Purification of Biologically Active Globin Messenger RNA by Chromatography on Oligothymidylic acid-Cellulose

A convenient technique for the partial purification of large quantities of functional, poly(adenylic acid)-rich mRNA is described. The method depends upon annealing poly(adenylic acid)-rich mRNA to oligothymidylic acid-cellulose columns and its elution with buffers of low ionic strength. Biologically active rabbit globin mRNA has been purified by this procedure and assayed for its ability to direct the synthesis of rabbit globin in a cell-free extract of ascites tumnor. Inasmuch as various mammalian mRNAs appear to be rich in poly(adenylic acid) and can likely be translated in the ascites cell-free extract, this approach should prove generally useful as an i -itial step in the isolation of specific mRNAs. Various questions regarding the expression of genetic information in higher organisms depend upon the detection and isolation of gene-specific messenger RNA (mRNA). In contrast to bacterial systems, in which transducing phage simplify these procedures, isolation and detection of mRNA in animal cells has relied largely upon studies of the kinetics of radioactive labeling of polysomal RNAs and upon techniques that exploit differences in their molecular weights. While these techniques are useful, methods that take advantage of other unique properties of mRNA should prove of value as well. Putative mRNAs isolated from animal cells differ from other RNA species in that they contain relatively long stretches of adenylic acid residues (1-4). The precise role of these poly(A)-rich regions in the metabolism of mRNA is not known, but it has been suggested that they are involved in the transport of mRNA from the cell nucleus to the cytoplasm, where protein synthesis occurs (4-5). Several workers have used the binding of poly(A)-rich RNA to oligo(dT)-cellulose (6), to poly(U)-cellulose (7), ohito nitrocellulose filters (4, 8) to detect poly(A) regions or putative mRNAs. Inasmuch as rabbit globin mRNA contains poly(A)-rich regions (1, 8, 9), it seemed likely that this feature, together with a highly-sensitive assay for the biological activity of the mRNA (10), would prove useful in the preparative purification of this and other biologically active mRNAs. In the present work, we show that oligo(dT)-cellulose chromatography can be used conveniently to separate globin mRNA from the bulk of ribosomal RNA in crude polysomal extracts. Oligo(dT)-cellulose seems to have several unique advantages for this purpose. Purification of the globin message in this and subsequent steps is greatly facilitated by a sensitive assay for the in vitro synthesis of rabbit globin. MATERIALS AND METHODS The sources of many of the reagents used in this study have been indicated (10). For the synthesis of oligo(dT)-cellulose, Whatman cellulose powder CC 41 was obtained from Reeve Angel; thymidine-5'-monophosphate, from Schwarz-Mann; and NN'-dicyclohexylearbodiimide, from Aldrich. [3H]Leucine (specific activity, 26 Ci/mmol); ['4C]leucine (specific activity, 263 Ci/mol) and [14C]valine (specific activity, 219 Ci/mol), from New England Nuclear; [14C]lysine (specific activity, 312 Ci/mol), from Amersham, ['4C]alanine (specific activity, 160 Ci/mol) from Schwarz BioResearch. Rabbit liver transfer RNA (tRNA) was obtained fom General Biochemicals. Trypsin ("chymotrypsin-free") was obtained from Worthingtoii. Chromo-Beads cation exchange resin, type P, was obtained from Technicon. Preparation of Oligo(dT)-Cellulose. Oligo(dT)-cellulose was prepared according to Gilham, using the N,N'-dicyclohexylcarbodiimide reaction for the polymerization of thymidine-5'monophosphate on cellulose (11). One gram (dry weight) of oligo(dr)-cellulose prepared in this way retained and was saturated by about 160-200 ,ug of poly(A) under the conditions noted below. Preparation of Rabbit Globin mRNA. Rabbit reticulocyte -lolysomes were isolated by described procedures (12). RNA was extracted from the polysomes by a modification of the procedure described by Lee, Mendecki, and Brawerman (4). The polysomes were suspended in 0.1 M Tris-HCl (pH 9.0)0.1 M NaCl-1 mM EI)TA at a concentration of 20 A 260 units/ ml and then were made 1% in sodium dodecyl sulfate (SDS). An equal volume of phenol-chloroform-isoamyl alcohol 50: 50: 1 was added, the mixture was shaken vigorously for 10 mill at room temperature and chilled to 50, and the phases were separated by centrifugation at 12,000 X g for 10 min. The aqueous phase was removed, extracted again as above, and made 2% in CH3COOK (pH 5.5). Crude polysomal RNA was precipitated by the addition of two volumes of ethanol and was allowed to stand at 20° overnight. The RNA was collected by centrifugation at 12,000 X g at -20° for 20 min. The RNA pellet was washed twice with ethanol-0.2 M NaCl 2: 1 and dissolved in H20 or, if oligo(dT)-cellulose chromatography was to be used, in 0.01 M Tris HCl (pH 7.5)-0.5 M KCl. 1408 Abbreviation: SDS, sodium dodecyl sulfate. Oligo(dT)-Cellulose Chromatography 1409 Oligo(dT)-Cellulose Chromatography. All operations were done at room temperature with glassware and reagents [except for oligo(dT)-cellulose] that had been autoclaved. 100 A260 units of crude rabbit reticulocyte polysomal RNA dissolved in application buffer containing 0.01 1\I TrisHCl (pH 7.5)-0.5M KCl was applied to a 2-ml (about 0.5 g, dry weight) oligo(dT)-cellulose column previously washed with application buffer. The nonabsorbed material was eluted by continued washing with the application buffer. The material retained by the column was eluted in two steps with buffers of reduced ionic strength (13). The first elution buffer contained 0.01 1\I Tris-HCl (pH 7.5)-0.1 M KCl; the second, 0.01 M Tris HC1 (pH 7.5). The material eluted in this way was immediately precipitated by the addition of CH3COOK and two volumes of ethanol as noted above, or, if sufficiently concentrated, it was frozen directly and stored in liquid nitrogen. The lrocedure can be scaled up 20-fold to accommodate 2000 A260 Units of polysomal RNA without difficulty, and the oligo(dT)-cellulose can be regenerated for repeated use by elution with 0.1 M KOH. Cell-Free Protein Synthesizing Systems and Assays. A cellfree protein synthesizing system (S30) that was tRNA dependent was prepared from Krebs II ascites tumor cells (10). Globin mRNA was assayed in 0.06-ml reaction mixtures that contained 30 mM Tris-HCl (pH 7.5), 3.5 mM magnesium acetate, 100 mM KCl, 7 mM 2-mereaptoethanol, 1 mM ATP, 0.1 mM GTP, 0.6 mM CTP, 10 mM creatine phosphate, 8 jig creatine kinase, 40 jiM (each) of 19 nonradioactive amino acids, 5 jiM radioactive amino acid of indicated high specific activity ([3H]leucine or [i4C]leucine, [14C]valine, [14C]lysine, and ['4C]alanine as indicated), 0.29 A260 Units tRNArabbit, 15 jil of previously incubated extract of aseites tumor cells containing 14 mg/ml of protein, and mRNA, as indicated. Incubation was at 370 for 60 min. Reactions were stopped by the addition of 0.2 ml of 0.1 M KOH. Incubation was continued for 20 min, and 1 ml of ice-cold 10% Cl3CCOOH was added. If the mixture was not to be used for analysis, it was cooled at 0° for 5 min, and the precipitate was collected over a 0.45-jim pore-size Millipore filter, washed three times with 3 ml (each) of 5% Cl3CCOOH, dried, and counted in a liquid scintillation counter at an efficiency of 20-25% for 3H and 50% for 14C. All determinations were done in duplicate. Rabbit globin labeled with [14C]leucine, used as a standard for tryptic peptide analysis, was synthesized in a rabbit reticulocyte lysate with endogenous rabbit globin mRNA (9).