RESUMEN
The identification of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has led to the identification of more than 225 presumed disease-causing mutations at the locus. The diagnosis of cystic fibrosis or the carrier state by direct DNA analysis is hindered by this large number. A practical assay must be able to detect enough mutations to achieve clinically significant sensitivity. The use of allele-specific oligonucleotide probes is the most promising of the available methods. However, to date this has generally involved tedious probe-by-probe hybridizations, due to variations in the oligonucleotides' denaturation temperatures caused by differences in their G-C base-pair content. We have developed a rapid, cost-effective assay that simultaneously detects 12 CFTR mutations after multiplex polymerase-chain-reaction amplification of genomic DNA. The test may be readily extended to detect additional mutations at minimal increase in the cost per test or the turnaround time. We improve specificity and avoid the need for individual hybridizations by the use of tetramethylammonium chloride to virtually eliminate the effects of G-C differences. Coupled with non-invasive sample-collection methods, this is an immediately practical assay for cystic fibrosis. More generally, it will serve as a model for the development of diagnostic tests in other genetic disorders involving complex mutation analysis.
Asunto(s)
Fibrosis Quística/diagnóstico , Análisis Mutacional de ADN/métodos , Proteínas de la Membrana/genética , Sondas de Oligonucleótidos , Fibrosis Quística/genética , Fibrosis Quística/prevención & control , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Tamización de Portadores Genéticos , Pruebas Genéticas , Humanos , Reacción en Cadena de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
Low-stringency hybridization with human glucocorticoid receptor (hGR) complementary DNA was used to isolate a new gene encoding a predicted 107-kilodalton polypeptide. Expression studies demonstrate its ability to bind aldosterone with high affinity and to activate gene transcription in response to aldosterone, thus establishing its identity as the human mineralocorticoid receptor (hMR). This molecule also shows high affinity for glucocorticoids and stimulates a glucocorticoid-responsive promoter. Together the hMR and hGR provide unexpected functional diversity in which hormone-binding properties, target gene interactions, and patterns of tissue-specific expression may be used in a combinatorial fashion to achieve complex physiologic control.
Asunto(s)
Receptores de Glucocorticoides/genética , Receptores de Esteroides/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromosomas Humanos Par 4 , Clonación Molecular , ADN/genética , Proteínas de Unión al ADN/genética , Humanos , Ratas , Receptores de Mineralocorticoides , Homología de Secuencia de Ácido Nucleico , Distribución Tisular , Transcripción GenéticaRESUMEN
Two assays were developed for identifying individual cells which bear murine leukemia virus receptors: an erythrocyte rosette assay for ecotropic receptors, and an efficient immune cytotoxic assay for cells with ecotropic or amphotropic receptors. Both assays indicate that ecotropic MuLV adsorbed to its cell surface receptor only slowly becomes internalized. Furthermore, attempts to isolate murine fibroblast variants lacking these ecotropic MuLV receptors were unsuccessful, suggesting either that mutations in the receptor gene are rare (less than 10(-7) per generation) or that the receptor is required for cell viability. These assays are rapid and can be used to identify receptor-bearing cells in mixed populations, a prerequisite for molecular genetic studies.