglobulina/genética , Regiões Promotoras Genéticas/imunologia , Fatores de Transcrição/fisiologia , Ativação Transcricional/imunologia , Animais , Linfócitos B/imunologia , Sequência de Bases , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida , Efrina-A2 , Interfase/imunologia , Ligantes , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fator 2 de Transcrição de Octâmero , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ets , Transativadores/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The intact SP6 kappa promoter stimulated transcription 30 times more efficiently than did a control promoter consisting of a TATA motif as the only promoter element. Mutation of the SP6 kappa promoter decamer in two positions reduced the transcriptional stimulation activity by over 90%. Promoters containing the SP6 kappa promoter octamer or a consensus octamer in front of a TATA box were ineffective immunoglobulin promoters and stimulated at the most 15% of maximal transcription. Identical results were obtained after transfection of untransformed mouse splenic B cells stimulated by lipopolysaccharide, that express high levels of Oct2A, or of S194 cells that express negligible levels of Oct2A. Selective mutations in the penta-decamer (pd), kappa Y or early B cell factor (EBF) elements of the promoter reduced transcriptional stimulation by 20-30% in untransformed B cells. In S194 plasmacytoma cells the EBF mutation was functionally silent while the kappa Y and pd mutations reduced transcriptional activation by 60-70% in this cell line. A mutation in a TATA-proximal E-box motif did not alter the functional activity of the promoter in either cell population. It can be concluded that kappa promoter function is highly dependent on complex interactions between individual promoter elements and that the decamer motif is pivotal for these interactions. The relative functional activity of a given promoter varied according to the target cell population used for the functional assay.
Assuntos
Cadeias kappa de Imunoglobulina/genética , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genética , Animais , Sequência de Bases , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Lipopolissacarídeos/farmacologia , Camundongos , Dados de Sequência Molecular , Transfecção/genéticaRESUMO
Promoter region sequences of human and mouse Igk-V genes were aligned and found to be conserved for about 200-300 base pairs (bp) within subgroups/families. No promoter similarity was found between IGKV promoters from different human subgroups. Related mouse Igk-V gene families were conserved in the promoter region but no similarity was evident when promoters from unrelated Igk-V gene families were compared. Most of the human IGKV promoter subgroups were shown to have mouse counterparts with a similarity region that extended about 150 bp upstream of the translational start codon. All promoters contained an octamer sequence element. The consensus octamer/decamer sequence was favored but only seven residues within the octamer element were strictly conserved. Furthermore, there was also sequence conservation immediately 3' of the octamer where either an A or a G residue was conserved. In addition, other DNA elements were also conserved both within the Igk-V subgroups/families and between mouse and human promoters from related subgroups/families. In several of the subgroups/families an E box of the E2A type was conserved 5' of the octamer and a CCCT element was conserved within the IGKV subgroup II and its related mouse Igk-V families. We conclude from this study that conservation of additional sequence elements besides the octamer is a common feature in Igk-V promoters but that distinct elements are conserved only within a given subgroup/family. Thus, the conservation appears to have operated at the level of function rather than at the level of recognition sequence for defined transcription factors.
Assuntos
Sequência Conservada , Genes de Imunoglobulinas , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Sequência de Bases , Mapeamento Cromossômico , Sequência Consenso , Humanos , Camundongos , Dados de Sequência Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
A protein interacting with an A-T-rich region that is a positive control element within the SP6 kappa promoter was purified and identified as CArG-box binding factor-A. The purified protein was shown to interact specifically with the coding strand of single-stranded DNA and, with lower affinity, with double-stranded DNA. A mutation that inhibited binding of the protein to the A-T-rich region also aborted the transcriptional stimulatory effect of the region. Two Ets proteins, PU.1 and elf-1, that have previously been shown to bind to an adjacent DNA element were shown to physically interact with CArG-box binding factor-A. An antiserum raised against the protein recognized two different forms indicating either that different splice-forms of CArG-box binding factor-A are expressed, or that the protein is subject to post-translational modification.
Assuntos
Proteínas de Ligação a DNA/isolamento & purificação , RNA Polimerases Dirigidas por DNA/metabolismo , Cadeias kappa de Imunoglobulina/genética , Regiões Promotoras Genéticas , Proteínas Repressoras/isolamento & purificação , Processamento Alternativo , Animais , Sequência de Bases , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
A novel Ets protein was isolated by yeast one-hybrid screening of a cDNA library made from lipopolysaccharide-stimulated mouse splenic B cells, using the SP6 kappa promoter kappaY element as a bait. The novel Ets protein was most closely related to PU.1 and Spi-B within the DNA binding Ets domain and was therefore named Spi-C. However, Spi-C may represent a novel subgroup within the Ets protein family, as it differed significantly from Spi-B and PU.1 within helix 1 of the Ets domain. Spi-C was encoded by a single-copy gene that was mapped to chromosome 10, region C. Spi-C interacted with DNA similarly to PU.1 as judged by methylation interference, band-shift and site selection analysis, and activated transcription of a kappaY element reporter gene upon co-transfection of HeLa cells. Spi-C RNA was expressed in mature B lymphocytes and at lower levels in macrophages. Furthermore, pre-B cell and plasma cell lines were Spi-C-negative, suggesting that Spi-C might be a regulatory molecule during a specific phase of B lymphoid development.
Assuntos
Linfócitos B/citologia , RNA Polimerases Dirigidas por DNA/metabolismo , Cadeias kappa de Imunoglobulina/genética , Regiões Promotoras Genéticas , Transativadores/química , Transativadores/genética , Sequência de Aminoácidos , Animais , Linfócitos B/metabolismo , Diferenciação Celular , Clonagem Molecular , DNA/metabolismo , Regulação para Baixo , Genes Reporter , Células HeLa , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/metabolismo , Baço/citologia , Baço/efeitos dos fármacos , Transativadores/isolamento & purificação , Transativadores/metabolismo , Ativação TranscricionalRESUMO
By mutational analysis of the octamer-TATA box intervening region in the mouse SP6 kappa promoter, we have mapped two octamer-dependent, costimulatory regions, A and B. The A region was active in late B cells only, while the B region was active throughout B cell differentiation. The B region was TATA proximal and contained a heptamer and an E box of the E2A type that is common in Vkappa promoters. Mutation of the heptamer element did not decrease transcriptional stimulation from this region, but mutations in, or immediately 5' of, the E box core sequence did. A protein binding to this region could be detected in nuclear extracts. The complex could only partially be competed with a muE5 binding site and could not be supershifted with Abs raised to E2A gene products, indicating that it may represent a novel E-box binding complex. The A region was located proximal to the octamer and contained a CCCT element that is conserved both with regard to position and sequence in human VkappaII promoters. By mutational analysis, the transcriptional stimulatory activity was mapped to the CCCT element that also is part of an early B cell factor (EBF) binding site. In late B cells, a novel protein (FA), which did not bind to the EBF binding site in the mb1 promoter, interacted with the A region. This protein was found to be expressed at lower levels in early B cells as well as in HeLa cells. Thus, the octamer-flanking sequence contains positive control elements that may act independently but that differ in the stage of B cell differentiation at which they are active. One of these factors is an example of an ubiquitously expressed transcription factor that participate in differentiation-specific transcriptional activation.
Assuntos
Linfócitos B/imunologia , Genes de Imunoglobulinas , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Diferenciação Celular , Linhagem Celular , DNA/genética , DNA/metabolismo , Humanos , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , TATA Box , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The GAGA box of the rat serine protease inhibitor 2 (spi 2) genes not only acts as a basal promoter element, but also mediates transcriptional activation by growth hormone and interleukin-6. The GAGA box is separated from the TATA box by only 12 bp, and this close association is required for efficient transcription. Hence, the GAGA box may influence transcription efficiency through interactions between GAGA box binding proteins and some components of the RNA polymerase II complex. Here we report the cloning of two GAGA box-binding proteins termed p38 and p40, that belong to the type A/B heterogeneous nuclear ribonucleoprotein subgroup. GAGA box mutations that diminish the affinity for p38 and p40 decrease basal and GH-induced reporter gene expression. Furthermore, nuclear extracts depleted of p38 and p40 can no longer support GAGA box-dependent in vitro transcription. Therefore, two polypeptides previously assigned to a family of RNA processing proteins also act as DNA-binding, promoter-specific transcription factors.
Assuntos
Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Ribonucleoproteínas/genética , Serpinas , Ativação Transcricional , Sequência de Aminoácidos , Animais , Fator de Ligação a CCAAT/genética , Sistema Livre de Células , Clonagem Molecular , Elementos Facilitadores Genéticos , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Fígado/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/genética , Ratos , Ribonucleoproteínas/isolamento & purificação , Ribonucleoproteínas/metabolismo , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Following antigen encounter, two distinct processes modify immunoglobulin genes. The variable region is diversified by somatic hypermutation while the constant region may be changed by class-switch recombination. Although both genetic events can occur concurrently within germinal centre B cells, there are examples of each occurring independently of the other. Here we compare the contributions of class-switch recombination and somatic hypermutation to the diversification of the serum immunoglobulin repertoire and review evidence that suggests that, despite clear differences, the two processes may share some aspects of their mechanism in common.