Rapid identification of DNA fragments containing promoters for RNA polymerase II.
We describe a direct procedure for screening genomic recombinant DNA libraries or restriction fragments of cloned DNA regions for RNA polymerase II promoters. Cellular polyadenylated mRNA is chemically de-capped by beta-elimination reaction and enzymatically re-capped with [alpha-32P]GTP by vaccinia guanylyl transferase. Since this enzyme only accepts di- or triphosphorylated 5' termini as a substrate, the mRNAs are labeled exclusively at the first nucleotide, irrespective of whether the mRNA was intact or fragmented before in vitro capping. By using in vitro-capped mRNA as a hybridization probe, recombinant DNA molecules or restriction fragments that carry a cap site (and thus likely an RNA polymerase II promoter) can directly be identified. Here, we demonstrate the applicability of this procedure by the isolation and characterization of several genomic DNA clones containing RNA polymerase II promoter sequences, that are highly active in liver.
Shaw
P H
PH
Institut de Pathologie, Université de Lausanne, Switzerland.
Walter-Sierra
R
R
Tamone
F
F
Schibler
U
U
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis / Q000235:genetics* / Q000302:isolation & purification; D015342:DNA Probes; D004274:DNA, Recombinant; D015723:Gene Library; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009710:Nucleotide Mapping; D009713:Nucleotidyltransferases / Q000378:metabolism; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D012315:RNA Caps; D012319:RNA Polymerase II / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics
D015342:DNA Probes; D004274:DNA, Recombinant; D012315:RNA Caps; D012333:RNA, Messenger; D004247:DNA; D009713:Nucleotidyltransferases; D012319:RNA Polymerase II; C020842:guanylyltransferase; D015719:Single-Strand Specific DNA and RNA Endonucleases