title: Identification of 3'-terminal exons from yeast artificial chromosomes.
abstract: We report an extension of 3'-terminal exon trapping technology to the identification of transcribed sequences from yeast artificial chromosomes (YACs). A 350-kb YAC containing mouse genomic DNA was gel-purified and used as the target DNA for the 3'-terminal exon trapping strategy. A novel direct ligation/transfection approach was employed to increase the efficiency of trapping 3'-terminal exons from recombinant vector-derived chimeric mRNA. The resulting RT-PCR product was then used to generate a plasmid library. Randomly chosen individual subclones from this library were sequenced, and the results indicate that 86% met sequence criteria characteristic of 3'-terminal exons, whereas 14% were background from identified sources. PCR mapping efforts suggest eight putative last exons present within this YAC, whereas RT-PCR studies demonstrate that three reside within valid expressed sequences.
authors.affiliation: Laboratory of Cancer Genetics, National Center for Human Genome Research, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: D B
authors.initials: DB
authors.lastname: Krizman
authors.forename: T A
authors.initials: TA
authors.lastname: Hofmann
authors.forename: U
authors.initials: U
authors.lastname: DeSilva
authors.forename: E D
authors.initials: ED
authors.lastname: Green
authors.forename: P S
authors.initials: PS
authors.lastname: Meltzer
authors.forename: J M
authors.initials: JM
authors.lastname: Trent
mesh_terms: D000818:Animals; D001483:Base Sequence; D002678:Chimera; D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D005091:Exons; D015870:Gene Expression; D016678:Genome; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000378:metabolism; D011897:Random Allocation
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger; D004247:DNA