RESUMEN
Tissue microarrays have been created from 326 lung tumours, including 173 squamous cell carcinomas (SCCs) and 132 adenocarcinomas (ADs). In order to evaluate the usefulness of this microarray series, the expression of p53, p16, and Rb proteins was compared by immunohistochemistry on both the tissue microarrays and the corresponding whole sections for all 326 tumours. The presence of replicate punches improved both the yield and the concordance of data relative to the whole section results, so that the consensus score from the replicates agreed with the whole section result in more than 90% of informative tumours. The large number of tumours in this series also allowed significant differences in protein expression patterns to be detected between SCC and AD, the major subtypes of non-small cell lung carcinoma (NSCLC). SCC had higher levels of p53 staining (67% vs 52% in AD) and substantially increased p16 loss (SCC 75%, AD 53%) combined with greater retention of pRB expression (SCC 86% vs 67% in AD). The strong inverse correlation between p16 and pRB seen in SCC was essentially absent in AD. This study represents the largest single immunohistochemical survey of protein expression for p53, p16, and RB in NSCLCs.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adulto , Distribución por Edad , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , ADN de Neoplasias/genética , Femenino , Humanos , Técnicas para Inmunoenzimas , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Estadificación de Neoplasias , Reproducibilidad de los Resultados , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
A putative novel murine serine protease, DISP, was identified by cDNA indexing and shown to be expressed primarily in distal gut. FISH analysis showed it to be localised to mouse chromosome 17A3. A possible human homologue for DISP has been identified. DISP is a novel member of clan SA/family S1 of the serine proteases, at present of unknown function.
Asunto(s)
Intestino Delgado/enzimología , Serina Endopeptidasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/análisis , Electroforesis en Gel de Agar , Células Epiteliales/metabolismo , Expresión Génica , Íleon/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina Endopeptidasas/análisis , Serina Endopeptidasas/químicaAsunto(s)
Análisis Citogenético , Genoma Humano , Proyecto Genoma Humano , Mapeo Físico de Cromosoma/métodos , Cromosomas Bacterianos/genética , Cromosomas Humanos Par 22/genética , Cromosomas Humanos Par 7/genética , Clonación Molecular , Mapeo Contig , Humanos , Hibridación Fluorescente in Situ , Lugares Marcados de SecuenciaRESUMEN
The hallmark of chronic myeloid leukemia (CML) is the BCR-ABL fusion gene, which is usually formed as a result of the t(9;22) translocation. Patients with CML show considerable heterogeneity both in their presenting clinical features and in the time taken for evolution to blast crisis. In this study, metaphase fluorescence in situ hybridization showed that a substantial minority of patients with CML had large deletions adjacent to the translocation breakpoint on the derivative 9 chromosome, on the additional partner chromosome in variant translocations, or on both. The deletions spanned up to several megabases, had variable breakpoints, and could be detected by microsatellite polymerase chain reaction in unfractionated bone marrow and purified peripheral blood granulocytes. The deletions were likely to occur early and possibly at the time of the Philadelphia (Ph) chromosome translocation: deletions were detected at diagnosis in 11 patients, were found in all Ph-positive metaphases, and were more prevalent in patients with variant Ph chromosomes. Kaplan-Meier analysis showed a median survival time of 36 months in patients with a deletion; patients without a detectable deletion survived > 90 months. The survival-time difference was significant on log-rank analysis (P =. 006). Multivariate analysis demonstrated that the prognostic importance of deletion status was independent of age, sex, percentage of peripheral blood blasts, and platelet count. Our data therefore suggest that an apparently simple, balanced translocation may result not only in the generation of a dominantly acting fusion oncogene but also in the loss of one or more genes that influence disease progression. (Blood. 2000;95:738-743)
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Eliminación de Secuencia , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Femenino , Humanos , Hibridación Fluorescente in Situ , Leucemia Mielógena Crónica BCR-ABL Positiva/mortalidad , Tablas de Vida , Masculino , Repeticiones de Microsatélite , Persona de Mediana Edad , Cromosoma Filadelfia , Reacción en Cadena de la Polimerasa , Pronóstico , Modelos de Riesgos Proporcionales , Análisis de SupervivenciaRESUMEN
We have established a landmark framework map over 20-25 Mb of the long arm of the human X chromosome using yeast artificial chromosome (YAC) clones. The map has approximately one landmark per 45 kb of DNA and stretches from DXS7531 in proximal Xq23 to DXS895 in proximal Xq26, connecting to published framework maps on its proximal and distal sides. There are three gaps in the framework map resulting from the failure to obtain clone coverage from the YAC resources available. Estimates of the maximum sizes of these gaps have been obtained. The four YAC contigs have been positioned and oriented using somatic-cell hybrids and fluorescence in situ hybridization, and the largest is estimated to cover approximately 15 Mb of DNA. The framework map is being used to assemble a sequence-ready map in large-insert bacterial clones, as part of an international effort to complete the sequence of the X chromosome. PAC and BAC contigs currently cover 18 Mb of the region, and from these, 12 Mb of finished sequence is available.
Asunto(s)
Mapeo Cromosómico/métodos , Cromosoma X/genética , Southern Blotting , Cromosomas Artificiales de Levadura/genética , Mapeo Contig , Electroforesis en Gel de Campo Pulsado , Femenino , Marcadores Genéticos/genética , Humanos , Hibridación Fluorescente in Situ , Análisis de Secuencia de ADNRESUMEN
X-linked lymphoproliferative syndrome (XLP or Duncan disease) is characterized by extreme sensitivity to Epstein-Barr virus (EBV), resulting in a complex phenotype manifested by severe or fatal infectious mononucleosis, acquired hypogammaglobulinemia and malignant lymphoma. We have identified a gene, SH2D1A, that is mutated in XLP patients and encodes a novel protein composed of a single SH2 domain. SH2D1A is expressed in many tissues involved in the immune system. The identification of SH2D1A will allow the determination of its mechanism of action as a possible regulator of the EBV-induced immune response.
Asunto(s)
Proteínas Portadoras/genética , Infecciones por Herpesviridae/complicaciones , Herpesvirus Humano 4 , Péptidos y Proteínas de Señalización Intracelular , Trastornos Linfoproliferativos/genética , Mutación , Dominios Homologos src/genética , Antígenos CD , Linfocitos B/inmunología , Linfocitos B/virología , Proteínas Portadoras/metabolismo , Clonación Molecular , Femenino , Ligamiento Genético , Glicoproteínas/metabolismo , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Humanos , Inmunoglobulinas/metabolismo , Trastornos Linfoproliferativos/complicaciones , Trastornos Linfoproliferativos/inmunología , Trastornos Linfoproliferativos/virología , Masculino , Datos de Secuencia Molecular , Linaje , Receptores de Superficie Celular , Alineación de Secuencia , Eliminación de Secuencia , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Linfocitos T/inmunología , Linfocitos T/virología , Cromosoma XRESUMEN
To investigate patterns of genetic recombination within a heterozygous paracentric inversion of chromosome 9 (46XY inv[9] [q32q34.3]), we performed sperm typing using a series of polymorphic microsatellite markers spanning the inversion region. For comparison, two donors with cytogenetically normal chromosomes 9, one of whom was heterozygous for a pericentric chromosome 2 inversion (46XY inv[2] [p11q13]), were also tested. Linkage analysis was performed by use of the multilocus linkage-analysis program SPERM, and also CRI-MAP, which was adapted for sperm-typing data. Analysis of the controls generated a marker order in agreement with previously published data and revealed no significant interchromosomal effects of the inv(2) on recombination on chromosome 9. FISH employing cosmids containing appropriate chromosome 9 markers was used to localize the inversion breakpoint of inv(9). Analysis of inv(9) sperm was performed by use of a set of microsatellite markers that mapped centromeric to, telomeric to, and within the inversion breakpoints. Three distinct patterns of recombination across the region were observed. Proximal to the centromeric breakpoint, recombination was similar to normal levels. Distal to the telomeric breakpoint, there was an increase in recombination found in the inversion patient. Finally, within the inversion, recombination was dramatically reduced, but several apparent double recombinants were found. A putative model explaining these data is proposed.
Asunto(s)
Inversión Cromosómica , Cromosomas Humanos Par 9 , Meiosis/genética , Recombinación Genética , Espermatozoides/fisiología , Adulto , Estudios de Casos y Controles , Variación Genética , Heterocigoto , Humanos , Masculino , Repeticiones de MicrosatéliteAsunto(s)
Cromosomas Humanos Par 22/genética , Proteínas Quinasas Activadas por Mitógenos , Proteínas Quinasas/genética , Animales , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/genética , Humanos , Hibridación Fluorescente in Situ , Proteína Quinasa 12 Activada por Mitógenos , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasRESUMEN
Our aim is to construct physical clone maps covering those regions of chromosome 6 that are not currently extensively mapped, and use these to determine the DNA sequence of the whole chromosome. The strategy we are following involves establishing a high density framework map of the order of 15 markers per Megabase using radiation hybrid (RH) mapping. The markers are then used to identify large-insert genomic bacterial clones covering the chromosome, which are assembled into sequence-ready contigs by restriction enzyme fingerprinting and sequence tagged site (STS) content analysis. Contig gap closure is performed by walking experiments using STSs developed from the end sequences of the clone inserts.
Asunto(s)
Cromosomas Humanos Par 6/genética , Mapeo Contig , Bases de Datos Factuales , Humanos , Análisis de Secuencia de ADNRESUMEN
We have identified a novel mouse gene encoding a protein that shows high homology to the dual-specificity tyrosine/threonine phosphatase family of proteins. The gene encodes a 5 kb transcript which is expressed predominantly in brain and lung and contains a translated complex trinucleotide repeat within the coding region. Using interspecific mouse backcross analysis, the gene has been localised to distal mouse chromosome 7. In human, homologous sequences are located in the syntenic region on distal chromosome 11p as well as to chromosome 10q11.2 and 10q22. The presence of a CG-rich trinucleotide repeat in the coding region provides a target for mutation which might result in loss of function or altered properties of this phosphatase.
Asunto(s)
Proteínas Tirosina Fosfatasas/genética , Repeticiones de Trinucleótidos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Química Encefálica , Cromosomas Humanos Par 10 , Cromosomas Humanos Par 11 , ADN Complementario/química , Humanos , Hibridación Fluorescente in Situ , Ratones , Microinyecciones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Transducción de Señal/genética , Fracciones Subcelulares/química , Transcripción Genética , TransfecciónRESUMEN
We have constructed a high-resolution clone map of human chromosome 22 which integrates the available physical and genetic information, establishing a single consensus. The map consists of all classes of DNA landmarks ordered on 705 yeast artificial chromosomes (YACs) at an average landmark density of more than one per 70 kilobases. This map represents the practical limits of currently available YAC resources and provides the basis for determination of the entire gene content and genomic DNA sequence of human chromosome 22.
Asunto(s)
Cromosomas Artificiales de Levadura , Cromosomas Humanos Par 22 , Mapeo Cromosómico/métodos , Marcadores Genéticos , Humanos , Lugares Marcados de SecuenciaRESUMEN
We report an infant with pre- and postnatal microcephaly and growth retardation, a distinctive face, and developmental delay. The initial diagnosis was of Seckel syndrome. He became pancytopenic at 16 months and died soon after. His bone marrow was of normal cellularity but had a small lymphocyte infiltration. Increased spontaneous chromosome breakage was seen in blood and fibroblasts. Mitomycin C induced chromosome damage was increased and comparable to that seen in Fanconi anaemia. Reports of similar patients are reviewed. This entity of severe intrauterine growth retardation and increased mitomycin C sensitivity is hypothesised to be a distinct chromosome breakage syndrome.
Asunto(s)
Aberraciones Cromosómicas/genética , Retardo del Crecimiento Fetal/etiología , Retardo del Crecimiento Fetal/genética , Mitomicina/farmacología , Adulto , Células Cultivadas , Trastornos de los Cromosomas , Fragilidad Cromosómica , Daño del ADN , Reparación del ADN/genética , Resistencia a Medicamentos , Femenino , Retardo del Crecimiento Fetal/complicaciones , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Edad Gestacional , Humanos , Lactante , Recién Nacido , Linfocitos/fisiología , Masculino , Fenotipo , Embarazo , Intercambio de Cromátides Hermanas , Piel/ultraestructura , Rayos XRESUMEN
We report the construction of a physical map based on cloned DNA within the candidate region for the tuberous sclerosis complex (TSC1) gene on chromosome 9q34, between the markers D9S149 and D9S66. The DNA clones form three contigs consisting of 7 YACs, bridged by P1 and cosmid clones, and cover more than 950 kb of 9q34. Despite intensive screening of all available libraries, two gaps remain. A detailed physical map of much of this region was derived, and restriction mapping of the YAC, P1, and cosmid clones reveals novel CpG islands in this region. This set of genomic clones provides a resource for characterizing candidates for the TSC1 gene, guided by the location of CpG islands.
Asunto(s)
Cromosomas Humanos Par 9 , Esclerosis Tuberosa/genética , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Artificiales de Levadura , Clonación Molecular , Cósmidos , Cartilla de ADN , Sondas de ADN , Marcadores Genéticos , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Mapeo RestrictivoRESUMEN
The tissue-specific expression of an antigen (P43) ubiquitously expressed at high levels in a variety of tumours of human and animal origin was investigated using a monoclonal antibody to P43. Whereas low amounts of P43 are expressed in the spleen, skeletal muscle and pancreas, P43 is abundantly produced in the liver and in other tissues such as the kidney, heart and brain which have levels of oxidative metabolism. Interestingly, a related protein of higher molecular weight is abundantly expressed in the lung and in amounts which were higher than those observed with other tissues. The human cDNA for P43 was isolated from a human liver cDNA library and mapped to chromosome 16 between p11.2 and 12 and also to a position near the centromere on the long arm of chromosome 17. The deduced amino acid sequence of P43 is remarkably similar to that of E. coli EF-Tu and the mitochondrial EF-Tu of S. cerevisiae with the structurally and functionally important amino acids of EF-Tu being completely conserved in P43. A comparison of the distribution of P43 and a mitochondrial protein Hsp 60 among different cellular fractions indicated a likely mitochondrial localisation for P43. Taken together these results suggest that P43 is a human mitochondrial elongation factor.
Asunto(s)
Antígenos de Neoplasias/biosíntesis , Mitocondrias/metabolismo , Factor Tu de Elongación Peptídica/biosíntesis , Factores de Elongación de Péptidos/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Células Cultivadas , Mapeo Cromosómico , Cromosomas Humanos Par 16 , Cromosomas Humanos Par 17 , ADN Complementario , Humanos , Hibridación in Situ , Masculino , Ratones , Proteínas Mitocondriales , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
A novel transcript containing homology to exons 5, 9, 10, and 11 of the beta-glucuronidase gene has been shown to be derived from Chromosome (Chr) 5. In situ hybridization analysis has shown that this transcript is homologous to four loci on Chr 5 (5p13.3, 5p15.1, 5q13.1, and 5q15), two loci on Chr 6 (6p11.2 and 6p21.3), and one on Chr 22 at 22q11.2. Analysis of cosmid clones from Chr 5 has defined three distinct contigs in which there are tandem genomic repeats of a unit containing sequences homologous to exons 5, 9, and 10 but not 11. Pulsed-field gel electrophoresis (PFGE) analysis has shown that the length of these repeats is highly variable between unrelated individuals, indicating that these regions of Chr 5 are prone to rearrangement. These sequences may be important with respect to stability around the Chr 5 centromere.
Asunto(s)
Cromosomas Humanos Par 5 , Reordenamiento Génico , Glucuronidasa/genética , Seudogenes , Secuencia de Bases , Cósmidos/genética , ADN Complementario , Electroforesis en Gel de Campo Pulsado/métodos , Exones , Expresión Génica , Humanos , Datos de Secuencia Molecular , Linaje , Polimorfismo de Longitud del Fragmento de Restricción , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido NucleicoRESUMEN
Laminin is a basement membrane glycoprotein composed of three nonidentical chains, A, B1, and B2. Variant chains such as merosin and S-laminin have been found in different tissues. We have isolated a cDNA encoding a novel laminin A variant that hybridizes to a 6.45-kb mRNA. Using amplification of genomic DNA and flow-sorted chromosomes we have assigned the gene (LAMA4) for this new laminin A variant to chromosome 6. Fluorescence in situ hybridization of a YAC clone further localized the gene to 6q21.
Asunto(s)
Cromosomas Humanos Par 6 , ADN Complementario/genética , Laminina/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Secuencia de Consenso , Cartilla de ADN/genética , Variación Genética , Humanos , Laminina/química , Datos de Secuencia Molecular , Estructura MolecularRESUMEN
Molecular genetic analysis of five cases of 3p- syndrome (del(3)(qter-->p25:)) was performed to investigate the relationship between the molecular pathology and clinical phenotype. Fluorescence in situ hybridization studies and analysis of polymorphic DNA markers from chromosome 3p25-p26 demonstrated that all four informative cases had distal deletions. However, the extent of the deletion was variable: in two patients with the most extensive deletions the deletion breakpoint mapped between RAF1 and D3S1250, in one patient the deletion breakpoint was between D3S1250 and D3S601, and in two patients the deletion commenced telomeric to D3S601 (and telomeric to D3S1317 in one of these). All five patients displayed the classical features of 3p- syndrome (mental retardation, growth retardation, microcephaly, ptosis and micrognathia) demonstrating that loss of sequences centromeric to D3S1317 is not required for expression of the characteristic 3p- syndrome phenotype. The three patients with the most extensive deletions had cardiac septal defects suggesting that a gene involved in normal cardiac development is contained in the interval D3S1250 and D3S18. The PMCA2 gene is contained within this region and deletion of this gene may cause congenital heart defects. At least three patients were deleted for the von Hippel-Lindau (VHL) disease gene although none had yet developed evidence of VHL disease. We conclude that molecular analysis of 3p- syndrome patients enhances the management of affected patients by identifying those at risk for VHL disease, and can be used to elucidate the critical regions for the 3p- syndrome phenotype.
Asunto(s)
Anomalías Múltiples/genética , Deleción Cromosómica , Cromosomas Humanos Par 3 , Adulto , Línea Celular , Niño , Mapeo Cromosómico , Femenino , Estudios de Seguimiento , Marcadores Genéticos , Humanos , Hibridación Fluorescente in Situ , Lactante , Linfocitos , Masculino , Fenotipo , Polimorfismo Genético , SíndromeRESUMEN
We have developed a novel coincidence cloning strategy, termed Coincidence Painting, which enables the rapid generation of large numbers of region specific sequences. Coincidence Painting utilises Degenerate Oligonucleotide Primed PCR (DOP-PCR) amplification of flow sorted derivative translocation chromosomes. The PCR products are hybridised in situ onto specific flow sorted chromosomes for coincident sequence selection. Eluted and reamplified material is then cloned using a novel insert end revelation and ligation technique. Cloned inserts range in size from 150-1300 bps of which approximately 54% appear to be single copy sequences. The cloning method permits the excision of vector free probe for library hybridisation screening and the small insert size facilitates analysis for the generation of sequence tagged sites (STSs). We have used such clones successfully for YAC screening by PCR and for cosmid screening by filter hybridisation. This new methodology should allow the rapid saturation with probes of regions defined by specific translocation breakpoints.