Use of molecular hybridization to purify and analyze albumin messenger RNA from rat liver

Abstract

A new procedure is described for purification of rat liver albumin m A. First a population of RNA molecules is enriched for albumin mRNA by immunoprecipitation of polysomes containing albumin nascent chains. Polyadenylylated RNA is prepared from immunoprecipitates, transcribed into complementary DNA, and shown to be enriched severalfold for a particular RNA frequency component. This enriched RNA component is then purified by molecular hybridization to a limited Rot value (product of RNA concentration and incubation time), under conditions in which only the most abundant sequence component is annealed. Potentially, this procedure can be employed for the purification of a wide variety of mRNAs present in lesser amounts in the cell.The isolated RNA appears to be a single frequency component by hybridization to complementary DNA transcribed from itself. This RNA is a 17S species and represents 5-8% of total cytoplasmic polyadenylylated RNA. In vitro translation of the purified RNA has shown that it codes for a single polypeptide that can be identified immunologically as albumin and migrates with rat serum albumin on sodium dodecyl sulfate/polyacrylamide gels. This albumin mRNA was determined to be essentially pure by comparing its kinetics of hybridization to those obtained with rabbit a + P globin mRNA and its DNA complement. The sequence complexity of purified rat albumin mRNA corresponds to 5.9 X 105 daltons. Various procedures have been employed for purification of eukaryotic mRNAs. Affinity chromatography with either oligo(dT)-cellulose or poly(U)-Sepharose has been most successful for separating mRNAs containing a 3'-poly(A) segment from the bulk of ribosomal and other RNA components (1, 2). However, this procedure cannot be used to separate specific polyadenylylated mRNAs from each other, and a portion of eukaryotic mRNA does not contain a 3'-poly(A) segment (3-7). Other commonly used methods for purification of eukaryotic mRNAs have been based either on a unique size for a specific mRNA-e.g., a and ,B globin mRNA(8), histone mRNA (3,9, 10), immunoglobin light chain mRNA (11)(12)(13)(14), ovalbumin mRNA (15), etc.-or an unusual nucleotide composition-e.g., silk fibroin mRNA (16). For purification of mRNAs that are average sized and are present as only a small portion of total cellular mRNA, the method of immunoprecipitation of polysomes containing nascent polypeptide chains for a specific protein is the only specific procedure available at the present time. Immunoprecipitation can enrich a polysome preparation for a specific mRNA by 5-to 10-fold (18-22). However, in order to obtain intact mRNA with good biological activity, the antibody must be highly purified to remove ribonuclease activity. Furthermore, large amounts of antibody are needed to generate sufficient amounts of material for use in molecular studies. In the present study, we have combined the techniques of imThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereb...

Direct regulation by calcium of cytoplasmic messenger ribonucleic acid coding for pre-proparathyroid hormone in isolated bovine parathyroid cells.

“…Plasmids were removed from tetracycline-resistant bacteria by alkaline lysis (8) and PTH cDNA inserts were identified by in situ hybridization and radioautography (7). The probe used for initial colony identification was a single-stranded cDNA copy labeled with 32P and purified by rapid hybridization and hydroxylapatite chromatography (9). Positive inserts were removed by cleav-age with Pst-1 and characterized for size on 5% polyacrylamide gel.…”

Presence of albumin mRNA precursors in nuclei of analbuminemic rat liver lacking cytoplasmic albumin mRNA.

“…RNA cDNA hybridization was performed as described (18) with nick-translated prAlb-1 or [3H]cDNA prepared from partially purified mRNA as described (18). Nick-translation was performed by the method of Rigby et al (22) with [32P]dCTP (2000-3000 Ci/ mmol) and the resulting [32P]cDNA was separated by electrophoresis on 5% polyacrylamide gel and purified further by hybridization with partially purified albumin mRNA and complete digestion with S1 nuclease as described by Strair et aL (23). The reactivity of [32P]cDNA was confirmed to be 100% by using normal cytoplasmic RNA.…”

Distribution of rat liver albumin mRNA membrane-bound and free in polyribosomes as determined by molecular hybridization

“…(20). Poly(A)+ RNA was prepared from total RNA extracts of membrane-bound and free polyribosomes by oligo(dT)-cellulose chromatography (20). RNA was resuspended in 10 mM Tris-Ha, pH 7.4/0.5% sodium dodecyl sulfate (NaDodSO4) and heated to 65°for 5 min.…”

“…This material was layered over a 12-ml 10-40% (wt/vol) exponential sucrose gradient Abbreviation: NaDodSO4, sodium dodecyl sulfate. (20). Poly(A)+ RNA was prepared from total RNA extracts of membrane-bound and free polyribosomes by oligo(dT)-cellulose chromatography (20).…”