STRUCTURAL-ANALYSIS OF THE HUMAN RET PROTOONCOGENE USING EXON TRAPPING

A genomic contig of the human ret proto-oncogene was created with four overlapping cosmid clones isolated from two libraries. After southern analysis with portions of the ret cDNA, eight cosmid fragments were a nalysed in detail for the presence of ret exons using exon trapping. P CR products corresponding to spliced exons were isolated and subcloned . Exon boundaries were delineated by comparison of the PCR product seq uence and the published ret cDNA sequence. The exons were initially po sitioned on a genomic map defined by BamHI, EcoRI and HindIII restrict ion sites. The positions of the exons were then refined by amplifying genomic DNA using primer pairs derived from one or more exons along th e ret gene, the length of the PCR product indicating the approximate g enomic distance between the exon sequences. The ret proto-oncogene is composed of at least 20 exons, ranging in size from 60 bp to 287 bp, d istributed along 30 kb of genomic DNA. The extracellular domain is enc oded by 10 exons and the cytoplasmic domain by 9 exons. The transmembr ane domain is encoded by a single exon.