Cloning exons of mapping of transcription: characterization of the Drosophila melanogaster alcohol dehydrogenase gene

Abstract

A novel method has been developed for determining the location of RNA termini and intron-exon boundaries using genomic clones. RNA is hybridized to a single-stranded M13 phage derivative of the genomic fragment of interest. S1 nuclease digestion results in an RNA-DNA hybrid corresponding to any transcript protected by the insert. Hydrolysis of the RNA with alkali, hybridization of the DNA with the opposite-strand M13 derivative of the genomic fragment and S1 nuclease digestion yields a mixture of pure exons. This mixture is analyzed by agarose gel electrophoresis and is cloned using excess blunt-ended M13 phage vector. Plaques that contain inserts are identified by transfer to nitrocellulose and hybridization to the genomic insert and are used without further purification. Cloning junctions are then determined by partial sequence analysis. These very nearly correspond to intron-exon boundaries or to either end of the transcript. When applied to the alcohol dehydrogenase gene from Drosophila, this method revealed clear differences between the 5' ends of embryo and adult transcripts both by blot hybridization and by analysis of 23 independent exon clones. In embryos, the mature transcript is apparently derived from three exons and in adults from four with the difference lying in the 5' untranslated portion of the transcript. The method should be particularly valuable for mapping and cloning transcripts that are rare or are not polyadenylated.