Nucleotide Sequences of Human Globin Messenger RNA

Globin messenger RNA, isolated from human peripheral blood reticulocytes, was transcribed into complementary DNA by use of the RNA-dependent DNA polymerase of avian myeloblastosis virus. The complementary DNA was then transcribed into (32)P-labeled complementary RNA by E. coli RNA polymerase in the presence of alpha-(32)P-labeled ribonucleoside triphosphates. The fingerprint pattern obtained from ribonuclease T1 digests of human globin complementary RNA was specific and reproducible. Different patterns were obtained from digests of duck, mouse, and rabbit globin complementary RNA. The fingerprint patterns obtained from digests of purified natural human 10S globin messenger RNA, labeled in vitro with (125)I or with [gamma-(32)P]ATP and polynucleotide kinase, were similar to that of the complementary RNA but contained some additional oligonucleotides. Sufficient nucleotide sequence information has been obtained from about 50% of the intermediate sized oligonucleotides (8-14 base residues long), to make possible examination of correspondence between these nucleotide sequences and globin amino-acid sequences. Approximately 70% of these oligonucleotide sequences can be matched to unique amino-acid sequences in the alpha- or beta-globin chains. The other 30% do not match known amino-acid sequences and presumably correspond to untranslated portions of the mRNA; some of these sequences, however, can be matched to amino-acid sequence in the abnormally long segment of the alpha chain of hemoglobin Constant Spring, which is thought to result from a chain-termination mutation.     

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.     

Formation of Sindbis Virus Capsid Protein in Mammalian Cell-Free Extracts Programmed with Viral Messenger RNA

Extracts from Krebs II ascites cells and rabbit reticulocytes effectively synthesize viral proteins with Sindbis viral mRNA isolated from Sindbis-infected BHK cells. The major product is identical to Sindbis capsid protein on the basis of its electrophoretic mobility in sodium dodecyl sulfate-acrylamide gels and two-dimensional tryptic-peptide fingerprints. Various amounts of several additional discrete polypeptides are formed, depending on the components of the cell-free extracts. One of these polypeptides may be a prematurely terminated part of the viral-capsid protein, while another is larger in molecular weight than capsid protein but contains the capsid tryptic peptides. Several of the proteins formed in vitro also are detected in extracts of Sindbis-infected BHK cells labeled with [(35)S]methionine.The three proteins found in Sindbis virions are postulated to originate by proteolytic cleavage from a larger molecular weight polypeptide precursor that is translated from a polycistronic mRNA presumed to contain a single site for initiation of protein synthesis. The two in vitro systems appear to translate this polycistronic viral mRNA to yield specific viral capsid although no evidence was found for post-translational proteolysis. Other mechanisms for production of the capsid protein in the cell-free extracts are considered, and some of these may function in the viral-infected cell where unusually large amounts of viral capsid proteins are frequently detected.     

Cell-Free Translation of Maternal Messenger RNA from Sea Urchin Eggs

The template activity of RNA extracted from unfertilized sea urchin eggs was demonstrated in a cell-free translation system, and, for the first time, specific proteins were identified among the products. All messenger RNA activity is recovered, under the conditions used, from the postribosomal supernatant. Histones, among many other proteins, were identified specifically as products of this "maternal" messenger RNA.     

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.     

Cell-Free Translation of Highly Purified Adenovirus Messenger RNA

Several polypeptides contained in the coat and the core of adenovirus type 2 particles have been identified as virus gene products. They are synthesized in vitro by a cell-free protein-making system programmed by adenovirus type 2 messenger RNA that has been hybridized with and eluted from virus DNA. Fractionation by size yields subpopulations of viral messenger RNA that differ in their coding specificities. The procedures outlined in this study may be used to establish a genetic map of adenovirus type 2.     

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.     

Mammalian Cells with Altered Forms of RNA Polymerase II

Mutants of Chinese hamster ovary cells that are resistant to α-amanitin can be isolated. At least some of these mutants contain an altered form of DNA-dependent RNA polymerase II, as indicated by its resistance to α-amanitin. These results indicate that mutation to α-amanitin resistance involves a change of a structural gene.     

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.     

Accessibility of DNA in Chromatin to DNA Polymerase and RNA Polymerase

The accessibility of DNA in chromatin to both exogenous DNA polymerase and RNA polymerase is slight when compared to isolated DNA. DNA in extracted chromatin is somewhat more accessible to these enzymes than is DNA in the chromatin of isolated nuclei; and the DNA template of chromatin is more accessible to DNA polymerase than to RNA polymerase. In these experiments we have given much attention to the technique of scintillation counting, since artifacts arising in this procedure can lead to erroneous conclusions.     

Release, Identification, and Isolation of Messenger RNA from Mammalian Ribosomes

The puromycin-induced dissociation, at high ionic strength, of active ribosomes from reticulocytes resulted in the release of protein-free, apparently undegraded, messenger RNA for globin, which was identified by centrifugation on a sucrose density gradient. This procedure should make messenger RNA from various cells available for isolation on a large scale, and has several advantages over procedures that use detergents or magnesium chelators.     

A Particle Associated with the Polyadenylate Segment in Mammalian Messenger RNA

A structure consisting of poly(A) complexed with other components is released from polysomes by ribonuclease treatment. The poly(A) complex has a sedimentation value of 12-15, while the corresponding sedimentation value for free poly(A) is 4. The complex does not appear to represent an artifact formed by interaction of free poly(A) with either cytoplasmic or polysomal proteins. The polynucleotide released from the complex by treatment with sodium dodecyl sulfate shows the same electrophoretic mobility as that of poly(A) isolated from deproteinized polysomal RNA. The poly(A) in the complex is partially protected from digestion by T(2) ribonuclease. At least part of the poly(A) is available for base pairing with poly(U). The components associated with the poly(A) cause it to bind to Millipore filters at low ionic strength. These components are removed from the complex by Pronase digestion. The findings indicate that the poly(A) segment in messenger RNA serves as a binding site for a particle. This particle appears to consist of proteins.     

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.     

Distribution of RNA Polymerase Binding Sites in Fractionated Chromatin

Calf-thymus chromatin was fractionated by ion-exchange chromatography on ECTHAM-cellulose and sucrose gradient sedimentation. [ECTHAM-cellulose is a cationic adsorbent prepared by coupling tris(hydroxymethyl)aminomethane to cellulose with epichlorohydrin.] The capacity of these fractionated chromatins to support RNA synthesis by DNA-dependent RNA polymerase of Escherichia coli was examined, using procedures that permit measurement of binding site frequency. Unfractionated calf thymus chromatin has 5-10% as many binding sites as protein-free DNA. By combination of the two fractionation methods, chromatin samples were obtained containing as few as 2% and as many as 47% of the number of binding sites found on protein-free DNA.     

Efficient Translation of Tobacco Mosaic Virus RNA and Rabbit Globin 9S RNA in a Cell-Free System from Commercial Wheat Germ

Extracts prepared from commercially available wheat germ efficiently translate messenger RNAs from either viral or eukaryotic origin. The addition of tobacco mosaic virus RNA stimulated amino-acid incorporation more than 100-times and rabbit globin 9S RNA between 20- and 30-times. The in vitro product directed by globin 9S RNA comigrated precisely with authentic rabbit globin on sodium dodecyl sulfate-polyacrylamide gels. Furthermore, during high voltage ionophoresis two [(35)S]methionine-labeled tryptic peptides synthesized in vitro comigrated in two dimensions with alphaT5 and betaT5 tryptic peptides from authentic [(35)S]methionine-labeled rabbit globin.     

Translation of Reovirus Messenger RNAs Synthesized In Vitro into Reovirus Polypeptides by Several Mammalian Cell-Free Extracts

Single-stranded reovirus RNA, synthesized in vitro by reovirus cores, functioned as messenger RNA in cell-free extracts prepared from several mammalian cells: Krebs II mouse ascites cells, mouse L cells, Chinese hamster ovary cells, HeLa cells, and rabbit reticulocytes. As shown by acrylamide gel electrophoresis, all eight polypeptides known to be specified by reovirus were synthesized in the reticulocyte system. In the other extracts, from 5 to 7 complete virus proteins were made.     

Cell-Free Translation of Messenger RNA of Simian Virus 40: Synthesis of the Major Capsid Protein

Extracts of wheat germ are capable of synthesizing the major capsid protein of simian virus 40. Poly(A)-containing RNA from BS-C-1 cells infected with simian virus 40 directed the synthesis of a novel polypeptide that migrates in polyacrylamide gels together with the major capsid polypeptide of simian virus 40, VP-1. The patterns of the major tryptic peptides of purified VP-1 and the novel polypeptide synthesized in vitro were identical after two-dimensional paper electrophoresis. The novel polypeptide was not synthesized in response to poly(A)-rich RNA from uninfected cells or from virus-infected cells treated with cytosine arabinoside. Messenger RNA from infected cells purified by selective hybridization to DNA of simian virus 40 directs the synthesis of a major polypeptide of electrophoretic mobility similar to that of VP-1 of simian virus 40. This approach should prove useful in identifying additional products specified by DNA tumor viruses.