RESUMEN
BACKGROUND: Congenital hypothyroidism is a frequent disease occurring with an incidence of about 1/1500 newborns/year. In about 75% of the cases, CH is caused by alterations in thyroid morphogenesis, defined "thyroid dysgenesis" (TD). TD is generally a sporadic disease but in about 5% of the cases a genetic origin has been demonstrated. Previous studies indicate that Dnajc17 as a candidate modifier gene for hypothyroidism, since it is expressed in the thyroid bud, interacts with NKX2.1 and PAX8 and it has been associated to the hypothyroid phenotype in mice carrying a single Nkx2.1 and Pax8 genes (double heterozygous knock-out). PURPOSE: The work evaluates the possible involvement of DNAJC17 in the pathogenesis of TD. METHODS: High-resolution DNA melting analysis (HRM) and direct sequencing have been used to screen for mutations in the DNAJC17 coding sequence in 89 patients with TD. RESULTS: Two mutations have been identified in the coding sequence of DNAJC17 gene, one in exon 5 (c.350A>C; rs79709714) and one in exon 9 (c.610G>C; rs117485355). The last one is a rare variant, while the rs79709714 is a polymorphism. Both are present in databases and the frequency of the alleles is not different between TD patients and controls. CONCLUSIONS: DNAJC17 mutations are not frequently present in patients with TD.
Asunto(s)
Biomarcadores/análisis , Proteínas del Choque Térmico HSP40/genética , Mutación , Factor de Transcripción PAX8/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Disgenesias Tiroideas/genética , Factor Nuclear Tiroideo 1/genética , Niño , Análisis Mutacional de ADN , Femenino , Humanos , Fenotipo , Pronóstico , Disgenesias Tiroideas/diagnósticoRESUMEN
Nation-wide community quarantines and social distancing are part of the new normal because of the global COVID-19 pandemic. Since extensive and prolonged lockdowns are relatively novel experiences, not much is known about the well-being of individuals in such extreme situations. This research effort investigated the relationship between well-being elements and resiliency of 533 Filipino adults who were placed under the nationwide enhanced community quarantine (ECQ) during the COVID-19 pandemic. Participants comprised of 376 females (70.56%) and 157 males (29.45%). The median and mode ages of the participants is 23 years, while 25 is the mean age. PERMA Profiler was used to measure participants' well-being elements, while Connor-Davidson Resiliency Scale-10 (CD-RISC-10) was used to measure their resiliency. Collected data were analyzed using the regression model and necessary condition analysis. This study corroborated that all the five pillars of well-being are significant positive correlates of resiliency (p < 0.00) in quarantined adults. The results shown accomplishment (ß = 0.447, p < 0.01) positively predicts resiliency, while negative emotions (ß = -0.171, p < 0.00) negatively predict resiliency. Lastly, the five pillars of well-being are necessary-but-not-sufficient conditions (ceiling envelopment with free disposal hull, CE-FDH p < 0.00) of resiliency. Our results cast a new light on well-being elements as constraints rather than enablers of resiliency. This novel result shows that optimum resiliency is only possible when all the five pillars of well-being are taken care of and when a person is at least minimally contented with their physical health. The present findings underscore the importance of a holistic as against an atomistic approach to maintaining good mental health, which suggests that deficiencies in certain areas of well-being may not be fully addressed by overcompensating on other areas, as all five pillars of well-being are necessary-but-not-sufficient conditions of resiliency. The study ends with the recommendation for the use of necessary condition analysis to study both classical and novel psychological research problems.
RESUMEN
A model substrate for the joining of Ig VH and DJH elements has been constructed in a retroviral vector carrying a selectable marker whose expression is independent of the arrangement of the resident Ig gene segments. The substrate was introduced into lymphoid and nonlymphoid cells, and site-specific recombination between the VH and DJH elements was monitored by a direct hybridization assay. Joining of the exogenous gene segments was observed in cell lines representative of three distinct stages in early B cell differentiation. Rearrangement was not observed in three cell lines derived from mature B cells, or in a fibroblastoid cell line. The VH and DJH elements were initially arranged so that the VH-DJH junction and the recombined flanking sequences could be recovered after rearrangement. By molecular cloning and nucleotide sequence determination, VH-DJH junctions formed upon rearrangement of the substrate were found to resemble closely similar junctions in functional H chain genes. The joining of VH and DJH elements was observed to be asymmetric; loss of nucleotides occurred at the coding joints, but not at the junctions between flanking sequences. Our results suggest that Ig H and L chain gene segments are joined by a common mechanism that is more active in B cell precursors than in mature B cells. These observations provide further evidence that the rearrangement of Ig gene segments occurs by a nonreciprocal recombinational mechanism. The model substrate described here is likely to be of use in defining the nucleotide sequences that mediate rearrangement and in examining the developmental specificity of this process.
Asunto(s)
Linfocitos B/metabolismo , Genes de Inmunoglobulinas , Virus de la Leucemia Murina/genética , Recombinación Genética , Transducción Genética , Animales , Linfocitos B/citología , Secuencia de Bases , Línea Celular Transformada , Clonación Molecular , Cadenas Pesadas de Inmunoglobulina/genética , Región de Unión de la Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Células Madre/microbiología , Linfocitos T Colaboradores-Inductores/metabolismo , Transfección , Replicación ViralRESUMEN
The stimulatory and inhibitory pathways initiated by engagement of stimulatory receptors such as the B cell receptor for antigen (BCR) and inhibitory receptors such as Fcgamma receptors of the IIB1 type (FcgammaRIIB1) intersect in ways that are poorly understood at the molecular level. Because the tyrosine kinase Csk is a potential negative regulator of lymphocyte activation, we examined the effects of BCR and FcgammaRIIB1 engagement on the binding of Csk to phosphotyrosine-containing proteins. Stimulation of a B lymphoma cell line, A20, with intact anti-IgG antibody induced a direct, SH2-mediated association between Csk and a 62-kD phosphotyrosine-containing protein that was identified as RasGTPase-activating protein-associated p62 (GAP-A.p62). In contrast, stimulation of A20 cells with anti-IgG F(ab')2 resulted in little increase in the association of Csk with GAP-A.p62. The effect of FcgammaRIIB1 engagement on this association was abolished by blockade of FcgammaRIIB1 with the monoclonal antibody 2.4G2. Furthermore, the increased association between Csk and GAP-A.p62 seen upon stimulation with intact anti-Ig was abrogated in the FcgammaRIIB1-deficient cell line IIA1.6 and recovered when FcgammaRIIB1 expression was restored by transfection. The differential effects of BCR and BCR-FcgammaRIIB1-mediated signaling on the phosphorylation of GAP-A.p62 and its association with Csk suggest that docking of Csk to GAP-A.p62 may function in the negative regulation of antigen receptor-mediated signals in B cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores de Antígenos de Linfocitos B/fisiología , Receptores de IgG/fisiología , Sitios de Unión , Proteína Tirosina Quinasa CSK , Línea Celular , Humanos , Fosforilación , Dominios Homologos src , Familia-src QuinasasRESUMEN
Antigen receptor genes are assembled by site-specific DNA rearrangement. The recombination activator genes RAG-1 and RAG-2 are essential for this process, termed V(D)J rearrangement. The activity and stability of the RAG-2 protein have now been shown to be regulated by phosphorylation. In fibroblasts RAG-2 was phosphorylated predominantly at two serine residues, one of which affected RAG-2 activity in vivo. The threonine at residue 490 was phosphorylated by p34cdc2 kinase in vitro; phosphorylation at this site in vivo was associated with rapid degradation of RAG-2. Instability was transferred to chimeric proteins by a 90-residue portion of RAG-2. Mutation of the p34cdc2 phosphorylation site of the tumor suppressor protein p53 conferred a similar phenotype, suggesting that this association between phosphorylation and degradation is a general mechanism.
Asunto(s)
Proteínas de Unión al ADN , Reordenamiento Génico , Proteínas/metabolismo , Receptores de Antígenos/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Proteína Quinasa CDC2/metabolismo , Línea Celular , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares , Fosforilación , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Proteína p53 Supresora de Tumor/genéticaRESUMEN
An unexpected immunoglobulin gene rearrangement, signal sequence replacement, was observed in which the recombinational signal sequences of a VH gene segment are fused intact to the 5' end of a DJH element. Nucleotides are not lost from the signal sequences, but they may be lost from the DJH coding sequence. Signal sequence replacement may result from the alternative resolution of an intermediate in VH-to-DJH recombination. This type of rearrangement provides a means to alter the targeting of immunoglobulin gene segments and suggests a mechanism for the occurrence of VH-JH junctions in vivo. Signal sequence replacement may represent an additional pathway for the generation of antibody diversity.
Asunto(s)
Reordenamiento Génico , Genes de Inmunoglobulinas , Señales de Clasificación de Proteína/genética , Animales , Secuencia de Bases , Línea Celular , ADN Recombinante , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ratones , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Recombinación Genética , Retroviridae/genéticaRESUMEN
Several pathways of transmembrane signaling in lymphocytes involve protein-tyrosine phosphorylation. With the exception of p56lck, a tyrosine kinase specific to T lymphoid cells that associates with the T cell transmembrane proteins CD4 and CD8, the kinases that function in these pathways are unknown. A murine lymphocyte complementary DNA that represents a new member of the src family has now been isolated and characterized. This complementary DNA, termed blk (for B lymphoid kinase), specifies a polypeptide of 55 kilodaltons that is related to, but distinct from, previously identified retroviral or cellular tyrosine kinases. The protein encoded by blk exhibits tyrosine kinase activity when expressed in bacterial cells. In the mouse and among cell lines, blk is specifically expressed in the B cell lineage. The tyrosine kinase encoded by blk may function in a signal transduction pathway that is restricted to B lymphoid cells.
Asunto(s)
Linfocitos B/enzimología , Expresión Génica , Proteínas Tirosina Quinasas/genética , Familia-src Quinasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Codón , ADN/genética , ADN/aislamiento & purificación , Escherichia coli/enzimología , Escherichia coli/genética , Ratones , Datos de Secuencia Molecular , Transducción de SeñalRESUMEN
Genetic and sequence information are reported for an angiotensin II-reactive antibody (Ab1, MAb 110) and an anti--anti-idiotypic antibody (Ab3, MAb 131) that have identical antigen binding properties and that are related by an anti-idiotypic antibody (Ab2-beta) that satisfies accepted biochemical criteria for an internal image-bearing antibody. The sequences of the variable regions of the Ab3 and of the Ab1 are nearly identical, even though the Ab1 is an antibody to a peptide and the Ab3 is an antibody to a globular protein. Significantly, amino acid residues that make critical contacts with antigen in the crystal structure of the Ab3-antigen complex are highly conserved in Ab1, suggesting that the epitopes of the Ab2-beta recognized by the Ab3 do indeed resemble the bound structure of the antigen.
Asunto(s)
Angiotensina II/inmunología , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/inmunología , Secuencia de Aminoácidos , Angiotensina II/química , Animales , Anticuerpos Antiidiotipos/química , Anticuerpos Antiidiotipos/genética , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Complejo Antígeno-Anticuerpo , Secuencia de Bases , Línea Celular , Hibridomas/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Ratones , Ratones Endogámicos BALB C/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Plasmacitoma , Conformación ProteicaRESUMEN
Patients with human severe combined immunodeficiency (SCID) can be divided into those with B lymphocytes (B+ SCID) and those without (B- SCID). Although several genetic causes are known for B+ SCID, the etiology of B- SCID has not been defined. Six of 14 B- SCID patients tested were found to carry a mutation of the recombinase activating gene 1 (RAG-1), RAG-2, or both. This mutation resulted in a functional inability to form antigen receptors through genetic recombination and links a defect in one of the site-specific recombination systems to a human disease.
Asunto(s)
Proteínas de Unión al ADN , Proteínas de Homeodominio , Proteínas/genética , Inmunodeficiencia Combinada Grave/genética , Linfocitos B/inmunología , Línea Celular , Consanguinidad , Femenino , Genes de Inmunoglobulinas , Genes Recesivos , Humanos , Inmunofenotipificación , Masculino , Mutación , Proteínas Nucleares , Polimorfismo Conformacional Retorcido-Simple , Receptores de Antígenos de Linfocitos T/genética , Recombinación Genética , Eliminación de Secuencia , Inmunodeficiencia Combinada Grave/inmunología , TransfecciónRESUMEN
Two structurally dissimilar transcriptional regulators, E2A and Pax-5, have been shown to play essential roles in B-cell development; the p50 subunit of NF kappa B, by contrast, is required for normal immune responses.
Asunto(s)
Linfocitos B/citología , Proteínas de Unión al ADN/fisiología , Hematopoyesis/fisiología , FN-kappa B/fisiología , Proteínas del Tejido Nervioso , Proteínas Nucleares/fisiología , Transactivadores/fisiología , Factores de Transcripción , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Secuencias Hélice-Asa-Hélice/fisiología , Hematopoyesis/genética , Síndromes de Inmunodeficiencia/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Familia de Multigenes , FN-kappa B/química , FN-kappa B/genética , Proteínas Nucleares/genética , Factor de Transcripción PAX5 , Factores de Transcripción TCF , Transactivadores/genética , Factor de Transcripción 4 , Proteína 1 Similar al Factor de Transcripción 7RESUMEN
During B-cell development, immature and mature forms of the B cell antigen receptor complex are deployed in a regulated fashion; thus, B cell antigen receptor complexes play essential roles in the transit of cells through ontogeny. The past year has seen progress in our understanding of how antigen receptor gene assembly is controlled and in defining the requirements for antigen receptor mediated signaling at specific developmental stages. The discovery that a defective protein tyrosine kinase is responsible for X-linked agammaglobulinemia in man and X-linked immunodeficiency in the mouse is particularly interesting, as it may provide the means to link a specific intracellular signaling pathway with a particular step in B-cell development.
Asunto(s)
Subgrupos de Linfocitos B/fisiología , Diferenciación Celular/fisiología , Sistema Inmunológico/crecimiento & desarrollo , Receptores de Antígenos de Linfocitos B/fisiología , Secuencia de Aminoácidos , Animales , Subgrupos de Linfocitos B/enzimología , Desarrollo Embrionario y Fetal , Citometría de Flujo , Humanos , Síndromes de Inmunodeficiencia/genética , Datos de Secuencia Molecular , Fosforilación , Proteínas Tirosina Quinasas/fisiología , Receptores de Antígenos de Linfocitos B/genética , Recombinación Genética/fisiología , Transducción de Señal/fisiologíaRESUMEN
Bruton's tyrosine kinase (Btk), the target of inactivating mutations in X-linked immunodeficiency diseases of mice and humans, is essential for normal B cell responsiveness. Recent studies have outlined a mechanism for the activation of Btk by B cell receptor engagement and have identified proximal and distal targets of Btk action.
Asunto(s)
Linfocitos B/citología , Síndromes de Inmunodeficiencia/enzimología , Proteínas Tirosina Quinasas/fisiología , Transducción de Señal/fisiología , Agammaglobulinemia Tirosina Quinasa , Animales , Antígenos T-Independientes/inmunología , Linfocitos B/enzimología , Diferenciación Celular , Activación Enzimática , Humanos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Ratones , Ratones Mutantes , Fosforilación , Procesamiento Proteico-Postraduccional , Cromosoma X/genética , Familia-src Quinasas/fisiologíaRESUMEN
Extraordinary progress has been made in refining our understanding of the B-cell antigen receptor complex, the role of protein-tyrosine phosphorylation as the key intermediary in immunoglobulin signal transduction, and in identifying candidate effectors of immunoglobulin-mediated signaling. In addition, the properties of a new group of G proteins may rekindle interest in older observations implicating such molecules as important in B-cell activation.
Asunto(s)
Linfocitos B/fisiología , Activación de Linfocitos/fisiología , Secuencia de Aminoácidos , Animales , Proteínas de Unión al GTP/fisiología , Humanos , Datos de Secuencia Molecular , Fosforilación , Proteínas Tirosina Quinasas/fisiología , Receptores de Antígenos de Linfocitos B/fisiología , Transducción de Señal/fisiología , Tirosina/fisiologíaRESUMEN
V(D)J recombination occurs at recombination signal sequences (RSSs) containing conserved heptamer and nonamer elements. RAG-1 and RAG-2 initiate recombination by cleaving DNA between heptamers and antigen receptor coding segments. RAG-1 alone contacts the nonamer but interacts weakly, if at all, with the heptamer. RAG-2 by itself has no DNA-binding activity but promotes heptamer occupancy in the presence of RAG-1; how RAG-2 collaborates with RAG-1 has been poorly understood. Here we examine the composition of RAG-RSS complexes and the relative contributions of RAG-1 and RAG-2 to heptamer binding. RAG-1 exists as a dimer in complexes with an isolated RSS bearing a 12-bp spacer, regardless of whether RAG-2 is present; only a single subunit of RAG-1, however, participates in nonamer binding. In contrast, multimeric RAG-2 is not detectable by electrophoretic mobility shift assays in complexes containing both RAG proteins. DNA-protein photo-cross-linking demonstrates that heptamer contacts, while enhanced by RAG-2, are mediated primarily by RAG-1. RAG-2 cross-linking, while less efficient than that of RAG-1, is detectable near the heptamer-coding junction. These observations provide evidence that RAG-2 alters the conformation or orientation of RAG-1, thereby stabilizing interactions of RAG-1 with the heptamer, and suggest that both proteins interact with the RSS near the site of cleavage.
Asunto(s)
Proteínas de Unión al ADN/genética , Genes de Inmunoglobulinas/genética , Proteínas de Homeodominio/genética , Secuencia de Bases , Proteínas Portadoras/genética , Línea Celular , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas de Unión a Maltosa , Datos de Secuencia Molecular , Proteínas Nucleares , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/genética , Recombinación Genética/genéticaRESUMEN
The genes that encode the variable regions of immunoglobulin (Ig) heavy chains are encoded by three DNA segments: VH, D, and JH. During B-cell development these segments are brought together by a pair of site-specific DNA rearrangements. The first of these joins a D segment to a JH segment; the second brings a VH segment in apposition to a DJH unit. B-cell precursors that have undergone D-to-JH joining express transcripts that initiate at the 5' flanks of rearranged D segments (DJH transcription). In this study we have examined the coordination of D-to-JH rearrangement and DJH transcription. The B-lymphoid progenitor cell line HAFTL-1 cell clone, joining of distal D segments (DSP2 and DFL16) to JH is accompanied by an increase in the steady-state level of transcripts initiating 5' of the D coding region. Steady-state transcription of a DSP2 gene segment was undetectable prior to rearrangement and was observed to increase at least 20-fold upon joining to JH. In contrast, transcription from the 5' flank of DQ52, which lies within 700 bp of the JH cluster, was detected prior to rearrangement and did not increase significantly after rearrangement. The 5' flank of a DSP2 segment was found to support expression of a heterologous gene upon transfection into B progenitor cell lines. Expression from this DSP2 promoter was at least 30-fold higher in the presence of the Ig heavy-chain enhancer, in either orientation, than in its absence. A DNA fragment spanning the interval from -165 to +19 bp relative to the major DSP2 transcriptional start site retained enhancer-dependent promoter activity. These observations imply that activation of DSP12JH and DFL16JH transcription is coordinated with D-to-JH rearrangement by approximation of enhancer-dependent D promoter elements to the Ig heavy-chain enhancer. This interpretation is consistent with our observation that the DQ52 segment, which is closely linked to the JH cluster, is transcribed both before and after rearrangement.
Asunto(s)
Elementos de Facilitación Genéticos , Reordenamiento Génico , Genes de Inmunoglobulinas , Región Variable de Inmunoglobulina/genética , Regiones Promotoras Genéticas , Transcripción Genética , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Cadenas Pesadas de Inmunoglobulina/genética , Ratones , Datos de Secuencia MolecularRESUMEN
Some patients with B-cell-negative severe combined immune deficiency (SCID) carry mutations in RAG-1 or RAG-2 that impair V(D)J recombination. Two recessive RAG-1 mutations responsible for B-cell-negative SCID, R621H and E719K, impair V(D)J recombination without affecting formation of single-site recombination signal sequence complexes, specific DNA contacts, or perturbation of DNA structure at the heptamer-coding junction. The E719K mutation impairs DNA cleavage by the RAG complex, with a greater effect on nicking than on transesterification; a conservative glutamine substitution exhibits a similar effect. When cysteine is substituted for E719, RAG-1 activity is enhanced in Mn(2+) but remains impaired in Mg(2+), suggesting an interaction between this residue and an essential metal ion. The R621H mutation partially impairs nicking, with little effect on transesterification. The residual nicking activity of the R621H mutant is reduced at least 10-fold upon a change from pH 7.0 to pH 8.4. Site-specific nicking is severely impaired by an alanine substitution at R621 but is spared by substitution with lysine. These observations are consistent with involvement of a positively charged residue at position 621 in the nicking step of the RAG-mediated cleavage reaction. Our data provide a mechanistic explanation for one form of hereditary SCID. Moreover, while RAG-1 is directly involved in catalysis of both nicking and transesterification, our observations indicate that these two steps have distinct catalytic requirements.
Asunto(s)
Reordenamiento Génico de Linfocito B , Proteínas de Homeodominio/genética , Mutación , Recombinación Genética , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/inmunología , Linfocitos B/inmunología , Secuencia de Bases , Cationes Bivalentes , Línea Celular , ADN/metabolismo , Cartilla de ADN/genética , Esterificación , HumanosRESUMEN
V(D)J recombination is initiated by RAG-1 and RAG-2, which introduce double-strand DNA breaks at recombination signal sequences (RSSs) of antigen receptor gene segments to produce signal ends, terminating in blunt, double-strand breaks, and coding ends, terminating in DNA hairpins. While the formation of RAG-RSS complexes has been documented, observations regarding the individual contributions of RAG-1 and RAG-2 to RSS recognition are in conflict. Here we describe an assay for formation and maintenance of functional RAG-RSS complexes in the course of the DNA cleavage reaction. Under conditions of in vitro cleavage, the RAG proteins sequester intact substrate DNA in a stable complex which is formed prior to strand scission. The cleavage reaction subsequently proceeds through nicking and hairpin formation without dissociation of substrate. Notably, the presence of both RAG-1 and RAG-2 is essential for formation of stable, functional complexes with substrate DNA under conditions of the sequestration assay. Two classes of substrate mutation are distinguished by their effects on RAG-mediated DNA cleavage in vitro. A mutation of the first class, residing within the RSS nonamer and associated with coordinate impairment of nicking and hairpin formation, greatly reduces the stability of RAG association with intact substrate DNA. In contrast, a mutation of the second class, lying within the RSS heptamer and associated with selective abolition of hairpin formation, has little or no effect on the half-life of the RAG-substrate complex.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos T/genética , ADN/química , ADN/genética , ADN/metabolismo , Humanos , Técnicas In Vitro , Cinética , Mutación , Proteínas Nucleares , Conformación de Ácido Nucleico , Recombinación GenéticaRESUMEN
Rapid analysis of mechanisms that regulate V(D)J recombination has been hampered by the lack of appropriate cell systems that reproduce aspects of normal prelymphocyte physiology in which the recombinase is activated, accessible antigen receptor loci are rearranged, and rearrangement status is fixed by termination of recombinase expression. To generate such a system, we introduced heat shock-inducible V(D)J recombination-activating genes (RAG) 1 and 2 into a recombinationally inert B-cell line. Heat shock treatment of these cells rapidly induced high levels of RAG transcripts and RAG proteins that were accompanied by a parallel induction of V(D)J recombinase activity, strongly suggesting that RAG proteins have a primary role in V(D)J recombination. Within hours after induction, these cells began to rearrange chromosomally integrated V(D)J recombination substrates but only if the substrates contained an active transcriptional enhancer; substrates lacking an enhancer were not efficiently rearranged. Activities necessary to target integrated substrates for rearrangement were provided by two separate lymphoid-specific transcriptional enhancers, as well as an active nonlymphoid enhancer, unequivocally demonstrating that such elements enhance both transcription and V(D)J recombinational accessibility.
Asunto(s)
Linfocitos B/metabolismo , ADN Nucleotidiltransferasas/metabolismo , Proteínas de Unión al ADN , Elementos de Facilitación Genéticos , Reordenamiento Génico de Linfocito B , Proteínas de Homeodominio , Animales , Secuencia de Bases , Northern Blotting , Línea Celular , ADN , Calor , Ratones , Datos de Secuencia Molecular , Proteínas/genética , Proteínas/metabolismo , Mapeo Restrictivo , Transfección , VDJ RecombinasasRESUMEN
The B-cell lymphocyte kinase (Blk) is a src-family protein tyrosine kinase specifically expressed in B-lineage cells of mice. The early onset of Blk expression during B-cell development in the bone marrow and the high expression levels of Blk in mature B cells suggest a possible important role of Blk in B-cell physiology. To study the in vivo function of Blk, mice homozygous for the targeted disruption of the blk gene were generated. In homozygous mutant mice, neither blk mRNA nor Blk protein is expressed. Despite the absence of Blk, the development, in vitro activation, and humoral immune responses of B cells to T-cell-dependent and -independent antigens are unaltered. These data are consistent with functional redundancy of Blk in B-cell development and immune responses.
Asunto(s)
Linfocitos B/enzimología , Linfocitos B/inmunología , Familia-src Quinasas/fisiología , Animales , Formación de Anticuerpos , Antígenos T-Independientes , Linfocitos B/citología , Secuencia de Bases , Diferenciación Celular , Cartilla de ADN/genética , Femenino , Marcación de Gen , Técnicas In Vitro , Activación de Linfocitos , Masculino , Ratones , Ratones Noqueados , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Linfocitos T/citología , Linfocitos T/enzimología , Linfocitos T/inmunología , Familia-src Quinasas/genéticaRESUMEN
It has been proposed that the helix-loop-helix (HLH) protein Id serves as a general antagonist of cell differentiation by inhibiting bHLH (HLH with an adjacent stretch of basic amino acids) proteins specifically required for developmental programs (such as MyoD). We show here that ectopic expression of Id represses in vivo activity of the bHLH protein E2-5 (encoded by the E2A gene) and of both the immunoglobulin heavy-chain (IgH) and kappa-light-chain gene enhancers to which E2-5 binds. Id does not affect the activity of the bHLH-zip protein, TFE3, which also binds these enhancers. We examined a large panel of B-cell lines that represent different stages of lymphoid development and found only two that express Id mRNA. The cell lines Ba/F3 and LyD9 have been categorized previously as early B-lymphoid-cell progenitors. Unlike their more mature B-lymphoid-cell counterparts, Ba/F3 and LyD9 cells do not express I mu sterile transcripts, which are indicative of IgH enhancer activity. Moreover, Ba/F3-derived nuclear extracts lack E2-box-binding activity, indicating the absence of free bHLH proteins, and transfected Ba/F3 cells fail to support the activity of the IgH enhancer. Hence, expression of Id correlates inversely with bHLH protein activity and enhancer function in vivo. These results suggest that Id may play a role early in B-lymphoid-cell development to regulate transcription of the IgH locus.