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

Abstract

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 ...

Translation of the messenger RNA for rabbit uteroglobin in Xenopus oocytes

“…The poly(A) rich RNA was prepared by a modification of the procedure af Aviv and Leder [7]. The RNA pellet was washed with 70%, 95% and absolute ethanol, dried and dissolved in 10 mM Tris, pH 7.6, containing 0.5 M NaCl, 0.2 mM MgCl* and 0.5% SDS.…”

Characterization of Rabbit Genes for Synovial Cell Collagenase

“…The rest of the transcribed regions found on the genomic clones were localized by hybridization with radiolabeled mRNA and pre-mRNA from rabbit synovial cells induced with PMA. This is also called "poly A+" RNA because of the polyadenosine tails added post-transcriptionally to the ends of transcripts from protein-coding genes in eukaryotes (31). Only exon portions of the collagenase gene are identified by this method, since intron sequences are not present in mRNA and only small amounts are present in premRNA from the nucleus-not enough to produce detectable hybridization signals.…”

“…Messenger RNA was prepared by 1 purification cycle on oligo-(dT) cellulose (31). It was 5' end-labeled by the following method, which was adapted from Biessmann et al (32).…”

Identification, characterization of levoglucosan kinase, and cloning and expression of levoglucosan kinase cDNA from Aspergillus niger CBX-209 in Escherichia coli

“…Preparation of mRNA: mRNA was separated by chromatography through oligo(dT)-cellulose (BRL, MD) [22,37].…”