RESUMEN
Most mammalian genes will soon be characterized as cDNA sequences with little information about their function. To utilize this sequence information for large-scale functional studies, a gene trap retrovirus shuttle vector has been developed to disrupt genes expressed in murine embryonic stem (ES) cells. A library of mutant clones was isolated, and regions of genomic DNA adjacent to 400 independent provirus inserts were cloned and sequenced. The flanking sequences, designated 'promoter-proximal sequence tags', or PSTs, identified 63 specific genes and anonymous cDNAs disrupted as a result of virus integration. The efficiency of tagged sequence mutagenesis suggests that many of the 10,000-20,000 genes expressed in ES cells can be targeted, providing defined mutations for the analysis of gene functions in vivo. In addition, PSTs provide the first expressed sequence tags derived from genomic DNA, and define gene features such as exon boundaries and promoters that are missing from cDNA sequences.
Asunto(s)
Técnicas Genéticas , Vectores Genéticos , Mutagénesis , Animales , Secuencia de Bases , ADN Complementario , Bases de Datos Factuales , Predicción , Expresión Génica , Marcación de Gen , Humanos , Ratones , Datos de Secuencia Molecular , Células MadreAsunto(s)
Vectores Genéticos , Mutagénesis Insercional , Retroviridae , Células Madre/citología , Animales , Células Cultivadas , Quimera , Células Clonales , Técnicas de Cultivo/métodos , Embrión de Mamíferos , Fibroblastos/citología , Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Fenotipo , Plásmidos , Células Madre/fisiología , Transcripción GenéticaAsunto(s)
Factor 1 de Crecimiento de Fibroblastos/metabolismo , Receptores de Superficie Celular/análisis , Animales , Unión Competitiva , Encéfalo/metabolismo , Carcinoma Hepatocelular , Bovinos , Línea Celular , Humanos , Radioisótopos de Yodo , Cinética , Neoplasias Hepáticas , Peso Molecular , Técnica de Dilución de Radioisótopos , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento de FibroblastosRESUMEN
Fibronectin and heparin-binding growth factors (HBGF) are essential for growth of cultured endothelial cells. The stimulation of endothelial cell growth by HBGF type one (HBGF-1) in particular requires heparin or a similar glycosaminoglycan. The requirement for fibronectin and heparin for HBGF-1-stimulated endothelial cell growth may be related. HBGF-1 absorbed to the natural subcellular matrix of endothelial cells supports cell growth. [125I]HBGF-1 specifically associates with a sequentially reconstituted matrix of collagen-fibronectin-heparin, and HBGF-1 absorbed to the reconstituted matrix supports growth of the endothelial cells. A reconstituted matrix of collagen-laminin-heparin neither supported binding of [125I]HBGF-1 nor HBGF-1-stimulated endothelial cell growth. Association kinetics of [125I]HBGF-1 to heparinlike sites and membrane receptor sites on endothelial cell monolayers suggest that fibronectin-heparinlike binding sites in the subcellular matrix may be an obligatory reservoir of active HBGF-1 that binds to specific cell membrane receptors.
Asunto(s)
Endotelio Vascular/metabolismo , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Fibronectinas/farmacología , Heparina/farmacología , Laminina/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Colágeno/farmacología , Endotelio Vascular/citología , Matriz Extracelular/metabolismo , Factor 1 de Crecimiento de Fibroblastos/farmacología , Humanos , Cinética , Receptores Mitogénicos/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Venas UmbilicalesRESUMEN
The detergent-soluble 125I-labeled receptor complex resulting after affinity cross-linking of 125I-heparin-binding growth factor type one (HBGF-1, m = 15.2-kDa) to HepG2 cells had an apparent molecular mass of 145-kDa, eluted from immobilized wheat germ lectin in the presence of N-acetylglucosamine, shifted to apparent mass of 128-kDa when treated with N-glycanase and shifted to apparent mass of 205-kDa after reduction, carboxymethylation and succinylation. Electrophoretic analysis of HepG2 cell membrane proteins revealed a major silver-stained protein of apparent molecular mass of 130-kDa that has correlative properties. These properties were used to purify the 130-kDa HepG2 glycoprotein to apparent homogeneity and suggest the glycoprotein as a candidate for the human HBGF receptor.
