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1.
J Exp Med ; 188(4): 627-34, 1998 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-9705945

RESUMEN

The products of recombination activating gene (RAG)1 and RAG2 initiate the lymphoid-specific phase of the V(D)J recombination by creating a DNA double-strand break (dsb), leaving hairpin-sealed coding ends. The next step uses the general DNA repair machinery of the cells to resolve this dsb. Several genes involved in both V(D)J recombination and DNA repair have been identified through the analysis of in vitro mutants (Chinese hamster ovary cells) and in vivo situations of murine and equine severe combined immunodeficiency (scid). These studies lead to the description of the Ku-DNA-dependent protein kinase complex and the XRCC4 factor. A human SCID condition is characterized by an absence of B and T lymphocytes. One subset of these patients also demonstrates an increased sensitivity to the ionizing radiation of their fibroblasts and bone marrow precursor cells. This phenotype is accompanied by a profound defect in V(D)J recombination with a lack of coding joint formation, whereas signal joints are normal. Functional and genetic analyses distinguish these patients from the other recombination/repair mutants, and thus define a new group of mutants whose affected gene(s) is involved in sensitivity to ionizing radiation and V(D)J recombination.


Asunto(s)
Antígenos Nucleares , Linfocitos B/inmunología , ADN Helicasas , Reparación del ADN , Reordenamiento Génico , Genes de Inmunoglobulinas , Tolerancia a Radiación , Inmunodeficiencia Combinada Grave/inmunología , Linfocitos T/inmunología , Animales , Línea Celular , Línea Celular Transformada , Cricetinae , Cricetulus , Reparación del ADN/efectos de la radiación , Proteína Quinasa Activada por ADN , Proteínas de Unión al ADN/metabolismo , Femenino , Rayos gamma , Reordenamiento Génico/efectos de la radiación , Humanos , Región de Unión de la Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Autoantígeno Ku , Ligandos , Masculino , Proteínas Nucleares/metabolismo , Linaje , Proteínas Serina-Treonina Quinasas/metabolismo , Inmunodeficiencia Combinada Grave/genética
2.
Nucleic Acids Res ; 20(2): 245-50, 1992 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-1346928

RESUMEN

High level transient gene expression in lymphoid cells has always been challenging because of the difficulty to efficiently transfect such cells. This has precluded any attempt to clone cDNA encoding proteins by means of their specific biological function in lymphoid cells. We have developed a very efficient transient eukaryotic expression system analogous to the well-known expression system in COS cells. Firefly luciferase and human CD2 genes were used as reporter genes and cloned into the eukaryotic shuttle vector pCDM8 which contains the strong cytomegalovirus promoter and the SV40 origin of replication for autonomous plasmid replication in permissive host cells that express the large SV40 T Antigen. Co-transfection of the reporter plasmids together with an SV40 T Ag expressing plasmid resulted in the several fold amplification of either the Luc activity or the cell surface expression of the CD2 marker in a transient assay. The level of amplification was dependent on the strength of the promoter used to drive the SV40 T Ag expression and was correlated with the extent of autonomous replication of the reporter plasmid in transfected cells. This highly efficient transient gene expression by SV40 T Ag boost was suitable to several human cell lines, making this system of general interest for expression cloning strategies or other gene transfer application that need high level expression.


Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Regulación de la Expresión Génica/genética , Transfección/genética , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos CD2 , Línea Celular , Replicación del ADN , Técnica del Anticuerpo Fluorescente , Humanos , Luciferasas/genética , Linfocitos , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Receptores Inmunológicos/genética
3.
Eur J Immunol ; 23(6): 1294-8, 1993 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-8388796

RESUMEN

The T cell receptor (TCR)-alpha and -delta loci are contained on the same chromosomal region, and yet are developmentally and genetically independent. The first element of the J alpha cluster (psi J alpha) is the site of an active rearrangement in the human thymus (delta Rec-psi J alpha rearrangement) and is localized downstream of a region expressed as a germ-line sterile transcript (TEA) in the human developing thymus. We hypothesized that the transcription of TEA could be indicative of (or responsible for) the opening of the J alpha to the V(D)J recombinase and undertook to analyze cis-acting sequences controlling the TEA transcription. The promoter of TEA was characterized. It was part of a region that is highly conserved between human and mouse and contained many sites for the putative binding of T cell-specific transcription factors. The in vitro activity of this promoter was dependent on the association with an enhancer. A strong DNase I hypersensitive site was found in the vicinity of this promoter again suggesting the possible presence of protein-DNA interactions in this region. The implications of these results in the general perspective of TCR-alpha/delta gene regulation is discussed.


Asunto(s)
Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Secuencia de Bases , Sitios de Unión , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa I , Regulación de la Expresión Génica , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Regiones Promotoras Genéticas , ARN Mensajero/genética , Transcripción Genética , Células Tumorales Cultivadas
4.
Eur J Immunol ; 29(12): 4072-80, 1999 12.
Artículo en Inglés | MEDLINE | ID: mdl-10602018

RESUMEN

TEA (T early alpha) is a genetic element located upstream of the TCR-Jalpha cluster. Thymocytes from mice carrying a targeted deletion of TEA do not rearrange their TCRalpha locus on a window spanning the first nine Jalpha segments. This led us to the hypothesis of TEA having a "rearrangement focusing" activity on the 5' side of the TCR-Jalpha region. We analyzed DNAseI and "phylogenetic" footprints within the TEA promoter in an attempt to identify trans-acting factors that could account for its regulatory function on DNA accessibility. One of these footprints corresponded to a putative DNA-binding site for an orphan nuclear receptor of the ROR / RZR family. The RORgammaT cDNA clone was isolated from a thymus library using a probe corresponding to the DNA-binding domain of RORgamma / TOR. RORgammaT is a thymus-specific isoform of RORgamma, expressed almost exclusively in immature double-positive thymocytes. RORgammaT binds, to the TEA promoter in vitro. Lastly, the expression of RORgammaT is stimulated in two situations that mimic activation through the pre-TCR and in which the thymocytes have their TCR-alpha locus in an "open", yet unrearranged DNA configuration. We propose that the expression of RORgammaT may be part of the pre-TCR activation cascade leading to the maturation of alpha / beta T cells and may participate in the regulation of DNA accessibility in the TCR-Jalpha locus.


Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos , Proteínas Portadoras/inmunología , Proteínas de la Membrana/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores Citoplasmáticos y Nucleares/inmunología , Receptores de Ácido Retinoico , Receptores de Hormona Tiroidea , Transducción de Señal/inmunología , Linfocitos T/inmunología , Timo/inmunología , Animales , Proteínas Portadoras/genética , Genes de Inmunoglobulinas , Humanos , Proteínas de la Membrana/genética , Ratones , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Regiones Promotoras Genéticas , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Regulación hacia Arriba/inmunología
5.
Int Immunol ; 3(12): 1301-5, 1991 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-1838004

RESUMEN

The nested location of the TCR-delta gene within the TCR-alpha locus is a common feature of both mouse and human. Yet alpha/beta and gamma/delta T cells represent two separate lineages. We have previously proposed that a specific rearrangement event (delta Rec--psi J alpha) resulting in the specific deletion of the TCR-delta gene could be responsible for the independent usage of these two loci. We used an in vitro model of T cell differentiation to test this hypothesis. We show that in culture conditions (IL-1 + sCD23) which promote the development of alpha/beta expressing T cells exclusively, the specific deletion of the TCR-delta locus occurs very rapidly, probably before the productive rearrangement of the TCR-alpha gene. These results clearly demonstrate that the specific deletion of the TCR-delta gene could be the initial regulatory event that imprints the irreversible commitment of T cell differentiation along the alpha/beta pathway.


Asunto(s)
Reordenamiento Génico de Linfocito T , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Linfocitos T/citología , Secuencia de Bases , Diferenciación Celular , Deleción Cromosómica , Genes , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Oligonucleótidos/química , Reacción en Cadena de la Polimerasa , Timo/citología
6.
Eur J Biochem ; 186(1-2): 181-8, 1989 Dec 08.
Artículo en Inglés | MEDLINE | ID: mdl-2574668

RESUMEN

Somatostatin receptors of plasma membranes from beta cells of hamster insulinoma were covalently labelled with 125I-[Leu8,D-Trp22,Tyr25]somatostatin-28 (125I-somatostatin-28) and solubilized with the non-denaturing detergent Triton X-100. Analysis by SDS/PAGE and autoradiography revealed three specific 125I-somatostatin-28 receptor complexes with similar molecular masses (228 kDa, 128 kDa and 45 kDa) to those previously identified [Cotroneo, P., Marie, J.-C. & Rosselin, G. (1988) Eur. J. Biochem. 174, 219-224]. The major labelled complex (128 kDa) was adsorbed to a wheat-germ-agglutinin agarose column and eluted by N-acetylglucosamine. Also, the binding of 125I-somatostatin-28 to plasma membranes was specifically inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate) (GTP[S]) in a dose-dependent manner. Furthermore, when somatostatin-28 receptors were solubilized by Triton X-100 as a reversible complex with 125I-somatostatin-28, GTP[S] specifically dissociated the bound ligand to a larger extent from the soluble receptors than from the plasma-membrane-embedded receptors, the radioactivity remaining bound after 15 min at 37 degrees C being 30% and 83% respectively. After pertussis-toxin-induced [32P]ADP-ribosylation of pancreatic membranes, a 41-kDa [32P]ADP-ribose-labelled inhibitory guanine nucleotide binding protein coeluted with the 128-kDa and 45-kDa receptor complexes. The labelling of both receptor proteins was sensitive to GTP[S]. The labelling of the 228-kDa band was inconsistent. These results support the conclusion that beta cell somatostatin receptors can be solubilized as proteins of 128 kDa and 45 kDa. The major labeled species corresponds to the 128-kDa band and is a glycoprotein. The pancreatic membrane contains a 41-kDa GTP-binding protein that can complex with somatostatin receptors.


Asunto(s)
Receptores de Neurotransmisores/metabolismo , Somatostatina/metabolismo , Animales , Células Cultivadas , Cromatografía en Gel , Cricetinae , Electroforesis en Gel de Poliacrilamida , Guanosina Trifosfato/metabolismo , Insulinoma/metabolismo , Radioisótopos de Yodo , Islotes Pancreáticos/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores de Somatostatina , Solubilidad , Somatostatina-28
7.
J Biol Chem ; 275(17): 12672-5, 2000 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-10777560

RESUMEN

The V(D)J recombination, which leads to the somatic rearrangement of variable, diversity, and joining segments, is the mechanism accountable for the diversity of T cell receptor- and Ig-encoding genes. The products of the RAG1 and RAG2 genes are the lymphoid-specific factors responsible for the initiation of the V(D)J recombination through the generation of a DNA double strand break. RAG1 or RAG2 gene inactivation in the mouse leads to abortion of the V(D)J rearrangement process, early block in both T and B cell maturation, and, ultimately, to severe combined immune deficiency (SCID). A human SCID condition is also characterized by an absence of mature T and B lymphocytes and is associated with mutations in either RAG1- or RAG2-encoding genes. Based on the predicted beta-propeller three-dimensional structure model for RAG2, we found that six out of the seven mutations described to date in T-B-SCID patients are clustered on one side of the propeller, in regions exposed to solvent. This finding reinforces the biological significance of this predicted model and suggests that RAG1 interacts with RAG2 on one of the side of the scaffold formed by the beta-propeller.


Asunto(s)
Proteínas de Unión al ADN/genética , Mutación , Inmunodeficiencia Combinada Grave/genética , Secuencia de Aminoácidos , Western Blotting , Línea Celular Transformada , Clonación Molecular , ADN Nucleotidiltransferasas/metabolismo , Proteínas de Unión al ADN/química , Fibroblastos/metabolismo , Genes RAG-1/genética , Células HeLa , Proteínas de Homeodominio/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas Nucleares , Fenotipo , Estructura Terciaria de Proteína , Recombinación Genética , Análisis de Secuencia de ADN , VDJ Recombinasas
8.
Cell ; 105(2): 177-86, 2001 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-11336668

RESUMEN

The V(D)J recombination process insures the somatic diversification of immunoglobulin and antigen T cell receptor encoding genes. This reaction is initiated by a DNA double-strand break (dsb), which is resolved by the ubiquitously expressed DNA repair machinery. Human T-B-severe combined immunodeficiency associated with increased cellular radiosensitivity (RS-SCID) is characterized by a defect in the V(D)J recombination leading to an early arrest of both B and T cell maturation. We previously mapped the disease-related locus to the short arm of chromosome 10. We herein describe the cloning of the gene encoding a novel protein involved in V(D)J recombination/DNA repair, Artemis, whose mutations cause human RS-SCID. Protein sequence analysis strongly suggests that Artemis belongs to the metallo-beta-lactamase superfamily.


Asunto(s)
Linfocitos B/fisiología , Reparación del ADN/genética , Proteínas Nucleares , Tolerancia a Radiación/genética , Recombinación Genética/genética , Inmunodeficiencia Combinada Grave/genética , Linfocitos T/fisiología , beta-Lactamasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Células Cultivadas , Cromosomas Humanos Par 10/genética , Clonación Molecular , Proteínas de Unión al ADN , Endonucleasas , Fibroblastos , Humanos , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Alineación de Secuencia , Inmunodeficiencia Combinada Grave/fisiopatología , Transfección , beta-Lactamasas/química , beta-Lactamasas/metabolismo
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