RESUMEN
The ASPP1 (Apoptosis Stimulating Protein of p53) protein is an important tumour-suppressor. We have detected a novel protein interaction between the human ASPP1 (hASPP1) protein and the predominantly nuclear adaptor protein SAM68. In the human testis, full-length endogenous hASPP1 protein is located in the nucleus like SAM68, predominantly within meiotic and postmeiotic cells. Mouse ASPP1 (mASPP1) protein is mainly expressed in the brain and testis. The interaction with nuclear SAM68 is likely to be restricted to human germ cells, since endogenous mASPP1 protein is exclusively cytoplasmic. The C-terminal region of hASPP1 efficiently targeted a fused GFP molecule to the nucleus, whereas the N-terminus of hASPP1 targeted GFP to the cytoplasm. In the context of the full-length molecule this cytoplasmic targeting sequence is dominant in HEK293 and Saos-2 cells, since full-length hASPP1-GFP is almost exclusively cytoplasmic. Despite its predominantly cytoplasmic location, we show that ASPP1-GFP expression in HEK293 cells can regulate the ratio of alternative spliced isoforms derived from a pre-mRNA regulated downstream of cytoplasmic signalling pathways, and our data suggest that ASPP1 may operate in this case downstream or parallel to RAS signalling pathways.
Asunto(s)
Empalme Alternativo , Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Exones/genética , Células Germinativas/metabolismo , Receptores de Hialuranos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/genética , Células Cultivadas , Citoplasma/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Riñón/metabolismo , Masculino , Osteosarcoma/metabolismo , Osteosarcoma/patología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Isoformas de Proteínas , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae , Transducción de Señal , Testículo/metabolismo , Testículo/patología , Proteínas Supresoras de Tumor/fisiología , Técnicas del Sistema de Dos HíbridosRESUMEN
The Delta Sxrb deletion interval of the mouse Y chromosome contains Spy, a spermatogenesis factor gene(s) whose expression is essential for the postnatal development of the mitotic germ cells, spermatogonia. The boundaries of Delta Sxrb are defined by the duplicated genes Zfy1 and Zfy2 and four further genes have previously been mapped within the interval: Ube1y and Smcy, linked with Zfy1 on a contig of 250 kb, and Dffry and Uty, which were unanchored. The interval was estimated to be >450 kb. In order to identify any further gene(s) that may underlie Spy, systematic exon trapping was performed on an extended contig, anchored on Zfy1, which covers 750 kb of the Delta Sxrb interval. Exons from two novel genes were isolated and placed together with Dffry and Uty on the contig in the order Dffry-Dby-Uty-Tspy-Eif2gammay-Smcy- Ube1y-Zfy1. All the genes, with the double exception of Tspy, are X-Y homologous and produce putatively functional, spliced transcripts. The tight linkage and order of Dffry, Dby and Uty was shown to be conserved in deletion intervals 5C/5D of the human Y chromosome by the construction of a contig of human PAC and YAC clones; this represents the first example of syntenic homology between Y chromosomes from two distinct mammalian orders. Interval 5C/5D contains the distal boundary of the AZFa interval, which, like Delta Sxrb, is believed to be necessary for spermatogonial development in the prepubertal testis. Our results therefore show that AZFa and Spy may be encoded by homologous genes.
Asunto(s)
Mapeo Cromosómico/métodos , Espermatogénesis/genética , Cromosoma Y , Animales , Cromosomas Bacterianos , Cromosomas Humanos , ARN Helicasas DEAD-box , Proteínas de Unión al ADN/genética , Exones , Femenino , Humanos , Factores de Transcripción de Tipo Kruppel , Ligasas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Proteínas Nucleares , Proteínas/genética , Ratas , Factores de Transcripción , Transcripción Genética , Ubiquitina-Proteína LigasasRESUMEN
The Delta Sxrb interval of the mouse Y chromosome is critical for spermatogenesis and expression of the male-specific minor transplantation antigen H-Y. Several genes have been mapped to this interval and each has a homologue on the X chromosome. Four, Zfy1 , Zfy2 , Ube1y and Dffry , are expressed specifically in the testis and their X homologues are not transcribed from the inactive X chromosome. A further two, Smcy and Uty , are ubiquitously expressed and their X homologues escape X-inactivation. Here we report the identification of another gene from this region of the mouse Y chromosome. It encodes the highly conserved eukaryotic translation initiation factor eIF-2gamma. In the mouse this gene is ubiquitously expressed, has an X chromosome homologue which maps close to Dmd and escapes X-inactivation. The coding regions of the X and Y genes show 86% nucleotide identity and encode putative products with 98% amino acid identity. In humans, the eIF-2gamma structural gene is located on the X chromosome at Xp21 and this also escapes X-inactivation. However, there is no evidence of a Y copy of this gene in humans. We have identified autosomal retroposons of eIF-2gamma in both humans and mice and an additional retroposon on the X chromosome in some mouse strains. Ark blot analysis of eutherian and metatherian genomic DNA indicates that X-Y homologues are present in all species tested except simian primates and kangaroo and that retroposons are common to a wide range of mammals. These results shed light on the evolution of X-Y homologous genes.
Asunto(s)
Compensación de Dosificación (Genética) , Factor 2 Eucariótico de Iniciación/genética , Evolución Molecular , Retroelementos , Cromosomas Sexuales , Secuencia de Aminoácidos , Animales , Northern Blotting , Mapeo Cromosómico , Cromosomas Humanos , Cromosomas Humanos Par 12 , Clonación Molecular , Factor 2 Eucariótico de Iniciación/metabolismo , Femenino , Humanos , Masculino , Mamíferos/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Endogámicos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Cromosoma X , Cromosoma YRESUMEN
The male-specific minor histocompatibility antigen H-Y plays an important role in both graft rejection and graft-versus-host disease following transplantation of male tissue into females that are completely matched at the major histocompatibility loci. The recent identification of two peptides that, in association with the mouse H-2Kk or human HLA B7 major histocompatibility class I molecules, are recognised by H-Y-specific T cells, has provided evidence for the molecular basis for such anti-H-Y responses. These peptides are encoded by the mouse and human homologues of a ubiquitously expressed Y chromosome gene, Smcy, whilst the equivalent peptides encoded by the X chromosome homologues of this gene fail to be recognised. Genetic studies have demonstrated that, as is the case for other minor histocompatibility antigens, peptide epitopes from several closely linked genes may be required to interact in order to elicit a response against H-Y. Definition of the peptides and the genes that encode these epitopes will allow the development of tolerogenic protocols that could specifically down-modulate the response to H-Y and perhaps even other minor histocompatibility antigens.
Asunto(s)
Epítopos de Linfocito T/genética , Rechazo de Injerto/genética , Antígeno H-Y/genética , Cromosoma Y , Secuencia de Aminoácidos , Mapeo Cromosómico , Epítopos de Linfocito T/inmunología , Femenino , Antígeno H-Y/inmunología , Antígeno H-Y/metabolismo , Humanos , Complejo Mayor de Histocompatibilidad/inmunología , Masculino , Datos de Secuencia Molecular , Trasplante de Órganos/fisiología , Mapeo Restrictivo , Linfocitos T , Trasplante de Tejidos/fisiologíaRESUMEN
Rejection of male tissue grafts by genotypically identical female mice has been explained by the existence of a male-specific transplantation antigen, H-Y (ref. 1), but the molecular nature of H-Y antigen has remained obscure. Hya, the murine locus controlling H-Y expression, has been localized to delta Sxrb, a deletion interval of the short arm of the Y chromosome. In mice, H-Y antigen comprises at least four distinct epitopes, each recognized by a specific T lymphocyte clone. It has recently been shown that one of these epitopes, H-YKk, is a peptide encoded by the Y-linked Smcy gene, presented at the cell surface with the H-2Kk major histocompatibility complex (MHC) molecule. However, deletion mapping and the analysis of variable inactivation of H-Y epitopes has suggested that the Hya locus may be genetically complex. Here we describe a novel mouse Y chromosome gene which we call Uty (ubiquitously transcribed tetratricopeptide repeat gene on the Y chromosome). We identify the peptide WMHHNMDLI derived from the UTY protein as an H-Y epitope, H-YDb. Our data formally demonstrate that H-Y antigen is the product of more than one gene on the Y chromosome.
Asunto(s)
Epítopos/genética , Antígeno H-Y/genética , Proteínas/genética , Cromosoma Y , Secuencia de Aminoácidos , Animales , Southern Blotting , Línea Celular , Mapeo Cromosómico , Clonación Molecular , Epítopos/biosíntesis , Femenino , Feto/metabolismo , Antígeno H-Y/biosíntesis , Masculino , Ratones , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas/inmunología , Caracteres Sexuales , TransfecciónRESUMEN
The male-specific transplantation antigen, H-Y, causes rejection of male tissue grafts by genotypically identical female mice and contributes to the rejection of human leukocyte antigen-matched male organ grafts by human females. Although first recognized 40 years ago, the identity of H-Y has remained elusive. T cells detect several distinct H-Y epitopes, and these are probably peptides, derived from intracellular proteins, that are presented at the cell surface with major histocompatibility complex (MHC) molecules. In the mouse, the gene(s) controlling H-Y expression (Hya) are located on the short arm of the Y chromosome between the zinc-finger genes Zfy-1 and Zfy-2. We have recently identified Smcy, a ubiquitously expressed gene, in this region and its X-chromosome homologue, Smcx. Here we report that Smcy encodes an H-YKk epitope that is defined by the octamer peptide TENSGKDI: no similar peptide is found in Smcx. These findings provide a genetic basis for the antigenic difference between males and females that contributes towards a tissue transplant rejection response.
Asunto(s)
Antígeno H-Y/genética , Cromosoma X , Cromosoma Y , Secuencia de Aminoácidos , Animales , Línea Celular , Mapeo Cromosómico , Clonación Molecular , Cósmidos , Proteínas de Unión al ADN/genética , Epítopos/genética , Femenino , Rechazo de Injerto/inmunología , Antígeno H-Y/inmunología , Masculino , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes , Caracteres Sexuales , Linfocitos T/inmunología , Factores de Transcripción , Dedos de Zinc/genéticaRESUMEN
A genetic map of the mammalian Y chromosome cannot be produced by standard Mendelian methods because the Y does not participate in meiotic exchange over the majority of its length. However, deletion mapping of the mouse Y chromosome is facilitated by the fact that its short arm carries the histocompatibility-Y (Hya) locus. This locus encodes male-specific (H-Y) antigens that can be selected against in tissue culture by the technique of immunoselection. To produce cells carrying deletions, cytotoxic T lymphocytes (CTLs) specific for H-Y antigens were cocultured with a lymphoblastoid cell line derived from a mouse carrying the portion of the short arm defined by the Sxra translocation on the distal end of its X chromosome. H-Y antigen-loss variant cells that contained Y-specific deletions were identified. Molecular, karyotypic, and immunological analysis of the deletion variants allowed us to define up to 16 ordered intervals and suggested an overall organization of Sxra. The analysis also suggests that at least two and up to five distinct loci encode H-Y antigens.
Asunto(s)
Antígeno H-Y/genética , Linfocitos T Citotóxicos/inmunología , Cromosoma Y , Animales , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Femenino , Antígeno H-Y/inmunología , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Datos de Secuencia Molecular , Eliminación de Secuencia , Cromosoma XRESUMEN
Bordetella pertussis adenylate cyclase (AC) toxin has the abilities to 1) enter target cells where it catalyzes cyclic AMP production and 2) lyse sheep erythrocytes, and these abilities require post-translational modification by the product of an accessory gene cyaC (Barry, E. M., Weiss, A. A., Ehrmann, E. E., Gray, M. C., Hewlett, E. L., and Goodwin, M. St. M. (1991) J. Bacteriol. 173, 720-726). In the present study, AC toxin has been purified from an organism with a mutation in cyaC, BPDE386, and evaluated for its physical and functional properties in order to determine the basis for its lack of toxin and hemolytic activities. AC toxin from BPDE386 is indistinguishable from wild-type toxin in enzymatic activity, migration on SDS-polyacrylamide gel electrophoresis, ability to bind calcium, and calcium-dependent conformational change. Although unable to elicit cAMP accumulation, AC toxin from BPDE386 exhibits binding to the surface of Jurkat cells which is comparable to that of wild-type toxin. This target cell interaction is qualitatively different, however, in that 99% of the mutant toxin remains sensitive to trypsin, whereas approximately 20% of cell-associated wild-type toxin enters a trypsin-resistant compartment. To evaluate the ability of this mutant AC toxin to function at its intracellular site of action, the cAMP-stimulated L-type calcium current in frog atrial myocytes was used. Extracellular addition of wild-type toxin results in cAMP-dependent events that include activation of calcium channels and enhancement of calcium current. In contrast, there is no response to externally applied toxin from BPDE386. When injected into the cell interior, however, the AC toxin from BPDE386 is able to produce increases in the calcium current comparable to those observed with wild-type toxin. Although AC toxin from BPDE386 is unaffected in its enzymatic activity, calcium binding, and calcium-dependent conformational change, the mutation in cyaC does result in a toxin which is able to bind to target cells but unable to elicit cAMP accumulation. In that AC toxin from BPDE386 is able to function normally when injected artificially to an intracellular site, we conclude that the disruption of cyaC produces a defect in insertion and transmembrane delivery of the catalytic domain.
Asunto(s)
Toxina de Adenilato Ciclasa , Adenilil Ciclasas/metabolismo , Bordetella pertussis/enzimología , Bordetella pertussis/genética , Genes Bacterianos , Genes Reguladores , Factores de Virulencia de Bordetella/genética , Adenilil Ciclasas/genética , Adenilil Ciclasas/aislamiento & purificación , Animales , Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Línea Celular , Células Cultivadas , AMP Cíclico/metabolismo , Electrofisiología/métodos , Corazón/efectos de los fármacos , Corazón/fisiología , Hemólisis , Humanos , Técnicas In Vitro , Modelos Biológicos , Conformación Proteica , Procesamiento Proteico-Postraduccional , Rana catesbeiana , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Espectrometría de Fluorescencia , Linfocitos T Colaboradores-Inductores , Factores de Virulencia de Bordetella/aislamiento & purificación , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Adenylate cyclase (AC) toxin from Bordetella pertussis enters cells to cause supraphysiologic increases in cAMP. AC toxin is also hemolytic. Substitution of Lys-58 with a methionine residue by site-directed mutagenesis of the structural gene for AC toxin, cyaA, and introduction of this mutation onto the B. pertussis chromosome results in an organism that synthesizes an enzyme-deficient AC toxin molecule. This mutant toxin molecule exhibits 1000-fold reduction in enzymatic activity relative to wild-type and has no toxin activity in J774 cells. The enzyme-deficient toxin molecule is not, however, impaired in its ability to lyse sheep red blood cells. In order to ascertain the importance of these two separate activities of AC toxin in vivo the enzyme-deficient organisms were used to infect infant mice. The hemolytic, enzyme-deficient mutant organisms are reduced in virulence relative to wild-type organisms after intranasal challenge indicating that, although the enzymatic activity of AC toxin does not contribute to hemolysis, it is this property of the toxin which is important for virulence of B. pertussis.
Asunto(s)
Toxina de Adenilato Ciclasa , Bordetella pertussis/patogenicidad , Hemólisis , Factores de Virulencia de Bordetella/farmacología , Animales , Animales Recién Nacidos , Bordetella pertussis/enzimología , Bordetella pertussis/genética , AMP Cíclico/metabolismo , Electroforesis en Gel de Poliacrilamida , Cinética , Pulmón/microbiología , Ratones , Mutagénesis Sitio-Dirigida , Mapeo Restrictivo , Factores de Virulencia de Bordetella/genética , Factores de Virulencia de Bordetella/metabolismo , Tos Ferina/microbiología , Tos Ferina/mortalidadRESUMEN
Adenylate cyclase (AC) toxin from Bordetella pertussis interacts with and enters eukaryotic cells to catalyze the production of supraphysiologic levels of cyclic AMP. Although the calmodulin-activated enzymatic activity (ability to convert ATP to cyclic AMP in a cell-free assay) of this molecule is calcium independent, its toxin activity (ability to increase cyclic AMP levels in intact target cells) requires extracellular calcium. Toxin activity as a function of calcium concentration is biphasic, with no intoxication occurring in the absence of calcium, low level intoxication (200-300 pmol of cyclic AMP/mg of Jurkat cell protein) occurring with free calcium concentrations between 100 nM and 100 microM and a 10-fold increase in AC toxin activity at free calcium concentrations above 300 microM. The molecule exhibits a conformational change when free calcium concentrations exceed 100 microM as demonstrated by shift in intrinsic tryptophan fluorescence, an alteration in binding of one anti-AC monoclonal antibody, protection of a fragment from trypsin-mediated proteolysis, and a structural modification as illustrated by electron microscopy. Thus, it appears that an increase in the ambient calcium concentration to a critical point and the ensuing interaction of the toxin with calcium induces a conformational change which is necessary for its insertion into the target cell and for delivery of its catalytic domain to the cell interior.
Asunto(s)
Toxina de Adenilato Ciclasa , Bordetella pertussis/metabolismo , Calcio/fisiología , Factores de Virulencia de Bordetella/metabolismo , Regulación Alostérica , Fluorescencia , Microscopía Electrónica , Conformación Proteica , Relación Estructura-Actividad , Tripsina , Factores de Virulencia de Bordetella/químicaRESUMEN
Adenylate cyclase (AC) toxin from B. pertussis enters eukaryotic cells where it produces supraphysiologic levels of cAMP. Purification of AC toxin activity [(1989) J. Biol. Chem. 264, 19279] results in increasing potency of hemolytic activity and electroelution of the 216-kDa holotoxin yields a single protein with AC enzymatic, toxin and hemolytic activities. AC toxin and E. coli hemolysin, which have DNA sequence homology [(1988) EMBO J. 7, 3997] are immunologically cross-reactive. The time courses of hemolysis elicited by the two molecules are strikingly different, however, with AC toxin eliciting cAMP accumulation with rapid onset, but hemolysis with a lag of greater than or equal to 45 min. Finally, osmotic protection experiments indicate that the size of the putative pore produced by AC toxin is 3-5-fold smaller than that of E. coli hemolysin.
Asunto(s)
Toxina de Adenilato Ciclasa , Bordetella pertussis/enzimología , Proteínas de Escherichia coli , Proteínas Hemolisinas , Hemólisis/efectos de los fármacos , Factores de Virulencia de Bordetella/toxicidad , Animales , Proteínas Bacterianas/genética , Western Blotting , Calcio/metabolismo , Reacciones Cruzadas , Eritrocitos/efectos de los fármacos , Ovinos , Factores de Virulencia de Bordetella/genéticaRESUMEN
In these studies, the Bordetella pertussis adenylate cyclase toxin-hemolysin homology to the Escherichia coli hemolysin is extended with the finding of cyaC, a homolog to the E. coli hlyC gene, which is required for the production of a functional hemolysin molecule in E. coli. Mutations produced in the chromosome of B. pertussis upstream from the structural gene for the adenylate cyclase toxin revealed a region which was necessary for toxin and hemolytic activities of the molecule. These mutants produced the 216-kDa adenylate cyclase toxin as determined by Western blot (immunoblot) analysis. The adenylate cyclase enzymatic activities of these mutants were equivalent to that of wild type, but toxin activities were less than 1% of that of wild type, and the mutants were nonhemolytic on blood agar plates and in in vitro assays. The upstream region restored hemolytic activity when returned in trans to the mutant strains. This genetic complementation defined a gene which acts in trans to activate the adenylate cyclase toxin posttranslationally. Sequence analysis of the upstream region defined an open reading frame with homology to the E. coli hlyC gene. In contrast to E. coli, this open reading frame is oriented oppositely from the adenylate cyclase toxin structural gene.