Human dbl proto-oncogene in 85 kb of Xq26, and determination of the transcription initiation site

The dbl oncogene is generated by substitution of the 5' portion of its norm al counterpart with an unrelated human sequence. To analyze the genomic str ucture and transcriptional regulation of the dbl proto-oncogene, we have is olated human genomic clones containing the entire human proto-dbl gene, loc alized in Xq26. Restriction mapping of a 600 kb YAC clone (yWXD311) placed proto-dbl about 50 kb telomeric to the coagulation Factor IX gene. The geno mic DNA fragment containing the 5' end of proto-dbl was subcloned into plas mid vectors and the nucleotide sequences of exon I, the flanking intronic r egion and genomic DNA 5' of the first codon were determined. Sequence analy sis of 85119 bp from the region revealed the genomic structure of proto-dbl . It contains 25 exons coding for a 4.7 kb transcript including large 5'- a nd 3'- (1218 bp and 701 bp, respectively) untranslated regions (UTRs). RNas e protection and primer extension assays on RNA from medullary thyroid carc inoma (TT) cells, which normally express dbl, revealed a transcription star t site 1218 bp upstream of the ATG of the first exon, A 1.6 kb genomic 5' o f the translation start sites drives the expression of a CAT-reporter in tr ansient transfections in the TT cell line, though lacking TATA or CAAT boxe s. (C) 2000 Elsevier Science B.V. All rights reserved.