Molecular cloning and characterization of the 5′ region of the mouse trkA proto-oncogene
[EN] The trkA proto-oncogene encodes a high-affinity NGF receptor that is essential for the survival, differentiation and maintenance of many neural and non-neural cell types. Altered expression of the trkA gene or trkA receptor malfunction have been implicated in neurode-generation, tumor progressi...
| Autores: | , , , |
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| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 1999 |
| País: | España |
| Institución: | Universidad de Salamanca (USAL) |
| Repositorio: | GREDOS. Repositorio Institucional de la Universidad de Salamanca |
| OAI Identifier: | oai:gredos.usal.es:10366/157114 |
| Acceso en línea: | http://hdl.handle.net/10366/157114 |
| Access Level: | acceso abierto |
| Palabra clave: | Protooncogén trk A Clonación molecular 2415 Biología Molecular 2407 Biología Celular 2302.04 Genética Bioquímica |
| Sumario: | [EN] The trkA proto-oncogene encodes a high-affinity NGF receptor that is essential for the survival, differentiation and maintenance of many neural and non-neural cell types. Altered expression of the trkA gene or trkA receptor malfunction have been implicated in neurode-generation, tumor progression and oncogenesis. We have cloned and characterized the 5' region of the mouse trkA gene and have identified its promoter. trkA promoter sequences are GC-rich, lack genuine TATA or CAAT boxes, and are contained within a CpG island which extends over the entire first coding exon. The mouse trkA transcription start site is located 70/71 bp upstream to the AUG translation initiation codon. Sequence analysis showed that the gene encoding the insulin receptor-related receptor, IRR, is located just 1.6 kbp upstream to the trkA gene and is transcribed in the opposite direction. We have used trkA-CAT transcriptional fusions to study trkA promoter function in transient transfection experiments. RNase protection assays and CAT protein ELISA analyses showed that a 150 bp long DNA segment, immediately upstream to the start site, is sufficient to direct accurate transcription in trkA-expressing cells. Dissection of this fragment allowed us to identify a 13 bp cis-regulatory element essential for both promoter activity and cell-type specific expression. Deletion of this 13 bp segment as well as modification of its sequence by site-directed mutagenesis led to a dramatic decline in promoter activity. Gel mobility shift assays carried out with double-stranded oligonucleotides containing the 13 bp element revealed several specific DNA-protein complexes when nuclear extracts from trkA-expressing cells were used. Supershift experiments showed that the Sp1 transcription factor was a component of one of these complexes. Our results identify a minimal trkA gene promoter, located very close to the transcription start site, and define a 13 bp enhancer within this promoter sequence. |
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