Direct mapping of rare messenger RNAs by means of oligomer-directed ribonuclease H cleavage.
A method has been developed for characterizing rare messenger RNAs in the bulk population by using oligodeoxyribonucleotide: RNA hybrids as substrates for Escherichia coli ribonuclease H. Two 1.3-kb mRNAs in lymphocyte cytoplasm, interferon-gamma (0.002% of polyadenylated mRNA), and prothymosin-alpha, have been studied. Interferon-gamma mRNA was cut virtually completely into two fragments, each about 0.6 kb in length, by using an interferon-specific 24-mer to direct cleavage. Prothymosin-alpha mRNA in the same bulk population was unaffected by this treatment. When the 24-mer was replaced by a 12-mer, whose sequence was based on an incomplete cDNA clone for prothymosin-alpha, the products included two fragments of prothymosin-alpha mRNA. The sum of the fragment lengths equaled the length of the mRNA. Although the reaction directed by the smaller oligomer did not go to completion, the 12-mer, and hence the cDNA clone from which it was derived, could nevertheless be oriented with respect to prothymosin-alpha mRNA. With this technique, sequences in mRNA can be mapped without first isolating full-length cDNA clones.
D001483:Base Sequence; D004247:DNA / Q000032:analysis; D004722:Endoribonucleases / Q000494:pharmacology*; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics; D009693:Nucleic Acid Hybridization; D009710:Nucleotide Mapping; D009838:Oligodeoxyribonucleotides / Q000032:analysis*; D011498:Protein Precursors / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis*; D016914:Ribonuclease H; D013947:Thymosin / Q000031:analogs & derivatives / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; D012333:RNA, Messenger; C040782:prothymosin alpha; D013947:Thymosin; D007371:Interferon-gamma; D004247:DNA; D004722:Endoribonucleases; D016914:Ribonuclease H