RESUMO
The WT1 gene is normally expressed in fetal kidney and mesothelium, and its expression has been suggested as a marker for Wilms tumor and mesothelioma. We examined WT1 expression levels by reverse-transcriptase polymerase chain reaction (RT-PCR) in 38 childhood small-cell tumors including Wilms tumor, embryonal and alveolar rhabdomyosarcoma, Ewing sarcoma, lymphoma, desmoplastic small round-cell tumor (DSRCT), synovial sarcoma, extrarenal rhabdoid tumor, and two tumors that were atypical for this group of tumors. WT1 expression was only detected in Wilms tumor, rhabdoid tumor, and in these two cases of uncertain histogenesis. Both arose in the peritoneal cavity and by immunohistochemistry were diffusely positive for vimentin, keratin, and desmin. Tonofilaments were identified by electron microscopy in one of the cases. RT-PCR failed to detect the t(11;22) translocation associated with DSRCT in either case. Our results suggest that WT1 expression is an unusual feature of childhood non-Wilms tumors and, in the right setting, it may indicate a mesothelial origin. The expression of WT1 may play a role in mesodermal cells acquiring epithelial characteristics, a concept supported by the mixed epithelial and mesenchymal phenotype of these two cases.
Assuntos
Carcinoma de Células Pequenas/metabolismo , Cromossomos Humanos Par 11 , Proteínas de Ligação a DNA/biossíntese , Fatores de Transcrição/biossíntese , Biomarcadores Tumorais , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Pré-Escolar , Proteínas de Ligação a DNA/genética , Epitélio/patologia , Humanos , Lactente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Linfoma/genética , Linfoma/metabolismo , Linfoma/patologia , Masculino , Mesotelioma/genética , Mesotelioma/metabolismo , Mesotelioma/patologia , Reação em Cadeia da Polimerase , Rabdomiossarcoma/genética , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Neoplasias Testiculares/genética , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologia , Fatores de Transcrição/genética , Proteínas WT1 , Tumor de Wilms/genética , Tumor de Wilms/metabolismo , Tumor de Wilms/patologia , Dedos de ZincoRESUMO
Rhabdomyosarcoma (RMS) is a malignancy of skeletal muscle derivation encompassing two major subtypes, embryonal and alveolar, which differ in clinical behavior and genetic markers. Because RMS is a relatively circumscribed tumor system for which the beginnings of a molecular genetic framework are in place, it becomes an ideal model for the application of improved methods of molecular genetic analysis. We have applied the technique known as differential display polymerase chain reaction (DD-PCR) to characterize expression of RNA in rhabdomyosarcoma subtypes. Our studies have shown that DD-PCR generates a characteristic electrophoretic profile that can be used to isolate subtype specific probes for fluorescence in situ hybridization (FISH) analysis. We have isolated two cDNA fragments and obtained clones suitable for FISH mapping to metaphase chromosomes. One probe was mapped to the centromeric region of human chromosome 22 and the other probe to the human chromosome band 6q25-26. This approach demonstrates the utility of DD-PCR as a technique for isolating novel cDNA expressed in tumors and their subsequent use as probes for FISH analysis. As more genes are identified by DD-PCR and their roles in tumorigenesis become defined, they are likely to provide novel targets for future molecular cytogenetic analysis.
Assuntos
Sondas de DNA/genética , DNA Complementar/genética , Hibridização in Situ Fluorescente , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/análise , RNA Neoplásico/análise , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Embrionário/genética , Sequência de Bases , Northern Blotting , Southern Blotting , Sondas de DNA/isolamento & purificação , DNA Complementar/isolamento & purificação , Humanos , Dados de Sequência MolecularRESUMO
The chromosome localizations for 159 gene and DNA segments have been refined to one of five intervals in the 7q21-132 region through hybridization analysis with a panel of somatic cell hybrid lines. Seventy-two of these chromosome 7 markers are also mapped on common or overlapping yeast artificial chromosome (YAC) clones. In addition, the breakpoints of chromosome rearrangement contained in five of the somatic cell hybrid lines have been defined by flanking probes within YAC contigs. To provide a framework for further mapping of the 7q21-q32 region, we have established the physical order of a set of reference markers: cen-(COL1A2-D7S15-CYP3A4-PON)-D7S456-(brea kpoint contained in cell hybrid 1EF2/3/K017)-GUSB-D7S186-ASL-(PGY1-PGY3 -GNB2-EPO-ACHE)-D7S238-(proximal breakpoint in GM1059-Rag5)-D7S240-(CUTL1-PLANH1)-(breakp oints in 1CF2/5/K016 and 2068Rag22-2)-(PRKAR2B-D7S13)-LAMB1-(breakpoint in JSR-17S)-DLD-D7S16-MET-WNT2-CFTR-D7S8-tel.
Assuntos
Passeio de Cromossomo , Cromossomos Fúngicos , Cromossomos Humanos Par 7 , Biblioteca Gênica , Genoma Humano , Sequência de Bases , DNA/genética , Sondas de DNA , Marcadores Genéticos , Humanos , Células Híbridas , Dados de Sequência MolecularRESUMO
We have cloned the mouse homolog of the human cystic fibrosis transmembrane conductance regulator (CFTR) using clones isolated from a mouse lung cDNA library and using amplification of cDNA to isolate specific regions. The cDNA was 6304 bp in length and encoded a polypeptide of 1476 amino acids. Comparison of the deduced amino acid sequence showed that the mouse protein has high homology to the human protein; overall identity was 78.3%. The amino acid identity was high for both transmembrane domains (first transmembrane domain, 86.7%; second transmembrane domain, 81.1%) and for both ATP-binding folds (first ATP-binding fold, 80.5%; second ATP-binding fold, 83.9%), suggesting the functional importance of these regions. On the other hand, the R domain was less well conserved (68.9% identity). All of the published missense mutation sites and the site of the common delta F508 mutation were conserved between human and mouse.
Assuntos
Proteínas de Membrana/genética , Camundongos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Regulador de Condutância Transmembrana em Fibrose Cística , DNA/genética , Genes , Humanos , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7. Transcripts, approximately 6500 nucleotides in size, were detectable in the tissues affected in patients with CF. The predicted protein consists of two similar motifs, each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in ATP (adenosine triphosphate) binding. A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients.
Assuntos
Fibrose Cística/genética , DNA/isolamento & purificação , Genes Recessivos , Genes , Proteínas de Membrana/genética , Peptídeos/genética , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Clonagem Molecular/métodos , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística , Humanos , Canais Iônicos/patologia , Proteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Peptídeos/isolamento & purificação , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
To facilitate mapping of the cystic fibrosis locus (CF) and to isolate the corresponding gene, we have screened a flow-sorted chromosome 7-specific library for additional DNA markers in the 7q31-q32 region. Unique ("single-copy") DNA segments were selected from the library and used in hybridization analysis with a panel of somatic cell hybrids containing various portions of human chromosome 7 and patient cell lines with deletion of this chromosome. A total of 258 chromosome 7-specific single-copy DNA segments were identified, and most of them localized to subregions. Fifty three of these corresponded to DNA sequences in the 7q31-q32 region. Family and physical mapping studies showed that two of the DNA markers, D7S122 and D7S340, are in close linkage with CF. The data also showed that D7S122 and D7S340 map between MET and D7S8, the two genetic markers known to be on opposite sides of CF. The study thus reaffirms the general strategy in approaching a disease locus on the basis of chromosome location.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 7 , Fibrose Cística/genética , Sondas de DNA , Marcadores Genéticos , Animais , Bandeamento Cromossômico , Clonagem Molecular , Humanos , Células Híbridas , Camundongos , Hibridização de Ácido Nucleico , Polimorfismo de Fragmento de Restrição , Sequências Repetitivas de Ácido NucleicoRESUMO
A linkage has been detected between the locus for cystic fibrosis (CF) and the pro alpha 2(I) collagen gene (COL1A2) which is located in the region q21.3----q22.1 of chromosome 7. Based on the combined linkage data derived from 50 informative two-generation nuclear families collected in Canada and Denmark, the distance between COL1A2 and CF is estimated to be 19 centiMorgans. Close linkage has also been detected between COL1A2 and the DNA marker D7S15 (formerly D0CRI-917) and the serum enzyme activity marker paraoxonase (PON), both of which have previously been found linked to CF. The results of the two-point and three-point linkage analyses indicate that the most probable order of these four genetic loci is COL1A2-D7S15 - PON - CF.
Assuntos
Cromossomos Humanos 6-12 e X , Fibrose Cística/genética , Genes , Ligação Genética , Pró-Colágeno/genética , Enzimas de Restrição do DNA , Feminino , Humanos , Masculino , Linhagem , Polimorfismo Genético , ProbabilidadeRESUMO
A polymorphic DNA marker has been found genetically linked, in a set of 39 human families, to an autosomal recessive gene that causes cystic fibrosis (CF), a disease affecting one in 2000 Caucasian children. The DNA marker (called D0CRI-917) is also linked to the PON locus, which by independent evidence is linked to the CF locus. The best estimates of the genetic distances are 5 centimorgans between the DNA marker and PON and 15 centimorgans between the DNA marker and the CF locus, meaning that the location of the disease gene has been narrowed to about 1 percent of the human genome (about 30 million base pairs). Although the data are consistent with the interpretation that a single locus causes cystic fibrosis, the possibility of genetic heterogeneity remains. The discovery of a linked DNA polymorphism is the first step in molecular analysis of the CF gene and its causative role in the disease.
