Demonstration of Globin Messenger Sequences in Giant Nuclear Precursors of Messenger RNA of Avian Erythroblasts

Highly purified globin mRNA from ducks was copied with RNA-directed DNA polymerase from avian myeloblastosis virus into anti-messenger DNA. With excess RNA, more than 90% of this DNA annealed back to its template with a C(o)t/2 value of 7.5 x 10(-4) mol.sec. liter(-1); the melting temperature of the hybrid was 86 degrees . Giant nuclear RNA fractions with sedimentation coefficients of more than 50 S formed hybrids of almost equal stability at C(o)t/2 values of 0.05-0.42 mol.sec. liter(-1), indicating amRNA content of 0.3-1.5%. 12S RNA from the same polyribosomes and nuclear giant RNA from HeLa cells did not cross-hybridize. Although a large part of the giant RNA broke down in 99% dimethylsulfoxide gradients, RNA fractions sedimenting faster than 28S rRNA still were found to consist of up to 0.03% globin mRNA sequences. Thus, the mRNA sequences are contained in the covalent structure of giant nuclear precursors, which are termed precursor-mRNA.     

Globin Messenger RNA in Hemoglobin H Disease

Functional messenger RNA (mRNA) forhuman globin synthesis was isolated fromreticulocytes of each of two patients withhemoglobin H disease. The RNA wastested for its capacity to direct globinsynthesis in a messenger RNA-dependentcell-free system derived from Krebs Type IImouse ascites tumor cells. In each case,hemoglobin H disease mRNA directed thesynthesis of a great excess of -globinchains relative to -globin chains of hemoglobin A. The / synthetic ratios obtained in the cell-free system at saturatingconcentrations of mRNA were >22 and>15, respectively, for the two hemoglobinH disease mRNA preparations, whereasthe / synthetic ratios obtained by incubation of intact reticulocytes from thesesame patients were 2.6 and 2.8, respectively. The / synthetic ratio obtained inthe cell-free system did not vary whenlower concentrations of hemoglobin Hdisease mRNA were used. A marked decrease in the amount of functional -globin-chain mRNA relative to -chainmRNA is therefore associated with the decreased -chain synthesis observed inhemoglobin H disease. This decrease in-chain-specific mRNA activity is greaterthan expected from the / synthetic ratioof intact reticulocytes in hemoglobin Hdisease.

Submitted on May 1, 1973 Revised on June 15, 1973 Accepted on June 16, 1973     

Beta Thalassemia and Translation of Globin Messenger RNA

To define the quality and relative quantity of beta and alpha messenger RNA in human nonthalassemic and thalassemic reticulocytes, intact cells were incubated with [(35)S]methionine. The relative amounts of beta- and alpha-nascent chains on polysomes of different sizes were measured by tryptic digestion of pooled polysomes and by determination of the specific activities of beta and alpha peptides that contain methionine. Betachain synthesis predominated on heavy polysomes in nonthalassemic, as well as in thalassemic cells. Since beta chains in thalassemia are made on normal-size polyribosomes, we conclude that the defect in thalassemia does not involve reduction in the rate of initiation of translation due to the production of an abnormal beta message. Such would lead to beta-chain synthesis on very small polysomes. We therefore suggest that the decreased production of beta-globin chains results from a decreased amount of functional beta-globin messenger RNA.     

Messenger RNA for Globin in the Postribosomal Supernatant of Rabbit Reticulocytes

10S RNA has been isolated from the postribosomal supernatant of rabbit reticulocyte lysate. This RNA is associated with protein in a ribonucleoprotein particle that sediments at approximately 20S. The 10S RNA extracted from this particle codes predominantly for alpha globin chains when it is translated by a mouse ascites cell-free extract. The product of the cell-free synthesis has been characterized by column chromatography and electrophoresis of the tryptic peptides. About 20% of the total alpha chain mRNA is present in the postribosomal supernatant, whereas beta chain mRNA is almost exclusively associated with polyribosomes.     

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 tumor. 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 initial step in the isolation of specific mRNAs.     

Globin Messenger RNA Activity in Erythroid Precursor Cells and the Effect of Erythropoietin

Cell populations enriched for erythroid precursor cells were fractionated from 13-day fetal-mouse livers by a method of immune hemolysis. These preparations of precursors, contaminated by less than 7% hemoglobinized erythroblasts, synthesize globin at a rate less than 6% that of the unfractionated liver erythroid cell population. RNA was isolated from these precursor cells and assayed for globin mRNA activity in a cell-free system from Krebs ascites tumor. The 6-16S RNA fraction from precursor cells has less than 5% of the globin mRNA activity of RNA isolated from unfractionated populations. Precursor cells incubated with erythropoietin show an increment in the rate of synthesis of globin only after 5-10 hr of incubation. After 10 hr of culture with this hormone, precursor cells show a 6- to 10-fold increase in globin mRNA activity. These results suggest that the precursor cells of hepatic erythropoiesis, responsive to erythropoietin, do not contain globin mRNA in a biologically active form. Erythropoietin-induced differentiation of these cells to erythroblasts is associated with an increase in globin mRNA.     

An Association Between Globin Messenger RNA and 60S RNA Derived from Friend Leukemia Virus

The association between certain cellular RNAs and purified RNA tumor viruses prompted us to examine the possibility that specific host messenger RNAs might also be incorporated into RNA tumor viruses. Using a mouse cell line infected with Friend leukemia virus, T-3-Cl-2, which can be induced to accumulate mouse-globin messenger RNA, we show that mouse-globin messenger RNA sequences are present in viral particles purified from the culture medium of globin-producing cells. These globin messenger RNA sequences are absent from viral particles derived from T-3-Cl-2 cells that are not producing globin messenger RNA. Virus-associated globin messenger RNA sequences sediment in association with the 60S viral RNA complex as well as in free, 9S form. However, under mild denaturing conditions which result in the conversion of viral 60S RNA to 30S and smaller forms, all the globin sequences sediment as 9S RNA. Appropriate control experiments indicate that the virus-associated globin messenger RNA is resistant to degradation by exogenous ribonuclease; that exogenously added globin messenger RNA does not become associated with the 60S viral RNA complex; and that globin messenger RNA can be detected in virions derived from cells both induced for and constitutively synthesizing globin messenger RNA.     

Use of Eukaryotic Native Small Ribosomal Subunits for the Translation of Globin Messenger RNA

A highly active in vitro system for the translation of globin mRNA, resulting in more than 10 rounds of translation, is described. The reconstituted system consists of native small ribosomal subunits of rabbit reticulocytes (as a source of initiation factors as well as small ribosomal subunits), large subunits derived from rat liver polysomes by the puromycin-KCl procedure, and a pH 5 fraction obtained from a Krebs ascites cell high speed supernatant. In this system no differences were found between globin messenger ribonucleoprotein and globin mRNA.     

Role of the Polyadenylate Segment in the Translation of Globin Messenger RNA in Xenopus Oocytes

The translations of native messenger RNA for rabbit globin and that of poly(A)-free globin messenger RNA have been compared after injection into Xenopus oocytes. The initial rate of translation of poly(A)-free mRNA is close to that found with intact mRNA. However, at longer incubation periods, the rate of globin synthesis with poly(A)-free mRNA is considerably lower than with native mRNA. Similar differences in the template activity of the two mRNA preparations were found with a cell-free extract of Krebs II ascites tumor. It is concluded that the presence of the 3' poly(A)-rich sequence in mRNA is required to ensure high functional stability.     

Unexpectedly Large Size of Globin Messenger Ribonucleic Acid

The globin messenger RNA of rabbit reticulocytes is identified on the basis of its presence in polysomes, absence from single ribosomes, and sedimentation coefficient of 9 S. Analysis of this RNA by electrophoresis in polyacrylamide gels provides evidence that its size is unexpectedly large (approximately 650 nucleotides) for a messenger coding for the 141 and 146 amino acids of a globin polypeptide.     

Nucleotide Sequences from Tryptophan Messenger RNA of Escherichia coli: The Sequence Corresponding to the Amino-Terminal Region of the First Polypeptide Specified by the Operon

Mutants with internal deletions that terminate near the operator end of the tryptophan operon of E. coli were used in studies on the nucleotide sequence at the 5' end of the messenger RNA transcribed on this operon. The findings obtained permitted identification of a sequence of 43 nucleotides corresponding to the aminoterminal 11 amino acids of anthranilate synthetase component I, the polypeptide specified by the operator-proximal structural gene of the operon. It was also shown that the translation initiation codon for this polypeptide is preceded on the messenger by a "leader" sequence of unknown function, at least 150 nucleotides in length.     

Hemoglobin Switching in Sheep and Goats: Change in Functional Globin Messenger RNA in Reticulocytes and Bone Marrow Cells

ANEMIA CAUSES A CHANGE IN THE TYPE OF CIRCULATING HEMOGLOBIN IN GOATS AND CERTAIN SHEEP: HbA (alpha(2)beta(2) (A)) is replaced by HbC (alpha(2)beta(2) (C)). We have isolated globin mRNA from erythroid cells of anemic and nonanemic animals to investigate the mechanism whereby anemia causes this switch. To study several stages in transition from beta(A) to beta(C) synthesis, active globin mRNA was isolated from bone marrow cells, as well as from reticulocytes. By assaying these globin mRNAs in a rabbit reticulocyte cell-free system, we have demonstrated that the switch from beta(A) to beta(C) globin synthesis is mediated via a change in functional globin mRNA.     

Quantitative Deficiency of Chain-Specific Globin Messenger Ribonucleic Acids in the Thalassemia Syndromes

A hybridization assay procedure was devised that makes possible quantitation of the ratio of mRNA of alpha to mRNA of beta globin chains in an RNA sample. The assay uses the radioactive synthetic DNA copies obtained by incubation of RNA-dependent DNA polymerase of avian myeloblastosis virus with rabbit globin mRNA that is 80-90% enriched in mRNA specific for synthesis of alpha or beta globin chains. The rabbit alpha-chain mRNA is obtained from the postribosomal supernatant of rabbit reticulocyte lysates; the rabbit beta-chain mRNA is obtained from the largest polysomes of rabbit reticulocytes treated with L-O-methylthreonine. Sufficient homology exists between rabbit and human globin chains and globin mRNAs that the synthetic DNA copies of chain-specific rabbit globin mRNA hybridize with human globin mRNA. Applied to the study of globin mRNA isolated from reticulocytes of humans with alpha and beta thalassemia, the technique revealed marked quantitative deficiency of alpha-chain mRNA relative to beta-chain mRNA in alpha thalassemia and similar deficiency of beta-chain mRNA relative to alpha-chain mRNA in beta thalassemia. The thalassemia syndromes are therefore characterized by true quantitative deficiency of the mRNA specific for the affected globin chain.     

Characterization of Messenger Ribonucleoprotein and Messenger RNA from KB Cells

Messenger ribonucleoprotein and mRNA from KB-cells were isolated under conditions designed to minimize nonspecific RNA-protein interaction and to minimize degradation by contaminating ribonucleases. A large fraction, 60-70%, of the messenger ribonucleoprotein from polysomes dissociated in vitro by either EDTA or puromycin sedimented faster than the large ribosome subunit. Messenger ribonucleoprotein particles with sedimentation coefficients up to 200 S were observed. Released mRNA was also large, with maximal molecular weights around 5 x 10(6).     

Messenger Characteristics of Nascent Bacteriophage RNA

The proteins initiated in vitro by nascent bacteriophage f2 RNA strands attached to isolated replicating structures have been analyzed. The observations confirm that coat protein is the major product initiated and completed. Nascent strands direct the initiation of viral maturation protein in amounts similar to the maximum levels observed in vivo; this synthesis is independent of translation of the coat protein gene. However, only a fraction of these maturation protein molecules initiated in vitro is completed. Nascent RNA molecules also direct the initiation of appreciable amounts of viral RNA polymerase protein, very little of which is completed. Certain constraints on the in vitro translation of the polymerase gene from single-stranded RNA appear to be relaxed in the nascent strands, as indicated by the reduced effect of a polar amber mutation in the coat cistron upon polymerase protein initiation from nascent RNA.     

The Identification of Collagen Messenger RNA

RNA isolated from calvaria of 16- to 18- day-old chick embryos, assayed in rabbit reticulocyte lysates, programs the synthesis of a collagenase-sensitive protein with the molecular weight of collagen pro-alpha-chains. When RNA labeled with [(3)H]uridine for 2 hr and chased for 1 or 2 hr was electrophoresed on aqueous polyacrylamide gels, most of the radioactivity not in 28S or 18S rRNA migrated with an apparent molecular weight of about 1,800,000. After oligo(dT)-cellulose chromotography and analysis in 99% formamide gels, this nonribosomal, rapidly labeled calvaria RNA species migrates at 28S-30S and thus has a molecular weight of at least 1,600,000.Both the ability to program the synthesis of collagenase-sensitive protein in reticulocyte lysates and the presence of a single prominent rapidly labeled 30S peak in acrylamide gels strongly support the deduction that there is only one major mRNA species in calvaria and that this species is collagen messenger RNA.