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 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.
Krizman
D B
DB
Laboratory of Cancer Genetics, National Center for Human Genome Research, National Institutes of Health, Bethesda, Maryland 20892, USA.
Hofmann
T A
TA
DeSilva
U
U
Green
E D
ED
Meltzer
P S
PS
Trent
J M
JM
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
D017931:DNA Primers; D012333:RNA, Messenger; D004247:DNA