RESUMEN
The 14 kb mRNA of the polycystic kidney disease gene PKD1 encodes a novel large (approximately 460 kDa) protein, polycystin-1, of unknown function that is responsible for autosomal dominant polycystic kidney disease (ADPKD). The unique organization of multiple adhesive domains of polycystin-1, including 16 Ig-like domains (or PKD domains) suggests that it may play an important role in cell-cell/cell-matrix interactions. Here we demonstrate the localization of polycystin-1 to epithelial cell-cell contacts in culture. These results along with structural predictions prompted us to propose that polycystin-1 is involved in cell-cell adhesion through its cluster of Ig-like repeats. We show that Ig-like domains II-XVI are involved in strong calcium-independent homophilic interactions in vitro. Domains XI-XVI form interactions with high affinity (K(d) = 60 nM) and domains II-V exhibit the lowest binding affinity (K(d) = 730 nM) in these studies. Most importantly, we show that antibodies raised against Ig-like domains of polycystin-1 disrupt cell-cell interactions in MDCK cell monolayers, thus indicating that polycystin-1 is directly involved in the cell-cell adhesion process. Collectively, these data suggest that interactions of the Ig-like repeats of polycystin-1 play an important role in mediating intercellular adhesion. We suggest that the loss of these interactions due to mutations in polycystin-1 may be an important step in cystogenesis.
Asunto(s)
Riñón Poliquístico Autosómico Dominante/genética , Proteínas/metabolismo , Animales , Anticuerpos/inmunología , Sitios de Unión , Unión Competitiva , Adhesión Celular , Línea Celular , Técnica del Anticuerpo Fluorescente , Cinética , Proteínas/genética , Proteínas/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Canales Catiónicos TRPPRESUMEN
The primary structure of polycystin predicts a large integral membrane protein with multiple cell recognition motifs, but its function remains unknown. Insight into polycystin's normal function and its role in the development of autosomal dominant polycystic kidney disease (PKD1) requires the assembly of an extensive collection of molecular reagents to examine its expression and create model systems for functional studies. Development of these crucial reagents has been complicated due to the presence of transcriptionally active homologous loci. We have assembled the authentic full-length PKD1 cDNA and demonstrated expression of polycystin in vitro. Polyclonal antibodies directed against distinct extra- and intracellular domains specifically immunoprecipitated in vitro translated polycystin. The panel of antibodies was used to determine localization of polycystin in renal epithelial and endothelial cell lines and tissues of fetal, adult, and cystic origins. In normal adult kidney and maturing fetal nephrons, polycystin expression was confined to epithelial cells of the distal nephron and vascular endothelial cells. Expression in the proximal nephron was only observed after injury-induced cell proliferation. Polycystin expression was confined to ductal epithelium in liver, pancreas, and breast, and restricted to astrocytes in normal brain. We report clear evidence for the membrane localization of polycystin by both tissue sections and by confocal microscopy in cultured renal and endothelial cells. Interestingly, when cultured cells made cell-cell contact, polycystin was localized to the lateral membranes of cells in contact. These data suggest that polycystin is likely to have a widespread role in epithelial cell differentiation and maturation and in cell-cell interactions.
Asunto(s)
Riñón/metabolismo , Biosíntesis de Proteínas , Adulto , Encéfalo/embriología , Encéfalo/metabolismo , Línea Celular , Células Cultivadas , ADN Complementario , Endotelio Vascular/metabolismo , Epitelio/metabolismo , Feto , Biblioteca de Genes , Humanos , Nefronas/embriología , Nefronas/metabolismo , Especificidad de Órganos , Riñón Poliquístico Autosómico Dominante , Reacción en Cadena de la Polimerasa , Proteínas/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Fracciones Subcelulares/metabolismo , Canales Catiónicos TRPPRESUMEN
As part of an effort to identify the gene responsible for the predominant form of polycystic kidney disease (PKD1), we used a gridded human P1 library for contig assembly. The interval of interest, a 700-kb segment on chromosome 16p13.3, can be physically delineated by the genetic markers D16S125 and D16S84 and chromosomally characterized as a GC-rich isochore enriched for CpG islands, genes, and Alu-like repeats. Our attempts to recover CEPH YACs that encode this region of chromosome 16 were unsuccessful. However, we screened an arrayed P1 library using 15 distinct probes from the D16S125-D16S84 interval and identified 56 independent P1 clones. Only one probe from the interval was unsuccessful in identifying a P1 clone. Forty-four P1 clones were determined to be unique based on restriction enzyme analysis, and 42 of these were found to originate from chromosome 16p13.3, based on FISH to metaphase chromosomes. The 700-kb interval could be defined by a single sequence-ready contig comprised of 12 P1 clones and 1 cosmid clone. Our studies support the use of multiple libraries to generate the requisite physical reagents for positional cloning and encourage the use of Escherichia coli-based large-insert cloning systems to recover clones from YAC-deficient chromosomal intervals.
Asunto(s)
Enfermedades Genéticas Congénitas/genética , Riñón Poliquístico Autosómico Dominante/genética , Bacteriófago P1/genética , Mapeo Cromosómico , Cromosomas Humanos Par 16/genética , Clonación Molecular , Cósmidos/genética , Biblioteca de Genes , Marcadores Genéticos/genética , Humanos , Hibridación Fluorescente in Situ , Lugares Marcados de SecuenciaRESUMEN
A 700-kb region of DNA in human chromosome 16p13.3 has been shown to contain the polycystic kidney disease 1 (PKD1) and the tuberous sclerosis type 2 (TSC2) disease genes. An estimated 20 genes are present in this region of chromosome 16. We have initiated studies to identify transcribed sequences in this region using a bacteriophage P1 contig containing 700 kb of DNA surrounding the PKD1 and TSC2 genes. We have isolated 96 unique exon traps from this interval, with 23 of the trapped exons containing sequences from five genes known to be in the region. Thirty exon traps have been mapped to additional transcription units based on data base homologies, Northern analysis, or their presence in cDNA or reverse transcriptase (RT)-PCR products. We have mapped the human RNPS gene to the cloned interval. We have obtained cDNAs or RT-PCR products from eight novel genes, with sequences from seven of these genes having homology to sequences in the data bases. Two of the newly identified genes represent human homologs for rat and murine genes identified previously. We have isolated three exon traps with homology to sequences in the data bases but have been unable to confirm the presence of these exon traps in expressed sequences. In addition, we have isolated 43 exon traps that do not map to our existing cDNAs or PCR products and have no homology to sequences in the data bases. In this report we present a transcriptional map for the 700 kb of DNA surrounding the PKD1 and TSC2 genes.
Asunto(s)
Mapeo Cromosómico , Riñón Poliquístico Autosómico Dominante/genética , Transcripción Genética/genética , Esclerosis Tuberosa/genética , Secuencia de Aminoácidos , Bacteriófago P1/genética , Northern Blotting , Cromosomas Humanos Par 16/genética , Clonación Molecular , ADN Complementario/genética , Bases de Datos Factuales , Exones/genética , Enfermedades Genéticas Congénitas/genética , Marcadores Genéticos , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas , Seudogenes , Alineación de Secuencia , Lugares Marcados de Secuencia , Canales Catiónicos TRPPRESUMEN
The complete genomic sequence of the gene responsible for the predominant form of polycystic kidney disease, PKD1, was determined to provide a framework for understanding the biology and evolution of the gene, and to aid in the development of molecular diagnostics. The DNA sequence of a 54 kb interval immediately upstream of the poly(A) addition signal sequence of the PKD1 transcript was determined, and then analyzed using computer methods. A leucine-rich repeat (LRR) motif was identified within the resulting predicted protein sequence of the PKD1 gene. By analogy with other LRR-containing proteins, this may explain some of the disease-related renal alterations such as mislocalization of membrane protein constituents and changes in the extracellular matrix organization. Finally, comparison of the genomic sequence and the published partial cDNA sequence showed several differences between the two sequences. The most significant difference detected predicts a novel carboxy-terminus for the PKD1 gene product.
Asunto(s)
Leucina/genética , Riñón Poliquístico Autosómico Dominante/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario , Genes Dominantes , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
The constituents of two cosmid contigs were analyzed by high resolution mapping using two-color fluorescence in situ hybridization (FISH) to extended DNA molecules. Samples were prepared by lysing the nuclei in situ followed by histone depletion. This treatment results in elongate DNA filaments appropriate for high resolution mapping. The hybridization signals appeared as a strong of fluorescent spots separated by non-fluorescing gaps. Probe-specific features of the hybridization patterns were detected and some of the non-fluorescing gaps were found to represent regions of repetitive DNA suppressed during hybridization.
Asunto(s)
Mapeo Cromosómico , Cósmidos , Amnios/química , Amnios/citología , Southern Blotting , Células Cultivadas , ADN/análisis , ADN/genética , Citometría de Flujo/métodos , Humanos , Hibridación Fluorescente in SituRESUMEN
Detection of chromosome aneuploidies in uncultured amniocytes is possible using fluorescence in situ hybridization (FISH). We herein describe the results of the first clinical program which utilized FISH for the rapid detection of chromosome aneuploidies in uncultured amniocytes. FISH was performed on physician request, as an adjunct to cytogenetics in 4,500 patients. Region-specific DNA probes to chromosomes 13, 18, 21, X, and Y were used to determine ploidy by analysis of signal number in hybridized nuclei. A sample was considered to be euploid when all autosomal probes generated two hybridization signals and when a normal sex chromosome pattern was observed in greater than or equal to 80% of hybridized nuclei. A sample was considered to be aneuploid when greater than or equal to 70% of hybridized nuclei displayed the same abnormal hybridization pattern for a specific probe. Of the attempted analyses, 90.2% met these criteria and were reported as informative to referring physicians within 2 d of receipt. Based on these reporting parameters, the overall detection rate for aneuploidies was 73.3% (107/146), with an accuracy of informative results for aneuploidies of 93.9% (107/114). Compared to cytogenetics, the accuracy of all informative FISH results, euploid and aneuploid, was 99.8%, and the specificity was 99.9%. In those pregnancies where fetal abnormalities had been observed by ultrasound, referring physicians requested FISH plus cytogenetics at a significantly higher rate than they requested cytogenetics alone. The current prenatal FISH protocol is not designed to detect all chromosome abnormalities and should only be utilized as an adjunctive test to cytogenetics. This experience demonstrates that FISH can provide a rapid and accurate clinical method for prenatal identification of chromosome aneuploidies.
Asunto(s)
Aneuploidia , Enfermedades Fetales/diagnóstico , Hibridación Fluorescente in Situ , Diagnóstico Prenatal/métodos , Adulto , Aberraciones Cromosómicas , Cromosomas Humanos Par 13 , Cromosomas Humanos Par 18 , Cromosomas Humanos Par 21 , Estudios de Evaluación como Asunto , Reacciones Falso Negativas , Reacciones Falso Positivas , Femenino , Humanos , Edad Materna , Valor Predictivo de las Pruebas , Embarazo , Embarazo de Alto Riesgo , Reproducibilidad de los Resultados , Cromosoma X , Cromosoma YRESUMEN
Two different mapping approaches were used to determine the human chromosomal location of the gene for protein S. A human protein S cDNA was used as a hybridization probe to analyze a panel of somatic cell hybrids containing different human chromosomes. Cosegregation of protein S-specific DNA restriction fragments with human chromosome 3 was observed. Three cell hybrids containing only a portion of chromosome 3 were analyzed in order to further localize protein S. Based on the somatic cell hybrid analysis, protein S is assigned to a region of chromosome 3 that contains a small part of the long arm and short arm of the chromosome including the centromere (3p21----3q21). In situ hybridization of the protein S cDNA probe to human metaphase chromosomes permitted a precise localization of protein S to the region of chromosome 3 immediately surrounding the centromere (3p11.1----3q11.2). Protein S is the first protein involved in blood coagulation that has been mapped to human chromosome 3.
Asunto(s)
Cromosomas Humanos Par 3 , Glicoproteínas/genética , Animales , Mapeo Cromosómico , ADN/análisis , Humanos , Células Híbridas/análisis , Ratones , Hibridación de Ácido Nucleico , Proteína SRESUMEN
Tissue-type plasminogen activator (tPA) cDNA derived from human uterine mRNA was inserted into different yeast expression vectors. All such expression plasmids carried a yeast acid phosphatase (PHO5) promoter, a 2-micron plasmid replication origin, transcription termination signals, and a selectable TRP1 gene. Plasmid pYBDT-10 contained the entire tPA coding region ("pre-pro-tPA"), pYBDT-10-PRO contained a sequence encoding the putative pro-tPA precusor, and pYBDT-6 contained only a mature tPA cDNA fused precisely in frame to the sequence encoding the entire signal peptide of acid phosphatase. All constructions directed the synthesis of single-chain tPA proteins that were readily precipitated with a specific antibody directed against human uterine tPA. Electrophoretic mobilities were approximately the same as those of the Bowes melanoma single-chain tPA and a 68-kD protein marker. Treatment of immunoprecipitates with endoglycosidase H resulted in increased electrophoretic mobilities, suggesting that these yeast products are glycosylated. Despite the use of either human or yeast signal sequences, however, tPA produced in yeast was not secreted into the culture medium, but rather was found only in cells following disruption with glass beads. Although this cellular tPA exhibited fibrinolytic activity, most of the activity was associated with large cellular debris.