IDENTIFICATION OF 3'-TERMINAL EXONS FROM YEAST ARTIFICIAL CHROMOSOMES

We report an extension of 3'-terminal exon trapping technology to the identification of transcribed sequences from yeast artificial chromeso mes (YACs). A 350-kb YAC containing mouse genomic DNA was gel-purfied and used as the target DNA for the 3'-terminal exon trapping strategy. A novel direct ligation/transfection approach was employed to Increas e the efficiency of trapping 3'-terminal exons from recombinant vector -derived chimeric mRNA. The resulting RT-PCR product war then used to generate a plasmid library. Randomly chosen individual subclones from this library were sequenced, and the results Indicate that 86% met seq uence criteria characteristic of 3'-terminal exons, whereas 14% were b ackground from identified sources. PCR mapping efforts suggest eight p utative last exons present within this YAC, whereas RT-PCR studies dem onstrate that three reside within valid expressed sequences.