Translation and characterization of poly(A)-lacking RNA from lupin root nodules

Total polysomal RNA from yellow lupin root nodules was fractionated by double oligo(dT)-cellulose chromatography. Poly(A)-containing and poly(A)-lacking RNA fractions showed considerable messenger activity in wheat germ and rabbit reticulocyte cell-free systems. The sizing of poly(A)-lacking RNA on sucrose-density gradient gives rise to separation of 14S mRNA from 22-24S mRNA species. A single polypeptide with molecular weight of 22,000 was coded for by 14S mRNA, while two polypeptides with an apparent mol. wt. of 90,000 and 87,000 were the main products of 22-24S mRNA fraction. High concentrations of unfractionated poly(A)-lacking RNA as well as the addition of poly(A) led to preferential synthesis of the 22,000 product. Preliminary results suggest the presence of m7GpppX cap structure at 5' terminus of the separated 14S and 22-24S mRNA species. This comes from the competition experiments with m7GMP and m7GTP as well as from the fact that the poly(A)-lacking RNA preparation was susceptible to methylation by methyl-transferase from vaccinia virus (methylated is the 2'-O-nucleotide adjacent to 7-methylguanosine). Digestion by T1 RNAase of methylated poly(A)-lacking RNA produced two short 5'-terminal oligonucleotides 10 and 17 nucleotides in length.     

Translation in a cell-free system of mRNA from term placenta extracted by guanidine hydrochloride

Human term placenta RNA and polyadenylated mRNA were prepared using guanidine HCl and oligo (dT)-cellulose affinity chromatography. Both fractions translated in a wheat germ cell-free system showed, under optimal condition of K+ and Mg++ ions and spermidine, about 9 times activity for RNA and 15-25 times for poly(A+) mRNA greater than the control. Homogenization of the tissue at high speed compared to that at low speed improved 4-fold activity. Analysis of tritiated products by SDS-Polyacrylamide gel electrophoresis and detected by fluorography showed more than ten different intensity bands ranging between 12 and 66 kD. According to the results obtained, guanidine HCl is an advantageous procedure for the extraction of RNA from this nuclease-rich tissue compared with that obtained with phenol extraction, in both activity and in larger translation products.     

Evidence for the presence of mRNA in the post-ribosomal cytoplasm of sheep lymphocytes.

Several fractions of RNA prepared from the post-ribosomal cytosol of sheep lymphoid cells were found to include messenger-like RNA as defined by the following criteria: a, template activity, i.e. the ability to promote the incorporation of radioactive amino acids into protein in cell-free protein-synthesising systems derived from wheat embryos or ascites tumour cells; b, a low magnesium optimum (1-2.5 mM) for template activity which is characteristic of many natural mRNAs; c, sensitivity of the template response to aurintricarboxylic acid, a specific inhibitor of the initiation of protein synthesis. The lymphoid post-ribosomal RNA fractions, however, were translated less efficiently than were rabbit reticulocyte globin mRNA or tobacco mosaic viral (TMV) RNA; no explanation for this relatively poor template activity was found. The major fraction of messenger-like RNA had an average sedimentation coefficient of 12 S; this fraction directed the translation of several discrete polypeptides in the molecular weight range 10 000-25 000. On average the products of 12 S RNA-directed protein synthesis appeared lysine rich compared with TMV RNA-directed products. It is suggested that the apparent pool of uncommitted mRNA in resting lymphocytes may be utilised during the early stages of lymphocyte activation, and that the mRNAs could be stored in forms similar to those evident in other dormant tissues.     

Translation and identification of the viral mRNA species isolated from subcellular fractions of vesicular stomatitis virus-infected cells.

The cytoplasm of vesicular stomatitis virus (VSV)-infected BHK cells has been separated into a fraction containing the membrane-bound polysomes and the remaining supernatant fraction. Total poly(A)-containing RNA was isolated from each fraction and purified. A 17S class of VSV mRNA was found associated almost exclusively with the membrane-bound polysomes, whereas 14,5S and 12S RNAs were found mostly in the postmembrane cytoplasmic supernatant. Poly(A)-containing VSV RNA synthesized in vitro by purified virus was resolved into the same size classes. The individual RNA fractions isolated from VSV-infected cells or synthesized in vitro were translated in cell-free extracts of wheat germ, and their polypeptide products were compared by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The corresponding in vivo and in vitro RNA fractions qualitatively direct the synthesis of the same viral polypeptides and therefore appear to contain the same mRNA species. By tryptic peptide analysis of their translation products, the in vivo VSV mRNA species have been identified. The 17S RNA, which is compartmentalized on membrane-bound polysomes, codes for a protein of molecular weight 63,000 (P-63) which is most probably a nonglycosylated form of the viral glycoprotein, G. Of the viral RNA species present in the remaining cytoplasmic supernatant, the 14.5S RNA codes almost exclusively for the N protein, whereas the 12S RNA codes predominantly for both the NS and M proteins of the virion.     

Translation of messenger RNA fractions extracted from free and membrane bound rat forebrain ribosomes in a rabbit reticulocyte cell-free system

Messenger RNA fractions were obtained from free and membrane bound rat forebrain ribosomes by alkaline phenol extractions. These RNA fractions stimulated protein synthesis in a cell-free rabbit reticulocyte system partially dependent on the addition of exogenous mRNA. The polypeptide products of protein synthesis with RNA fractions derived from free and membrane bound brain ribosomes and reticulocyte ribosomes were compared by polyacrylamide gel electrophoresis and found to have different distributions.     

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.     

Changes in the sequence content of albumin mRNA and in its translational activity in the rat liver with age

To investigate the regulation of age-related changes in albumin synthesis in the rat liver, total postnuclear RNA and polyribosomes, both membrane-bound and free, were prepared from livers of rats of different ages. By the use of a specific complementary DNA probe, the albumin mRNA sequence content was quantitated in these RNA fractions. These studies showed a specific increase in albumin mRNA sequence content in total postnuclear RNA and membrane-bound polyribosomes at between 12 and 24 months of age. Between 24 and 36 months of age, the increase in the amount of albumin mRNA in these two fractions was due only to an increase in liver weight. The increase in albumin mRNA sequence content was not found in the poly(A)⁺ fraction but in the RNA extracted from the void of oligo(dT)-cellulose column chromatography. The isolated polyribosomes were translated in a cell-free system to assess age-related changes in total protein and albumin synthesis due to translational control. No changes with age were found in the translational capacity of membrane-bound and free polyribosomes per RNA unit. Immunoprecipitation of the synthesized albumin in the translation products revealed that albumin synthesis in the cell-free system is not increased proportionally with the elevated albumin mRNA level between 12 and 24 months of age. This indicates that albumin mRNAs present in the livers of old rats are biologically less active than those found in younger animals.     

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.     

The course of photooxidation of menadione.

The highly purified (> 95%) mRNA coding for immunoglobulin light (L)-chain yields on acrylamide gels a discrete 15.5S band and a “shoulder” ranging in size from 15.5 to 9.5S. The “shoulder” was isolated and found to be fragmented mRNA as judged from: 1. hybridization kinetic analysis, using the complementary-DNA to the L-chain mRNA; 2. capacity to form aggregates, similarly to the intact 15.5S mRNA. Partial cleavage of the mRNA probably occurs during mechanical disruption of the myeloma cells. Fragments with intact 3′ end are selected due to binding via the poly(A) moiety to oligo(dT)-cellulose, i.e., the fragments should be deficient at the 5′ end where mRNA translation is initiated. In agreement, the fragmented mRNA is essentially untranslatable in a cell-free system. The size of the L-chain mRNA is rather uncertain. A value of 15.5S is obtained from migration in acrylamide gels made in water or formamide, a value of 12S is obtained from sucrose gradient centrifugation.     

The synthesis of presumptive procollagen messenger ribonucleic acid in the calvaria of the developing chick embryo

The presumptive messenger RNAs for type I procollagen were isolated from chick embryo calvaria at various stages of development. Poly(A)-containing RNA fractions from denaturing sucrose gradients directed protein synthesis in a cell-free system derived from wheat germ. Procollagen mRNA activity was detected in a region of about 26 S. Approx. 80% of the labeled proline incorporated into cell-free product was susceptible to digestion by purified bacterial collagenase. The synthesis of procollagen mRNAs was followed during development. Comparison of the in vitro labeled mRNAs from calvaria of day 12--16 embryos indicated that the 26 S component was most pronounced at day 13 and decreased progressively towards day 16. In addition, incubation of calvaria with tritiated nucleosides for 1.5--25 h revealed that 26 S mRNA was significantly labeled only after prolonged periods. The results suggest that procollagen mRNA is a relatively stable species with a prolonged half-life compared to the majority of mRNAs in this tissue.     

Poly(adenylic acid)-containing and -deficient messenger RNA of mouse liver

RNA was isolated and fractionated into poly(A)-containing and -deficient classes by oligo(dT) chromatography. Approximately 99% of the poly(A) material bound to the oligo(dT); that which did not bind contained substantially shorter poly(A) chains. All RNA fractions retained an ability to initiate cell-free translation, with the poly(A)-deficient fraction containing half the total translational activity, i.e., mRNA. Two-dimensional polyacrylamide gel analysis of the cell-free translation products revealed three classes of mRNA: 1, mRNA preferentially containing poly(A), including the abundant liver mRNA species; 2, poly(A)-deficient mRNA, including many mid- and low-abundant mRNAs exhibiting less than 10% contamination in the poly(A)-containing fraction fraction; and 3, bimorphic species of mRNA proportioned between both the poly(A)-containing and -deficient fractions. Poly(A)-containing and bimorphic mRNA classes were further characterized by cDNA hybridizations. The capacity of various RNA fractions to prime cDNA synthesis was determined. Compared to total RNA, the poly(A)-containing RNA retained 70% of the priming capacity, while 20% was found in the poly(A)-deficient fraction. Poly(A)-containing, poly(A)-deficient, and total RNA fractions were hybridized to cDNAs synthesized from (+)poly(A)RNA. Poly(A)-containing RNA hybridized with an average R0t 1/2 approximately 20 times faster than total RNA. Poly(A)-deficient RNA hybridized with an average R0t 1/2 approximately 3-4 times slower than total RNA. These R0t 1/2 shifts indicated that in excess of three-quarters of the total hybridizable RNA was recovered in the poly(A)-containing fraction and that less than one-quarter was recovered in the poly(A)-deficient RNA fraction. Abundancy classes were less distinct in heterologous hybridizations. In all cases the extent of hybridization was similar, indicating that while the amount of various mRNA species varied among the RNA fractions, most hybridizing species of RNA were present in each RNA fraction. cDNA to the abundant class of mRNAs was purified and hybridized to both (+)- and (-)poly(A)RNA. Messenger RNA corresponding to the more abundant species was enriched in the poly(A)-containing fraction at least 2-fold over the less abundant species of mRNA, with less than 10% of the abundant mRNAs appearing inthe poly(A)-deficient fraction.     

Isolation and partial purification of catfish pancreatic islet messenger RNA.

Poly(a)-rich mRNA has been isolated from catfish pancreatic islet total nucleic acid. Cell-free translation of the mRNA by wheat germ extracts yielded a protein of 11 000-12 000 molecular weight, estimated by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis. This peptide is larger than catfish proinsulin, but contains tryptic peptides of proinsulin. Its synthesis comprises up to 23% of the cell-free product, depending on the conditions of cell-free synthesis. Synthesis is inhibited by 7-methylguanosine 5'-monophosphate suggesting the presence of a 7-methylguanosine cap on the 5' end of catfish proinsulin mRNA. Sucrose gradient centrifugation of the islet poly(A)-rich mRNA yielded 8S and 12S peaks. These fractions were translated with wheat germ extracts and it was determined that over 60% of the islet mRNA-dependent protein from the 8S fraction was preproinsulin. The 8S mRNA fraction was electrophoresed on 3% agarose-6 M urea gels and demonstrated to be several bands, ranging from 100 000-200 000 molecular weight.     

Wheat Storage Proteins : ISOLATION AND CHARACTERIZATION OF THE GLIADIN MESSENGER RNAs

A total RNA extract was prepared from developing wheat seeds using guanidine-HCl to eliminate endogenous RNase activity. The RNA preparation, substantially free of protein, carbohydrate and DNA, was chromatographed on either a poly uridylic acid-agarose or poly guanylic acid-agarose column to yield a gliadin-enriched mRNA fraction. Only slight differences were observed for the products synthesized in a wheat germ cell-free translation system when either poly adenylic acid-enriched or cytosine-rich RNA was used as a template. These results are consistent with the high proline content of the gliadins and indicate that a large proportion of the mRNA activity in these RNA preparations is directed toward gliadin synthesis. After a second affinity chromatography step, the gliadin-enriched mRNA fraction was fractionated by two cycles on sucrose-density gradient centrifugation under denaturing conditions. The RNA sedimented as a broad band with a peak at 14S and a shoulder at the 11S region of the sucrose gradient. RNA from the peak 14S fraction translated predominantly the two major gliadin polypeptides which had molecular weights of 34,000 and 36,000. Analysis of the 14S RNA by methylmercury hydroxide-agarose gel electrophoresis revealed the presence of a predominant RNA species with a molecular size of 415,000 (1,200 nucleotides).     

A specific dimerization of rabbit beta-globin messenger ribonucleic acid.

Rabbit globin mRNA, when layered in low salt on 0.1 M-NaCl/sucrose gradients, separates into two peaks of material. Translation of these two RNA fractions in the wheat-germ cell-free system, hybridization against globin complementary DNA (cDNA) and cross-hybridization against cDNA species prepared from each fraction show that the first peak sedimenting at 10S is a alpha-globin mRNA and the second peak, sedimenting at approx. 15S, is beta-globin mRNA. The sedimentation rate of the beta-globin mRNA is concentration-dependent. By changing concentration and pH, it is indicated that in low-salt beta-globin mRNA adopts a conformation that leads to specific, but weak, self-dimerization during centrifugation in 0.1M-NaCl. This property permits rapid preparation of intact and relatively pure alpha- and beta-globin mRNA species.     

Isolation of ribonucleic acids possessing template activity from Crithidia oncopelti mitochondria

Three RNA fractions (12SE, 9SE and 8SE) were isolated from highly purified mitochondrial preparations of Crithidia oncopelti. Using inhibitory analysis and competitive hybridization, it was shown that the mitochondrial fractions under study are synthesized in the kinetoplasts and do not belong to ribosomal RNA. It was demonstrated that these fractions possess template activity, since they stimulate protein synthesis in a cell-free system of E. coli. Each fraction is subjected to translation to form one predominant polypeptide with molecular weight of about 20 000, 10 000 and 7000, respectively.     

Degradation of Bowman-Birk protease inhibitor mRNA with a cell-free extract.

A cell-free mRNA degradation system consisting of the polysomal and postpolysomal fractions was obtained from cultured soybean cotyledons for studying Bowman-Birk protease inhibitor (BBPI) mRNA stability. This in vitro system reflected the shorter in vivo half-life of BBPI mRNA of cotyledons cultured in basal-medium as compared to cotyledons cultured in methionine-supplemented medium. Most of the BBPI mRNA degradative activity was found to be present in the postpolysomal supernatant fraction. The higher rate of BBPI mRNA degradation in basal medium-cultured cotyledons was due to an increased destabilizing activity specific to BBPI mRNA in the postpolysomal fraction from basal-medium cultured cotyledons. The specificity was absent when purified RNA was used as the substrate. Degradation of the mRNA was not divalent cation-dependent and was inhibited in the presence of higher concentrations of monovalent and divalent cations.     

Histone messenger RNAs of the mouse testis

A 6-12S RNA fraction has been isolated following sucrose gradient fractionation of mouse testis RNA, and further resolved into poly A+ and poly A- RNA fractions by oligo-(dt)-cellulose chromatography. Polyacrylamide gel electrophoresis of products formed in a reticulocyte lysate-dependent cell-free translation system has enabled identification of histone variants, H1t, H2S, H2A . X, an H4-like protein and a low Mr protein (presumably TP and/or protamine). Cell-free synthesis of a number of these histone variants appears to be directed by poly A+ mRNAs.     

On the existence of polyadenylated histone mRNA in Xenopus laevis oocytes

In a variety of systems, histone mRNA has been shown to lack poly(A) (Adesnik and Darnell, 1972;Grunstein et al., 1973). We have found, however, that in Xenopus laevis oocytes, poly(A)-containing mRNA codes for histones, in a wheat germ cell-free system, based on the following criteria: first, co-migration with authentic X. laevis oocyte histones on polyacrylamide gels; second, no detectable incorporation of tryptophan; third, differential incorporation of lysine and methionine into histone fraction H2A; fourth, resistance of histone fraction H2A to cleavage with cyanogen bromide; and fifth, correspondence of tryptic peptide maps of partially purified cell-free products with authentic X. laevis oocyte histone. RNA which directs the synthesis of histones in the cell-free system is retained on oligo(dT)-cellulose, even after denaturation in 80% DMSO at 70°C, thereby demonstrating the covalent attachment of polyadenylic acid sequences to the mRNA. Poly(A)^? RNA (7S–14S fraction) was also found to code for histones using the same criteria. We discuss the significance of the finding that X. laevis oocytes contain two classes of histone mRNA as well as the potential developmental implications of this observation.     

Assay of protamine messenger RNA from rainbow trout testis.

A low molecular weight RNA fraction possessing protamine mRNA activity was prepared from rainbow trout testis polysomes. Addition of low molecular weight RNA to a Krebs II ascites S-30 cell-free protein synthesis system strongly stimulated [14C]arginine incorporation into acid-insoluble material. This stimulation was completely abolished by 10-4 M aurintricarboxylic acid, an inhibitor of eukaryotic protein synthesis at the level of initiation. Starch gel electrophoresis showed that labeled arginine was incorporated in vitro into products identical with both authentic protamine and histones as found previously (Gilmour, R. S., and Dixon, G. H. (1972) J. Biol. Chem. 247, 4621-4627). The 4 to 6 S RNA fraction, isolated from the polysomal low molecular weight RNA by sucrose gradient fractionation, enhanced the incorporation of [14C]arginine into acid-insoluble material and when this product was examined by starch gel electrophoresis, it co-migrated with authentic rainbow trout protamine.