In vitro selection of exonic splicing enhancer sequences: identification of novel CD44 enhancers.
We have developed an in vitro selection procedure that allows the identification and isolation of functional splicing enhancer sequences from any cDNA. It is based on the enhancement of general splicing activity of a pre-mRNA reporter derived from the Drosophila dsx gene. Short DNase I fragments are cloned into a cassette in the second exon of the reporter construct, replacing the natural dsx enhancer. After splicing and reverse transcription-PCR, fragments are recovered from the mRNA product. Applying this selection to the CD44 gene, which undergoes extensive alternative splicing processes, we have identified several novel exonic enhancers. Two of them, which reside in CD44 variable exon 6, were further characterized by mutational analysis and confirmed to function within their natural CD44 context.
Woerfel
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Institut für Biochemie, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.
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D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D003850:Deoxyribonuclease I / Q000378:metabolism; D004330:Drosophila / Q000235:genetics; D029721:Drosophila Proteins; D005091:Exons / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D018960:Hyaluronan Receptors / Q000235:genetics*; D019476:Insect Proteins / Q000235:genetics; D051379:Mice; D009154:Mutation / Q000235:genetics; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C070369:DSX protein, Drosophila; D029721:Drosophila Proteins; D018960:Hyaluronan Receptors; D019476:Insect Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; D003850:Deoxyribonuclease I