Purification of low-abundance messenger RNAs from rat liver by polysome immunoadsorption.
We have purified three low-abundance hepatic mRNAs to near homogeneity by polysome immunoadsorption. The mRNAs coding for the precursor of ornithine transcarbamoylase [carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3], the precursor of the beta-subunit of propionyl-CoA carboxylase [propionyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.3], and cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22], representing approximately 0.20, 0.02, and 0.015% of total hepatic mRNA, respectively, were purified 450- to 6,300-fold. We used the following steps: interaction of rat liver polysomes with an IgG fraction of monospecific antisera raised against each polypeptide; immobilization of polysome-antibody complexes on a protein A-Sepharose column; removal of the bulk of polysomes by extensive washing; dissociation of ribosomal subunits and elution of specific mRNA with EDTA; and isolation of the eluted mRNA by chromatography on an oligo(dT)-cellulose column. It seems likely that this procedure will permit isolation of other low-abundance mRNAs and subsequent cloning of their respective cDNAs.
D000818:Animals; D000936:Antigen-Antibody Complex; D002262:Carboxy-Lyases / Q000235:genetics*; D003541:Cystathionine beta-Synthase / Q000235:genetics*; D004792:Enzyme Precursors / Q000235:genetics; D006836:Hydro-Lyases / Q000235:genetics*; D007106:Immune Sera; D008099:Liver / Q000201:enzymology*; D008297:Male; D043586:Methylmalonyl-CoA Decarboxylase; D009954:Ornithine Carbamoyltransferase / Q000235:genetics*; D011061:Poly A / Q000235:genetics; D011132:Polyribosomes / Q000378:metabolism*; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D051381:Rats
D000936:Antigen-Antibody Complex; D004792:Enzyme Precursors; D007106:Immune Sera; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D009954:Ornithine Carbamoyltransferase; D002262:Carboxy-Lyases; D006836:Hydro-Lyases; D003541:Cystathionine beta-Synthase; D043586:Methylmalonyl-CoA Decarboxylase