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 METHODSThe sources of many of the reagents used in this study have been indicated (10). For the synthesis of oligo (dT) 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 ...

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A cell-free system derived from Krebs II ascites tumor has been used to assay biologically active mRNA for myeloma Our initial attempts have been directed toward purifying a mouse kappa-chain mRNA derived from myeloma tumor MOPC-41 (9). Stavnezer and Huang have recently translated this mRNA in a reticulocyte cell-free system (10). Our strategy has been to use the ascites tumor cell-free system that is dependent on tRNA (11) to assay biologically active light-chain mRNA, and to use oligo(dT)-cellulose chromatography (12) and sucrose gradient centrifugation as initial steps in its purification. In previous work, we have shown that this approach can be used successfully for the isolation of poly(A)-rich rabbit globin mRNA (12). This mRNA, in turn, can be converted into its highly labeled DNA complement with RNA-directed DNA polymerase of avian myeloblastosis virus (13)(14)(15). In the present work, we show that the ascites tumor cell-free system provides a sensitive and convenient assay for the translation of light-chain mRNA, that light-1967 chain mRNA is retained by oligo(dT)-cellulose, suggesting that it is a poly(A)-rich mRNA, and that biologically active light-chain mRNA sediments during sucrose gradient centrifugation as a discrete peak, of s2o,w about 13, roughly corresponding to a molecule containing about 200 more bases than are necessary to encode both the variable and the constant regions of an immunoglobulin light chain. MATERIALS AND METHODSThe sources of many of the reagents used in this study have been indicated (11,12).Preparation of Myeloma Polysomes and Myeloma mRNA. Myeloma tumors, the generous gift of Dr. M. Potter, were grown and harvested according to standard procedures (9), and were frozen and stored in liquid N2 before processing. The microsomal fraction was obtained according to a modified procedure (16). 70 g of dissected, nonnecrotic tumor was suspended in 170 ml of buffer A containing 0.05 M Tris * HCl (pH 7.7)-0.025 M KC1-5 mM MgCl2-7 mM 2-mercaptoethanol-0.88 M sucrose, disrupted for 30 sec in a chilled Waring Blendor at medium speed, and further disrupted with eight strokes in a loose-fitting, Teflon-glass, motordriven homogenizer. The homogenate was centrifuged at 20,000 X g for 20 min; the supernatant was diluted to 0.62 M sucrose with buffer A minus sucrose, and layered over a discontinuous sucrose gradient composed of 5 ml of buffer A containing 2.0 M sucrose and 7 ml of buffer A containing 1.5