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Título
Molecular cloning and characterization of the 5′ region of the mouse trkA proto-oncogene
Autor(es)
Palabras clave
Protooncogén trk A
Clonación molecular
Clasificación UNESCO
2415 Biología Molecular
2407 Biología Celular
2302.04 Genética Bioquímica
Fecha de publicación
1999
Editor
nature.com
Citación
Sacristán, M. P., de Diego, J. G., Bonilla, M., & Martín-Zanca, D. (1999). Molecular cloning and characterization of the 5′ region of the mouse trkA proto-oncogene. Oncogene, 18(42), 5836-5842.
Resumen
[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.
URI
ISSN
0950-9232
DOI
10.1038/sj.onc.1202963
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