Messenger RNA of human immune interferon: isolation and partial characterization

Suspension cultures of total and lymphocyte-enriched peripheral white blood cells were used as a source for the preparation of mRNA for human γ-interferon. The cells were lysed and total RNA was extracted with the phenol method. A poly(A)-rich RNA fraction was isolated by affinity chromatography on oligo(dT)-cellulose and further purified on a preparative sucrose gradient. The RNA preparations were translated by oocytes into (a) protein(s) that showed biological activity in the interferon-assay. On sucrose gradient centrifugation the translatable fractions of the RNA migrated with a sedimentation coefficient of approximately 15 S, a value compatible with the molecular weight of human γ-interferon. The translation product was further characterized by serological cross reactivity with purified γ-interferon in neutralization reactions.     

Insulin antagonism of glucocorticoid induction of tyrosine aminotransferase in cultured foetal hepatocytes

Polyadenylated RNA from developing Artemia salina cysts was fractionated by centrifugation through a sucrose gradient containing methylmercuric hydroxide (CH3HgOH). Aliquots of each fraction were directly added to a rabbit reticulocyte lysate to program protein synthesis in vitro. The translation products were assayed for eukaryotic elongation factor Tu (eEF-Tu) by immunoprecipitation with an antibody raised in rabbits and purified by affinity chromatography. The immunoprecipitated radioactivity was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. Sequences coding for eEF-Tu sediment in the 20-S region of the gradient and form a major component of the poly(A)-containing RNA. The mRNA of the 20-S region, comprising about 10% of the poly(a)-containing RNA fractionated on the gradient, has been translated in vitro and 30% of the translation products represent immunoprecipitable eEF-Tu protein chains with an Mr of 50000.     

Utilization of polyadenylate mRNA during growth and starvation in Physarum polycephalum

The effect of growth on the efficiency of utilization of poly(A)-containing mRNA for translation has been investigated in microplasmodia of Physarum polycephalum. Measurement of the relative proportions of poly(A)-rich mRNA in polysomal and post-polysomal fractions isolated by sucrose density gradient centrifugation reveals that newly synthesized poly(A)-rich mRNA is present in increasing proportions in the polysomal region during exponential growth. However, the proportion of long-lived poly(A)-rich mRNA observed in actively-translating polysomes declines as starvation approaches. The ribonuclease content and morphology of the microplasmodia were monitored during growth and starvation in an effort to related this phenomenon to the onset of spherulation.     

Partial Purification and Characterization of Messenger RNA Coding 14-3-2 Protein from Rat Brain

14-3-2 Protein (neuron-specific enolase) is a neuron-specific protein. Using a reticulocyte lysate cell-free system for translation of 14-3-2 protein mRNA, we have partially purified this mRNA by several procedures, including formamide sucrose density centrifugation, formamide polyacrylamide gel electrophoresis (PAGE) and polyuridylic acid (poly(U))-Sepharose affinity chromatography. Using mRNA obtained by these procedures, we could increase the translation ratio of 14-3-2 protein synthesized/total soluble protein synthesized to 7.31%. The overall purification was 37.8-fold. The size of 14-3-2 protein mRNA appears to be about 19-20S, because translation activity of mRNA obtained by sucrose density gradient centrifugation or formamide PAGE was the most active in this RNA size.     

Cell-free synthesis of rat alpha 2-macroglobulin and induction of its mRNA during experimental inflammation

Poly(A)-rich RNA was isolated from the livers of acutely inflamed rats by extraction with guanidinium HCl and oligo(dT)-cellulose chromatography. After translation in a recticulocyte lysate and immunoprecipitation with a specific antiserum to alpha 2-macroglobulin a polypeptide with an apparent molecular weight of 162000 could be detected. The cell-free synthesis of alpha 2-macroglobulin was stimulated 8-fold by the addition of RNase inhibitor. Full-length alpha 2-macroglobulin polypeptide chains appeared after 35 min in the presence of 1.85 mM Mg2+ and 100 mM K+. A nucleotide number of about 5100 was estimated for alpha 2-macroglobulin by means of sucrose gradient centrifugation of poly(A)-rich RNA followed by translation in vitro and immunoprecipitation of alpha 2-macroglobulin. In normal liver alpha 2-macroglobulin mRNA represented about 0.0007% of total translatable RNA. Acute inflammation generated by intramuscular injection of turpentine led to a 66-fold increase in translatable alpha 2-macroglobulin mRNA after 18 h, followed by a rapid decrease. In accordance to the induction of alpha 2-macroglobulin mRNA serum concentrations of alpha 2-macroglobulin increased to about 2 mg/ml. Unlike alpha 2-macroglobulin mRNA serum alpha 2-macroglobulin levels remained unchanged up to 60 h.     

Regulation of the synthesis of lactate dehydrogenase-X during spermatogenesis in the mouse

Total mouse testis RNA directs the synthesis of the sperm-specific C subunit of lactate dehydrogenase-X (LDH-X) when translated in a cell- free system derived from rabbit reticulocytes. The newly synthesized C subunits were isolated by immunoprecipitation with antibody specific for this isozyme, and quantitated by electrophoresis on SDS polyacrylamide gels. The amount of radioactivity incorporated into the enzyme subunit was directly proportional to the amount of testis RNA added to the translational system, thereby providing a sensitive and reliable method for assessing relative LDH-X mRNA activity. A combination of sucrose gradient centrifugation and oligo(dT)-cellulose chromatography resulted in a 23-fold purification of LDH-X mRNA over total cytoplasmic testis RNA. Analysis of LDH-X mRNA activity in the developing testis indicated that the appearance of functional LDH-X mRNA activity coincides with the appearance of LDH-X catalytic activity at 14 d postpartum. Measurement of LDH-X mRNA levels in separated testis cell populations prepared by centrifugal elutriation demonstrated that LDH-X mRNA represents 0.17-0.18% of the total functional mRNA activity in fractions enriched in pachytene spermatocytes and round spermatids, but only 0.09-0.10% of the translation products of elongated spermatids.     

核仁含磷蛋白B_(23)mRNA的部分提纯

用蔗糖梯度离心法对Novikoff肝癌细胞的多聚(A)~+mRNA进行了链长分部。沉降在13S和15S的组分6和组分7富集了B_(23)mRNA,其体外转译产物可特异地被抗蛋白B_(23)的抗体免疫吸附,被吸附的蛋白在SDS-PAGE中迁移到大约37,000道尔顿的区带。另外,用多聚核糖体免疫吸附技术提纯了少量B_(23)mRNA,它在体外转译系统中也指导合成了分子量约为37,000道尔顿的蛋白质。     

Wheat embryo ribonucleates. XIV. Mass isolation of mRNA from wheat germ and comparison of its translational capacity with that of mRNA from imbibing wheat embryos

Commercially milled wheat germ is shown to be a convenient source material for facile recovery of mass (milligram) quantities of highly purified poly(A)-rich RNA. This poly(A)-rich RNA is efficiently translated in a nuclease-treated extract of rabbit reticulocytes. By sucrose density gradient fractionation of bulk poly(A)-rich RNA from wheat germ, it has been possible to show that there is a direct relationship between the molecular weights of the polypeptide products of cell-free synthesis and the molecular weights of the wheat mRNA molecules which program their synthesis. As assessed by SDS -- polyacrylamide gel electrophoresis, the same array of polypeptides is synthesized when nuclease-treated reticulocyte extract is programmed by poly(A)-rich RNA from either commercially supplied or laboratory-prepared wheat embryos. Significantly, there are gross quantitative if not qualitative differences between the translational capacities of poly(A)-rich RNA from dry and imbibing wheat embryos, and the possible importance of these differences for interpreting a changing pattern of polypeptide synthesis in imbibing wheat embryos is the subject of a brief discussion.     

Translation in vitro of Tetrahymena pyriformis polyadenylated mRNA. Identification of tubulin amongst the translated products and demonstration of its heterogeneity.

The capacity of poly(A)-containing RNA of the protozoan ciliate Tetrahymena pyriformis to direct the synthesis of proteins in vitro has been tested using two cell-free systems: a wheat germ extract and a rabbit reticulocyte lysate. The results obtained with these two systems are compared and the identification of alpha and beta tubulins among the products of protein synthesis in vitro, after separation by one-dimensional and two-dimensional electrophoresis, is described. By isoelectric focusing in polyacrylamide gels, each species of tubulin is resolved into several bands, suggesting that the main subunits are more heterogeneous than has been generally described. Poly(A)-containing RNA has also been fractionated on a 70% formamide/sucrose gradient and it is shown that alpha and beta tubulins are coded by separate mRNAs.     

Separation and characterization of low-molecular-weight nuclear RNA species that inhibit cell-free protein synthesis

Various RNA fractions were isolated from nuclei of 12-day lactating rat mammary glands and examined for their ability to inhibit cell-free protein synthesis. Although total nuclear RNA was generally inactive, material contained in the poly(A)+ nuclear RNA fraction and the low-molecular-weight RNA derived from total nuclear RNA by sucrose gradient centrifugation, inhibited the translation of several mRNAs but not poly(U) or poly(A). Separation of the small nuclear RNAs by preparative polyacrylamide-urea gel electrophoresis allowed the identification of at least three active inhibitor RNA species. These differed both with respect to their ability to inhibit protein synthesis, and in their mechanism of action. While two of the RNA species inhibited elongation the other inhibited initiation of protein synthesis.     

Isolation and partial characterization of the messenger RNA encoding the B880 holochrome protein of Rhodospirillum rubrum

The B880 holochrome messenger RNA was extracted from cultures of the photosynthetic bacterium Rhodospirillum rubrum. It was purified by chromatography on Sepharose 4B followed by sucrose density gradient centrifugation. The purified fractions were shown to program an Escherichia coli cell-free system into synthesizing both the alpha and the beta polypeptides of the holochrome. The translation products were identified by immunoprecipitation with specific antibodies raised against these polypeptides. The latter are effective competitors with the translation products for antigen-antibody complex formation. The purest mRNA preparations contained approximately 33% holochrome messenger RNA activity. Its most probable size, as determined by agarose gel electrophoresis in the presence of 6 M urea or methylmercuric hydroxide, is approximately 620 nucleotides. Since the combined sizes of the alpha and beta polypeptides add up to only 106 amino acid residues, we conclude that the holochrome mRNA is most probably polycistronic.     

Nuclear steady-state RNA from chicken immature red blood cells. Distribution of globin-coding and poly (A) sequences

Nuclear steady-state RNA and polysomal RNA of chicken immature red blood cells were isolated and separated on formamide sucrose gradients. For comparison the distribution of 9 S globin mRNA was investigated by gradient centrifugation of 125I-labelled mRNA. The material was either pooled into two fractions (less than 20 S; greater than 20 S) and translated in an Ehrlich ascites cell-free system or each gradient fraction was analyzed by hybridization with [3H]-poly (U) or [3H]-labelled DNA complementary to purified 9 S globin mRNA (globin cDNA). In neither case could evidence be obtained for the existence of a high molecular weight RNA as a probable globin mRNA precursor. Further analysis was performed by electrophoresis of RNA on exponential polyacrylamide gels in formamide and subsequent hybridization with cDNA. The results are consistent with those of gradient centrifugation and demonstrate that the distribution of globin-coding sequences in nuclear steady state RNA corresponds to that of cytoplasmic 9 S globin mRNA.     

Characterization of messenger RNA for chick-embryo DNA polymerase beta and its translation product in vitro

A specific immunoprecipitation method, using rabbit anti-(chick DNA polymerase beta) IgG was applied to detect the polypeptide of DNA polymerase beta among translation products obtained in vitro with mRNA extracted from chick embryos. A polypeptide of Mr = 40 000 was specifically immunoprecipitated from [35S]methionine-labeled translation products and was competitive with the purified DNA polymerase beta for the antibody. Furthermore, the 40 000-Mr translation product obtained in vitro had DNA polymerase activity, which was detected by assay in situ after electrophoresis in a polyacrylamide gel containing DNA. The mRNA for DNA polymerase beta was polyadenylated and its content was estimated as the range of 0.001% of total poly(A)-rich RNA on the basis of [35S]methionine incorporation in the translation in vitro. The size of this mRNA was determined to be about 1800 nucleotides by zone sedimentation and agarose gel electrophoresis under denaturating conditions.     

Separation and characterization of low-molecular-weight nuclear RNA species that inhibit cell-free protein synthesis

Various RNA fractions were isolated from nuclei of 12-day lactating rat mammary glands and examined for their ability to inhibit cell-free protein synthesis. Although total nuclear RNA was generally inactive, material contained in the poly(A)⁺ nuclear RNA fraction and the low-molecular-weight RNA derived from total nuclear RNA by sucrose gradient centrifugation, inhibited the translation of several mRNAs but not poly(U) or poly(A). Separation of the small nuclear RNAs by preparative polyacrylamide-urea gel electrophoresis allowed the identification of at least three active inhibitor RNA species. These differed both with respect to their ability to inhibit protein synthesis, and in their mechanism of action. While two of the RNA species inhibited elongation the other inhibited initiation of protein synthesis.     

Purification of messenger RNA coding for glycine methyltransferase from rat liver

Rat liver messenger RNA coding for glycine methyltransferase was associated preferentially with free polysomes. The mRNA was purified about 1000-fold over the total poly(A)-containing RNA by specific immunoadsorption of polysomes to protein A-Sepharose followed by oligo(dT)-cellulose column chromatography. Sodium dodecyl sulfate-gel electrophoresis of the in vitro translation products in a rabbit reticulocyte lysate system revealed only one major band which migrated to the position of the purified glycine methyltransferase subunit. The result shows that the mRNA isolated is nearly homogeneous and suggests that no precursor form of the enzyme existed. The mRNA sedimented at the position slightly smaller than 18 S rRNA in a sucrose density-gradient centrifugation and was shown to contain about 1,300 nucleotides by the Northern blot hybridization analysis with a cDNA probe.     

Cell-free synthesis of a larger-molecular-weight precursor of cytochrome c oxidase subunit V from rat liver and the distribution of its mRNA between free and membrane-bound polysomes

Poly(A)-rich RNA from phenol-extracted rat liver polysomes was translated in a heterologous cell-free system derived from wheat germs. The labeled translation products were incubated with an antiserum against cytochrome c oxidase subunit V. After immunoprecipitation and affinity chromatography with protein-A-Sepharose, the isolated antigen-immunoglobulin complexes were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. Only one protein with an apparent molecular weight of 15 500 was visualized. In immunocompetition experiments with unlabeled individual cytochrome c oxidase subunits IV, V, VI or VII only subunit V could compete with the 15 500-Mr protein synthesized in vitro. Two-dimensional fingerprints of cytochrome c oxidase subunit V and the polypeptide synthesized in vitro showed a high degree of similarity. It is concluded that the cytochrome c oxidase subunit V is synthesized as a precursor with an amino-terminal extension of about 25 amino acids. It was possible to convert the precursor of cytochrome c oxidase subunit V synthesized in vitro to its mature form by intact mitochondria as well as by submitochondrial particles. A chain length of 830 +/- 70 nucleotides was estimated for the poly(A)-rich mRNA of the higher-molecular-weight precursor of rat liver cytochrome c oxidase subunit V. Assuming a molecular weight of 15 500 for the precursor a non-coding region of about 300 nucleotides must exist. In experiments on the site of synthesis it is shown that the poly(A)-rich RNA for the higher-molecular-weight precursor of cytochrome c oxidase subunit V is found in free, loosely and tightly membrane-bound polyribosomes.