Molecular cloning and characterization of the 5' region of the mouse trkA proto-oncogene.
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 neurodegeneration, 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.
Sacristán
M P
MP
Instituto de Microbiología Bioquímica, CSIC/Universidad de Salamanca, Avda. Campo Charro s/n, 37007 Salamanca, Spain.
de Diego
J G
JG
Bonilla
M
M
Martín-Zanca
D
D
D020121:5' Untranslated Regions / Q000737:chemistry* / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011401:Promoter Regions, Genetic; D020917:Receptor, trkA / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*
D020121:5' Untranslated Regions; D010446:Peptide Fragments; D020917:Receptor, trkA