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1.
Scand J Immunol ; 57(5): 446-52, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12753501

RESUMEN

The development of T cells is thought to be independent of B cells. However, defects in cell-mediated immunity in individuals with B-cell deficiency suggest the contrary. To test whether B cells affect T-lymphocyte development, we constructed mice with a monoclonal T-cell compartment (MT) and monoclonal B- and T-cell compartments (MBTs). In these mice, the T cells expressed a DO 11.10 transgenic (DO-T) cell receptor restricted to major histocompatibility complex (MHC) class IId. While CD4+ DO-T lymphocytes are rare in transgenic H-2b MT mice, we found that in H-2b MBT mice under the influence of B cells, DO-T lymphocytes mature into large numbers of CD4+ peripheral T cells. H-2b MBT mice have more CD4+ thymocytes than H-2b MT mice. These data are consistent with the view that B cells play some role in thymocyte development.


Asunto(s)
Linfocitos B/inmunología , Linfocitos T/inmunología , Animales , Subgrupos de Linfocitos B/inmunología , Recuento de Linfocito CD4 , Supresión Clonal/inmunología , Antígenos H-2/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunidad Celular , Cadenas lambda de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/inmunología , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/patología , Inmunofenotipificación , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Inmunológicos , Ovalbúmina/inmunología , Bazo/citología , Bazo/inmunología , Linfocitos T/citología
2.
Eur J Immunol ; 31(8): 2467-75, 2001 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-11500831

RESUMEN

Protein SWAP-70 was initially isolated from nuclei of activated B cells and was implicated in the immunoglobulin class switch process. After B cell activation the protein translocates from the cytoplasm to the nucleus, and may serve to signal nuclear processes. We have generated mice deficient in SWAP-70 and found three main differences when compared to wild-type mice: (i) their B lymphocytes are two- to threefold more sensitive to gamma-irradiation than B cells of wild type; (ii) SWAP-70-deficient mice developed autoantibodies at a much higher frequency; and (iii) the CD40 signaling pathway is compromised in the mutant mice. CD40-dependent switching to the IgE isotype is reduced five- to eightfold in vitro. In SWAP-70-deficient mice, IgE levels prior to immunization were six- to sevenfold lower than in wild-type mice, and after immunization three- to fourfold lower. CD40-induced proliferation was transiently increased in the mutant. LPS-induced switching to other isotypes, however, and LPS-induced proliferation were normal. We propose that SWAP-70 serves a specific role in the CD40 signaling pathway, in particular in the IgE response.


Asunto(s)
Autoanticuerpos/inmunología , Linfocitos B/inmunología , Antígenos CD40/inmunología , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/fisiología , Factores de Intercambio de Guanina Nucleótido , Inmunoglobulina E/inmunología , Proteínas Nucleares/deficiencia , Proteínas Nucleares/fisiología , Animales , Apoptosis , Autoanticuerpos/biosíntesis , Linfocitos B/citología , Linfocitos B/metabolismo , Linfocitos B/efectos de la radiación , División Celular , Proteínas de Unión al ADN/genética , Rayos gamma , Marcación de Gen , Cambio de Clase de Inmunoglobulina/genética , Cambio de Clase de Inmunoglobulina/inmunología , Inmunoglobulina E/biosíntesis , Inmunoglobulina E/genética , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Lipopolisacáridos/inmunología , Activación de Linfocitos , Ratones , Ratones Noqueados , Antígenos de Histocompatibilidad Menor , Mutación/genética , Proteínas Nucleares/genética , Tolerancia a Radiación , Transducción de Señal
3.
J Immunol ; 166(2): 855-60, 2001 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-11145660

RESUMEN

The quasi-monoclonal (QM) mouse has a functionally rearranged H chain gene inserted into its natural position in the IgH locus. In this position, the H chain gene is subject to many of the same activities as normally arranged H chain genes, including somatic hypermutation, V(H) gene replacement, and class switch recombination. Here, we have used this mouse strain to determine some of the rules that govern the V(D)J recombination activity of the IgH locus in thymus. We focused on the requirements for V(H) gene replacement. In normal mice, thymic DJ(H) rearrangements are common, but VDJ(H) rearrangements are not. We found intermediate products of V(H) replacement in double-positive CD4(+)CD8(+) cells of the QM thymus, demonstrating that the inserted V(H) gene was accessible and ruling out the possibility that a V(H) gene per se cannot be rearranged in the thymus. We found transcripts from the knocked-in H chain gene of QM, but no mu H chain protein was detectable in thymocytes. Cloning and sequencing of these transcripts revealed that some had been generated by V(H) gene replacement. Corresponding signal joints could also be identified. These results suggest that neither a B cell-specific signal nor an Ig protein are necessary to activate V(H)-to-VDJ(H) joining in thymocytes. Possible mechanisms remaining to account for overcoming the barrier to V(H) joining in thymocytes include the insertion of a transcriptionally active gene segment and/or the inactivation of a silencer.


Asunto(s)
Reordenamiento Génico de Linfocito T , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Timo/inmunología , Timo/metabolismo , Animales , Secuencia de Bases , Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Cadenas Pesadas de Inmunoglobulina/aislamiento & purificación , Región Variable de Inmunoglobulina/aislamiento & purificación , Cadenas mu de Inmunoglobulina/análisis , Cadenas mu de Inmunoglobulina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Recombinación Genética , Timo/citología , Transcripción Genética/inmunología
4.
Eur J Immunol ; 30(11): 3121-31, 2000 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11093126

RESUMEN

This study tracks the fate of antigen-reactive B cells through follicular and extrafollicular responses and addresses the function of CD40 in these processes. The unique feature of this system is the use of transgenic B cells in which the heavy chain locus has been altered by site-directed insertion of a rearranged V(H) DJ(H) exon such that they are able to clonally expand, isotype-switch and follow a normal course of differentiation upon immunization. These Ig transgenic B cells when adoptively transferred into non-transgenic (Tg) mice in measured amounts expanded and differentiated distinctively in response to T cell-independent (TI) or T cell-dependent (TD) antigens. The capacity of these Tg B cells to faithfully recapitulate the humoral immune response to TI and TD antigens provides the means to track clonal B cell behavior in vivo. Challenge with TI antigen in the presence of agonistic anti-CD40 mAb resulted in well-defined alterations of the TI response. In vivo triggering of Tg B cells with TI antigen and CD40 caused an increase in the levels IgG produced and a broadening of the Ig isotype profile, characteristics which partially mimic TD responses. Although some TD characteristics were induced by TI antigen and CD40 triggering, the Tg B cells failed to acquire a germinal center phenotype and failed to generate a memory response. Therefore, TD-like immunity can be only partially reconstituted with CD40 agonists and TI antigens, suggesting that there are additional signals required for germinal center formation and development of memory.


Asunto(s)
Formación de Anticuerpos , Linfocitos B/inmunología , Antígenos CD40/inmunología , Genes de Inmunoglobulinas , Traslado Adoptivo , Animales , Cambio de Clase de Inmunoglobulina , Ratones , Ratones Transgénicos , Linfocitos T/inmunología
5.
Eur J Immunol ; 30(8): 2404-11, 2000 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-10940932

RESUMEN

V(H)-gene replacement is a recombination event in which a pre-existing immunoglobulin heavy chain gene can be altered by the replacement of the rearranged V(H) gene segment with another V(H) gene segment. Although this event has been demonstrated in various model systems, its role in generating antibody diversity is still unsettled. We have used a genetically modified mouse strain, QM, with a quasi monoclonal primary B cell repertoire specific for NP to determine whether V(H) gene replacement can generate a new antigen specificity. Hybridomas generated from QM splenocytes after immunization with different antigens, gave rise to antibodies with specificity to the immunizing antigen or with new specificities. We found V(H)-gene replacement was used to change the original heavy chain gene rearrangement specific for NP into a heavy chain gene encoding the new antigen specificity. V(H)-gene replacement intermediates were detected both before and after the immunization, suggesting that the event was selective rather than instructive. These results demonstrate that V(H)-gene replacement can generate a new antibody heavy chain gene with a different functional and selectable antigen specificity.


Asunto(s)
Formación de Anticuerpos , Especificidad de Anticuerpos , Reordenamiento Génico , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Animales , Secuencia de Bases , Eritrocitos/inmunología , Hemocianinas/inmunología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Ovinos
6.
Immunogenetics ; 51(1): 16-9, 2000 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-10663557

RESUMEN

The protein SWAP-70 was isolated as part of a DNA recombination complex in B lymphocytes, where it is predominantly expressed. In resting B cells, SWAP-70 is found in the cytoplasm; upon B-cell activation, it is transported both into the nucleus and to the cell membrane, where it is associated with the B-cell receptor complex and may play a role in signal transduction. In the nucleus, its involvement in heavy-chain class switch recombination has been suggested. In this report, using restriction fragment length polymorphism, simple sequence length polymorphism, and fluorescence in situ hybridization, we map the chromosomal localization of the mouse and the human genes to syntenic regions of mouse mid Chromosome (Chr) 7 and human Chr 11p15.


Asunto(s)
Cromosomas Humanos Par 11/genética , Proteínas de Unión al ADN/genética , Factores de Intercambio de Guanina Nucleótido , Proteínas Nucleares/genética , Mapeo Físico de Cromosoma , Animales , Haplotipos , Humanos , Hibridación Fluorescente in Situ , Ratones , Ratones Endogámicos BALB C , Repeticiones de Microsatélite , Antígenos de Histocompatibilidad Menor , Polimorfismo de Longitud del Fragmento de Restricción
7.
J Exp Med ; 191(3): 485-94, 2000 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-10662794

RESUMEN

Germinal centers are critical for affinity maturation of antibody (Ab) responses. This process allows the production of high-efficiency neutralizing Ab that protects against virus infection and bacterial exotoxins. In germinal centers, responding B cells selectively mutate the genes that encode their receptors for antigen. This process can change Ab affinity and specificity. The mutated cells that produce high-affinity Ab are selected to become Ab-forming or memory B cells, whereas cells that have lost affinity or acquired autoreactivity are eliminated. Normally, T cells are critical for germinal center formation and subsequent B cell selection. Both processes involve engagement of CD40 on B cells by T cells. This report describes how high-affinity B cells can be induced to form large germinal centers in response to (4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll in the absence of T cells or signaling through CD40 or CD28. This requires extensive cross-linking of the B cell receptors, and a frequency of antigen-specific B cells of at least 1 in 1,000. These germinal centers abort dramatically at the time when mutated high-affinity B cells are normally selected by T cells. Thus, there is a fail-safe mechanism against autoreactivity, even in the event of thymus-independent germinal center formation.


Asunto(s)
Linfocitos B/fisiología , Centro Germinal/fisiología , Linfocitos T/fisiología , Animales , Antígenos CD/análisis , Antígeno B7-1/análisis , Antígeno B7-2 , Antígenos CD40/análisis , Ficoll , Haptenos , Glicoproteínas de Membrana/análisis , Ratones , Ratones Desnudos , Ratones Transgénicos , Nitrofenoles , Aglutinina de Mani , Fenilacetatos , Transducción de Señal , Bazo/efectos de los fármacos
8.
Proc Natl Acad Sci U S A ; 97(5): 2180-4, 2000 Feb 29.
Artículo en Inglés | MEDLINE | ID: mdl-10681448

RESUMEN

SWAP-70 is a component of an enzyme complex that recombines Ig switch regions in vitro. We report here the cloning of the human cDNA and its B lymphocyte-specific expression. Although its sequence contains three nuclear localization signals, in small resting B cells, SWAP-70 is mainly found in the cytoplasm. On stimulation, SWAP-70 translocates to the nucleus. In activated, class-switching B cell cultures, it is associated with membrane IgG, but not IgM. The membrane Ig association requires a functional pleckstrin homology domain and is controlled by the C terminus. We suggest that SWAP-70 is involved not only in nuclear events but also in signaling in B cell activation.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factores de Intercambio de Guanina Nucleótido , Proteínas Nucleares/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Antígenos CD40/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Mutagénesis , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Conejos , Distribución Tisular , Células Tumorales Cultivadas
9.
Int Immunol ; 12(1): 29-35, 2000 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-10607747

RESUMEN

We describe a new population of non-naive B cells in the peripheral blood of quasimonoclonal (QM) mice. Surface Ig of switched isotypes is expressed, but not B220 nor CD19. These cells are larger and denser than naive B cells but smaller than blasts or plasma cells; they do not stain with syndecan, a marker for plasma cells. Telomerase, which is usually expressed in B cell blasts, was not present in this population. We sorted the switched, idiotype-positive, B220(-) B cells from the peripheral blood of QM mice and sequenced Ig H chain and lambda L chain cDNA. There were many point mutations but no V gene replacements, gene conversions or other type of diversifications. As they express switched isotypes and have mutated their Ig genes, cells in the B220(-), CD19(-) population must have been in an immune response and we suggest that it includes the memory B cell subset.


Asunto(s)
Antígenos CD19/análisis , Subgrupos de Linfocitos B/inmunología , Cambio de Clase de Inmunoglobulina/genética , Antígenos Comunes de Leucocito/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Genes de Inmunoglobulinas , Ingeniería Genética , Células Madre Hematopoyéticas , Cadenas Pesadas de Inmunoglobulina/genética , Idiotipos de Inmunoglobulinas , Cadenas gamma de Inmunoglobulina/genética , Glicoproteínas de Membrana , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Peritoneo/citología , Proteoglicanos , Receptores de Antígenos de Linfocitos B , Sindecanos , Telomerasa
10.
Eur J Immunol ; 29(11): 3712-21, 1999 11.
Artículo en Inglés | MEDLINE | ID: mdl-10556827

RESUMEN

A subset of myeloid dendritic cells is described which is associated with the ability of splenic and lymph node plasmablasts to survive and differentiate into plasma cells. Plasmablast-associated dendritic cells (PDC) are CD11c(high), DEC-205(-) and unlike conventional dendritic cells do not associate with T cells. The following findings suggest a requirement for PDC if plasmablasts are to differentiate to plasma cells. First, when large numbers of B cells are recruited into antibody responses and plasmablasts outgrow the PDC stroma, only those associated with PDC survive and differentiate into plasma cells. Conversely, if the number of PDC is increased by ligating their CD40, more plasmablasts survive on the expanded PDC stroma and differentiate into plasma cells. Finally, in T cell-deficient mice, the plasma cells that develop atypically in the T zones in response to thymus-independent antigens are associated with ectopic PDC.


Asunto(s)
Apoptosis/inmunología , Células Dendríticas/inmunología , Bazo/citología , Animales , Diferenciación Celular , División Celular , Supervivencia Celular , Integrina alfaXbeta2/inmunología , Ganglios Linfáticos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL
11.
Eur J Immunol ; 29(6): 1812-22, 1999 06.
Artículo en Inglés | MEDLINE | ID: mdl-10382743

RESUMEN

SWAP-70 is part of a protein complex that catalyzes cell-free DNA recombination between immunoglobulin heavy chain gene switch region substrates. This report studies the expression pattern of SWAP-70 in mouse tissues, sorted cells, and cultured primary cells. SWAP-70 RNA is strongly increased upon switch-induction of spleen cells, and very weakly expressed in thymus and bone marrow. SWAP-70 protein is specifically expressed in B cells, and levels increase rapidly after stimulation. Tissue staining shows strong expression in germinal center B cells, while macrophages and T lymphocytes do not stain. SWAP-70 is not detected in early B cells in the bone marrow. Its expression during mouse ontogeny after birth correlates with the appearance of non-IgM isotypes. While SWAP-70 localizes to the cell nucleus in activated B cells, it is not tightly associated with the chromatin and is found in the cytoplasm as well. SWAP-70 expression is not increased by gamma or UV irradiation of spleen cells, nor does it depend on p53. These characteristics are consistent with the putative role of SWAP-70 in immunoglobulin class switching.


Asunto(s)
Proteínas de Unión al ADN/genética , Factores de Intercambio de Guanina Nucleótido , Proteínas Nucleares/genética , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Diferenciación Celular , Células Cultivadas , Proteínas de Unión al ADN/biosíntesis , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos C57BL , Antígenos de Histocompatibilidad Menor , Proteínas Nucleares/biosíntesis , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Recombinación Genética , Fracciones Subcelulares/metabolismo , Distribución Tisular
12.
J Exp Med ; 189(11): 1791-8, 1999 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-10359583

RESUMEN

Variable (V) region gene replacement was recently implicated in B cell repertoire diversification, but the contribution of this mechanism to antibody responses is still unknown. To investigate the role of V gene replacements in the generation of antigen-specific antibodies, we analyzed antiviral immunoglobulin responses of "quasimonoclonal" (QM) mice. The B cells of QM mice are genetically committed to exclusively express the anti-(4-hydroxy-3-nitrophenyl) acetyl specificity. However, approximately 20% of the peripheral B cells of QM mice undergo secondary rearrangements and thereby potentially acquire new specificities. QM mice infected with vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus, or poliovirus mounted virus-specific neutralizing antibody responses. In general, kinetics of the antiviral immunoglobulin responses were delayed in QM mice; however, titers similar to control animals were eventually produced that were sufficient to protect against VSV-induced lethal disease. VSV neutralizing single-chain Fv fragments isolated from phage display libraries constructed from QM mice showed VH gene replacements and extensive hypermutation. Thus, our data demonstrate that secondary rearrangements and hypermutation can generate sufficient B cell diversity in QM mice to mount protective antiviral antibody responses, suggesting that these mechanisms might also contribute to the diversification of the B cell repertoire of normal mice.


Asunto(s)
Diversidad de Anticuerpos , Linfocitos B/inmunología , Reordenamiento Génico de Linfocito B , Mutación , Animales , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/genética , Secuencia de Bases , ADN/genética , Marcación de Gen , Ratones , Ratones Endogámicos C57BL , Pruebas de Neutralización , Homología de Secuencia de Ácido Nucleico , Virus de la Estomatitis Vesicular Indiana/inmunología
13.
Curr Opin Immunol ; 11(2): 186-9, 1999 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-10322147

RESUMEN

By studying the role of mismatch repair in hypermutation at the immunoglobulin loci, the field of antibody hypermutation has been integrated into the larger area of DNA repair. Trans-acting factors - Ku70, Ku80 and possibly SWAP-70 - have been identified for the temporally related but not mechanistically related immunoglobulin heavy-chain class-switch.


Asunto(s)
Afinidad de Anticuerpos , Reparación del ADN , Genes de Inmunoglobulinas , Mutación , Animales , Humanos , Cambio de Clase de Inmunoglobulina , Cadenas Pesadas de Inmunoglobulina/genética
14.
Dev Immunol ; 7(1): 43-50, 1999.
Artículo en Inglés | MEDLINE | ID: mdl-10636478

RESUMEN

We have generated a monoclonal B-cell mouse by introducing homozygous, nonfunctional RAG-2 alleles and a lambda1 light-chain transgene into the quasi-monoclonal (QM) mouse, which contains a "knocked-in" V(H)DJ(H) rearrangement. Thus, this mouse, which we call MonoB, is devoid of T cells and contains preformed heavy- and light-chain genes encoding immunoglobulin with an anti-NP specificity. The MonoB mouse allows us to examine immunoglobulin diversity in the absence of processes mediated by V(D)J recombination and T cells. Here we report that not only is the MonoB's primary immunoglobulin repertoire monoclonal, but also that its secondary repertoire is not further diversified by V-gene replacement or gene conversion. Among 99 heavy-chain and 41 lambda light-chain genes from peripheral B cells of the MonoB mouse, there were no V-gene replacements. When compared to the QM mouse, which has RAG activity, and for which V-gene replacement is the major diversifying mechanism, these data suggest that V-gene replacement is mediated by V(D)J recombination and not by other recombination systems.


Asunto(s)
Linfocitos B/inmunología , Reordenamiento Génico de Linfocito B , Isotipos de Inmunoglobulinas/genética , Isotipos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/genética , Animales , Variación Genética , Ratones , Ratones Noqueados , Ratones Transgénicos
15.
J Biol Chem ; 273(27): 17025-35, 1998 Jul 03.
Artículo en Inglés | MEDLINE | ID: mdl-9642267

RESUMEN

We have purified and biochemically characterized a multiprotein complex designated SWAP. In a DNA transfer assay, SWAP preferentially recombines ("swaps") sequences derived from Ig heavy chain switch regions. We identified four of the proteins in the SWAP complex: B23 (nucleophosmin), C23 (nucleolin), poly(ADP-ribose) polymerase (PARP), and SWAP-70. The first three are proteins known to be present in most cells. B23 promotes single-strand DNA reannealing and the formation of joint molecules in a D-loop assay between homologous, but also between Smu and Sgamma sequences. SWAP-70 is a novel protein of 70 kDa. Its cDNA was cloned and sequenced, and the protein was overexpressed in Escherichia coli. SWAP-70 protein expression was found only in B lymphocytes that had been induced to switch to various Ig isotypes and in switching B-cell lines. SWAP-70 is a nuclear protein, has a weak affinity for DNA, binds ATP, and forms specific, high affinity complexes with B23, C23, and poly(ADP-ribose) polymerase. These findings are consistent with SWAP being the long elusive "switch recombinase" and with SWAP-70 being the specific recruiting element that assembles the switch recombinase from universal components.


Asunto(s)
Linfocitos B/metabolismo , ADN Recombinante/genética , Proteínas de Unión al ADN/genética , Factores de Intercambio de Guanina Nucleótido , Proteínas Nucleares/genética , Recombinación Genética , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Calor , Cinética , Ratones , Ratones Endogámicos C57BL , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Nucleofosmina , Fosfoproteínas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Unión Proteica
16.
Proc Natl Acad Sci U S A ; 95(5): 2396-9, 1998 Mar 03.
Artículo en Inglés | MEDLINE | ID: mdl-9482896

RESUMEN

Hypermutation at the Ig loci is confined to the area between the promoter and the intronic enhancer, which includes the rearranged variable region gene segment. We identified factors that contribute to the site-specificity at the heavy chain locus. We found that distance from both the promoter and the intronic enhancer is crucial in hypermutation. The presence of the enhancer is required, and, in contrast to its definition for transcriptional activity, its effect is orientation-sensitive.


Asunto(s)
Elementos de Facilitación Genéticos , Genes de Inmunoglobulinas , Intrones , Animales , Composición de Base , Secuencia de Bases , Línea Celular , Citosina , Guanina , Humanos , Ratones , Mutagénesis , Mapeo Restrictivo , Transfección
17.
Science ; 279(5354): 1207-10, 1998 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-9469811

RESUMEN

Mice homozygous for a disrupted allele of the mismatch repair gene Pms2 have a mutator phenotype. When this allele is crossed into quasi-monoclonal (QM) mice, which have a very limited B cell repertoire, homozygotes have fewer somatic mutations at the immunoglobulin heavy chain and lambda chain loci than do heterozygotes or wild-type QM mice. That is, mismatch repair seems to contribute to somatic hypermutation rather than stifling it. It is suggested that at immunoglobulin loci in hypermutable B cells, mismatched base pairs are "corrected" according to the newly synthesized DNA strand, thereby fixing incipient mutations instead of eliminating them.


Asunto(s)
Adenosina Trifosfatasas , Enzimas Reparadoras del ADN , Reparación del ADN , Proteínas de Unión al ADN , Genes de Inmunoglobulinas , Región Variable de Inmunoglobulina/genética , Mutación , Proteínas/genética , Alelos , Secuencia de Aminoácidos , Animales , Linfocitos B/inmunología , Composición de Base , Secuencia de Bases , Clonación Molecular , Cruzamientos Genéticos , Femenino , Reordenamiento Génico , Heterocigoto , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/química , Cadenas lambda de Inmunoglobulina/química , Cadenas lambda de Inmunoglobulina/genética , Masculino , Ratones , Ratones Noqueados , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto , Datos de Secuencia Molecular , Proteínas/fisiología
18.
Biol Chem ; 378(6): 559-63, 1997 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-9224938

RESUMEN

The Na,K-ATPase, or sodium pump, a ubiquitous transmembrane enzyme in higher eukaryotes, consists of an alpha and a beta subunit. Here we investigate the expression pattern of the two beta isotypes in mouse B cell lines. Neither primary cells nor cell lines express beta 2. Abelson virus-transformed pre-B cells express beta 1, while B lymphomas and plasmacytomas do not. Thus, beta 1 expression in transformed cells follows that of their untransformed counterparts. Some subclones of pre-B cell line 70Z/3 express beta 1, and others do not, but lipopolysaccharide induces the beta 1-negative cells to become beta 1-positive.


Asunto(s)
Linfocitos B/enzimología , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Virus de la Leucemia Murina de Abelson , Animales , Linfocitos B/efectos de los fármacos , Encéfalo/citología , Línea Celular , Transformación Celular Viral , Inducción Enzimática , Regulación de la Expresión Génica , Isoenzimas/biosíntesis , Isoenzimas/genética , Linfoma de Células B/enzimología , Linfoma de Células B/genética , Ratones , Datos de Secuencia Molecular , Plasmacitoma/enzimología , Plasmacitoma/genética , ATPasa Intercambiadora de Sodio-Potasio/genética
19.
Dev Immunol ; 5(2): 105-14, 1997.
Artículo en Inglés | MEDLINE | ID: mdl-9587710

RESUMEN

The Abelson-virus-transformed mouse pre-B-cell line 18-81 switches almost exclusively from mu to gamma 2b. From nuclear extracts of this cell line, we have isolated a factor that specifically binds to S gamma 2b. After an eight-step purification scheme, in which different types of DNA-affinity chromatography were used as key elements, we obtained a preparation with two narrowly spaced bands at approximately 69 kD on a silver-stained SDS gel. Binding specificity of main-peak fractions of affinity-purified proteins was analyzed by gel shift assays, in which S gamma 2b, but not S mu, competes. The results are consistent with this factor being part of the switch recombinase.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Inmunoglobulina G/genética , Región de Cambio de la Inmunoglobulina , Animales , Especificidad de Anticuerpos , Autorradiografía , Secuencia de Bases , Línea Celular Transformada , Transformación Celular Viral , Cromatografía de Afinidad , Huella de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/aislamiento & purificación , Ratones , Datos de Secuencia Molecular , Peso Molecular , Recombinación Genética , Secuencias Repetitivas de Ácidos Nucleicos
20.
Eur J Immunol ; 27(12): 3398-403, 1997 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-9464828

RESUMEN

In hypermutation at the immunoglobulin loci, some bases are much more mutable than others. The increased mutability of the hot spots has been attributed to their being embedded in short sequence motifs. Among the suggested motifs are palindromes, TAA and RGYW (i.e. A/G G C/T A/T). We have tested these proposed motifs in a transfection system in vitro, which ordinarily uses the hypermutable stop codon TAG. The stop codon TAA is not hypermutable in our system, even when embedded in the pentamer and hexamer palindromes TAATA and ATTAAT; in fact, the revertants isolated were due to deletions. Single or double base changes in an RGYW motif containing a hypermutable stop codon result in a reduction of one order of magnitude or more in point mutation frequency. When the nonamer GACTAGTAT, which includes the same RGYW motif, was moved over hundred base pairs upstream, hypermutability was reduced by an order of magnitude. Thus, while RGYW apparently is a hypermutability motif, it cannot be the sole determinant of mutability.


Asunto(s)
Genes de Inmunoglobulinas , Inmunoglobulinas/genética , Mutación , Animales , Humanos
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