Polyadenylic Acid Research Articles

Alkylation of RNA by vinyl bromide metabolites in vitro and in vivo

[1,2-14C]Vinyl bromide was incubated with rat liver microsomes, NADPH, and polyadenylic acid, polycytidylic acid, or RNA, respectively. Part of the adenosine moieties in RNA or in polyadenylic acid were alkylated and labelled 1,N6-ethenoadenosine structures were formed. Part of the cytidine moieties were converted into 3,N4-ethenocytidine. In addition, a further unidentified cytidine alkylation product was observed which was not seen in experiments using [1,2-14C]vinyl chloride. When rats were exposed to [1,2-14C]vinyl bromide, radioactive ethenoadenosine and ethenocytidine were present in hydrolysates of liver RNA. A further alkylation product was observed in the RNA hydrolysates which did not occur in experiments using [14C]vinyl chloride. The data show that vinyl bromide metabolites alkylate nucleic acids; although in general in this respect vinyl bromide and vinyl chloride behave similarly, some differences are observed in the alkylation behaviour of both compounds.

Synthesis of a Possible Precursor of α-Amylase in Wheat Aleurone Cells

alpha-Amylase from wheat aleurone (Triticum aestivum) was synthesized in a S-150 wheat germ readout system using polysomes, and a messenger RNA-dependent reticulocyte lysate system using polyadenylic acid [poly(A)]-enriched RNA. The product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, precipitation by specific lambda-globulin for alpha-amylase, and proteolysis. Two immunoprecipitated products were synthesized from the readout system, the predominant species migrating coincidentally with authentic alpha-amylase on sodium dodecyl sulfate-polyacrylamide gels. A putative precursor, 1,500 daltons larger, was evident but was less abundant. The relationship between the two polypeptides was established by proteolytic analysis using Staphylococcus aureus V8 protease. At least nine fragments were generated and were identical in both species. The poly(A)-enriched RNA synthesized only the putative precursor in the reticulocyte lysate system. Attempts to process the precursor to the mature size of alpha-amylase failed. These findings are discussed in connection with the signal hypothesis (proposed for the transport of proteins across membranes) and the mode of secretion of alpha-amylase in aleurone cells.

The effects of cytochrome P-450-448 inhibitors on the binding of benzo(a)pyrene and derivatives to DNA upon microsomal activation.

1. [3H]Benzo(a)pyrene and 6-substituted derivatives of [3H]benzo(a)pyrene are covalently bound to calf thymus DNA upon reaction with microsomal preparations from rats pretreated with 3-methylcholanthrene in the presence of NADPH. Two different types of cytochrome P-450-448 inhibitors, alpha-naphthoflavone and 1-benzylimidazole, show greater than 80% inhibition of the binding of benzo(a)pyrene to DNA. 2. In the presence of these inhibitors, 6-hydroxymethylbenzo(a)pyrene, 6-methylbenzo(a)pyrene and 6-formylbenzo(a)pyrene show varying degrees of inhibition of binding to DNA depending upon the inhibitor employed. 3. Polyguanylic acid is the most effective substrate for the binding of each activated polynuclear aromatic hydrocarbon; polyadenylic acid and DNA show essentially equivalent binding.

In Vitro Replication of Cowpea Mosaic Virus RNA III. Template Recognition by Cowpea Mosaic Virus RNA Replicase

Cowpea mosaic virus (CPMV) RNA replicase has been purified about 200-fold from CPMV-infected Vigna unguiculata leaves. Optimal reaction conditions for replicase activity have been established that allow RNA synthesis to proceed for at least 15 h. Using a polymerase assay under conditions optimal for CPMV RNA-directed RNA synthesis, all natural RNA species tested appeared to be able to direct the incorporation of labeled ribonucleotides, whereas synthetic homoribopolymers were either inactive or only slightly active. Using a nitrocellulose membrane filter assay to measure complex formation between the replicase preparation and various RNA species, all natural RNA species tested, except that of the comovirus radish mosaic virus, appeared to be unable to compete with (32)P-labeled CPMV RNA in binding to replicase. We propose that CPMV replicase is actually template specific but does not display this property in a polymerase assay, since labile complexes between heterologous templates and replicase become stabilized by the formation of phosphodiester bonds. From homoribopolymer competition binding experiments we conclude that the polyadenylic acid on the CPMV genome might be a part of the replicase binding site.

Changes in two barley leaf ribonuclease fractions during infection by the powdery mildew fungus

We have isolated two ribonuclease (RNase) fractions from barley leaves ( Hordeum vulgare L.) of variety Prior which is susceptible to race 3 of the powdery mildew fungus ( Erysiphe graminis f. sp. hordei). These are termed soluble RNase representing the major RNase fraction of these leaves and pH 5 insoluble RNase which is quantitatively a minor RNase component and is apparently associated with ribosomes. These two enzymes are quite different from each other with respect to the effects of various reagents and in their substrate preference as determined by the relative rates of hydrolysis of polyadenylic, polyuridylic and polycytidylic acids. Both enzymes exhibit a predominantly endonucleolytic mode of action although the patterns of yeast RNA hydrolysis by the two enzymes are different. These RNases undergo remarkable changes in their substrate specificity during the infection of barley leaves by the powdery mildew fungus. The changes in the two enzymes are detectable at different stages of disease development. The catalytic properties of the pH 5 insoluble RNase from inoculated barley leaves at the early and later stages of disease development are quite different from those of the enzyme from healthy leaves and the mildew fungus. These changes in the properties of the pH 5 insoluble RNase in barley leaves represent one of the earliest detectable biochemical symptoms of mildew infection.

Poliovirus Polyuridylic Acid Polymerase and RNA Replicase Have the Same Viral Polypeptide

A poliovirus-specific polyuridylic acid [poly(U)] polymerase that copies a polyadenylic acid template complexed to an oligouridylic acid primer was isolated from the membrane fraction of infected HeLa cells and was found to sediment at 4 to 5S on a linear 5 to 20% glycerol gradient. When the poly(U) polymerase was isolated from cells labeled with [(35)S]methionine and was analyzed by glycerol gradient centrifugation and polyacrylamide gel electrophoresis, the position of only one viral protein was found to correlate with the location of enzyme activity. This protein had an apparent molecular weight of 62,500 based on its electrophoretic mobility relative to that of several molecular weight standards and was designated p63. When the poly(U) polymerase was isolated from the soluble fraction of a cytoplasmic extract, the activity was found to sediment at about 7S. In this case, however, both p63 and NCVP2 (77,000-dalton precursor of p63) cosedimented with the 7S activity peak. When the 7S polymerase activity was purified by phosphocellulose chromatography, both p63 and NCVP2 were found to co-chromatograph with poly(U) polymerase activity. The poliovirus replicase complexed with its endogenous RNA template was isolated from infected cells labeled with [(35)S]methionine and was centrifuged through a linear 15 to 30% glycerol gradient. The major viral polypeptide component in a 26S peak of replicase activity was p63, but small amounts of other poliovirus proteins were also present. When the replicase-template complex was treated with RNase T1 before centrifugation, a single peak of activity was found that sedimented at 20S and contained only labeled p63. Thus, p63 was found to be the only viral polypeptide in the replicase bound to its endogenous RNA template, and appears to be active as a poly(U) polymerase either as a monomer protein or as a 7S complex.

Characterization of anti-ApApA antibodies.

Antibodies to a trinucleotide, ApApA, were prepared by injecting bovine serum albumin conjugated with ApApA into rabbits. The specificities of the antibodies were determined by estimating the inhibiton of the binding of [14C]ApApA to the antibodies by various nonradioactive mono-, oligo-, and polynucleotides, using the ammonium sulfate precipitation method. The antibodies were found to react with ApApN sequences and oligoadenylic acids, but also reacted slightly with polyadenylic acid, RNA and DNA. Significant crossreactions were observed with other oligonucleotides containing adenosine.

Polyadenylate-containing RNA in dormant and germinating sporangiospores of Rhizopus stolonifer

Polyadenylic acid [poly(A)]segments and polyadenylate-containing [poly(A+)] RNAs were isolated from dormant and germinating sporangiospores of Rhizopus stolonifer . At all stages of germination the poly(A) segments (i) composed about 0.1% of the total RNA, (ii) averaged about 33 residues in length, and (iii) were attached to large heterodisperse RNA molecules. From this information and knowing the weight of RNA per spore, we estimated that each dormant spore contained about 6.6 × 10 5 molecules of poly(A+) RNA. Translation of the spore poly(A+) RNA in vitro revealed that at least some of these molecules were functional messenger RNAs and that they coded for the synthesis of many discrete polypeptides.

Absence of circularly permuted and largely redundant sequences in the genome of visna virus

A previous study of the infectivity of visna virus proviral DNA suggested that the genetic information of the virus is distributed over at least two of the RNA subunits. Because the genetic complexity of visna virus corresponds to the size of one subunit, this result may imply that sequence redundancies exist within each subunit. In the present article we have examined this question by constructing a map of the large RNase T1-resistant oligonucleotides of the viral genome. Our principal results are as follows: (i) all 36S RNA subunits have the same genetic content regardless of their polyadenylic acid [poly(A)] content; (ii) the poly(A) tract is present at the 3' end of the molecule; and (iii) the recoveries of 19 large RNase T1-resistant oligonucleotides from poly(A)-tagged RNA fragments of various sizes demonstrate that the oligonucleotides are organized in the same linear order within all subunits. Our results, therefore, exclude the existence of large sequence redundancies in the genome of visna virus.

Measurement of polyadenylic acid by hybridization with polyuridylic acid: A source of error due to the lability of tritiated polyuridylic acid in trichloroacetic acid

During brief exposure to trichloroacetic acid at 0°C, significant amounts of tritiated polyuridylic acid are converted to acid-soluble products. The loss of polymeric radioactivity is dependent on both acid concentration and the time of exposure to acid. When trichloroacetic acid precipitation is used to recover the tritiated polyuridylic acid present in a hybrid with polyadenylic acid, significant underestimates of the hybridized radioactivity can occur because of the lability of tritiated polyuridylic acid. Conditions are given which minimize the lability and permit quantitative recovery of tritiated polyuridylic acid by trichloroacetic acid precipitation.

The genomes of three coronaviruses

A number of studies on the genomic RNAs of coronaviruses have been reported. The genome of avian infectious bronchitis virus (IBV) is a single single-stranded molecule of high mol. wt [l-4] , which is prone to degradation into smaller molecules [ 1,5] . There is some disagreement concerning its mol. wt with estimates varying from 9.0 X lo6 [l] to 5.6 X lo6 [4]. Similar studies on the human coronavirus (HCV) genome have also shown it to be a large molecule of mol. wt varying from 6.1 X lo6 for strain OC43 [6] to 5.8 X IO6 for strain 2298 [7]. Again, as with IBV, the HCV genome is prone to degradation [6,7]. The genomes of two porcine coronaviruses, transmissible gastroenteritis virus (TGEV) and haemagglutinating encephalomyelitis virus (HEV), have also been shown to be large single single-stranded RNA molecules of about 60 S that dissociate into 35 S and 4 S material on heating above 60°C [8] ~ Coronavirus genomes possess certain mRNA characteristics. Polyadenylic acid [poly(A)] sequences are found in the genomes of IBV [2,4,9] and HCV [7,10] at or near their 3’-termini [7] . Furthermore, the genome of IBV has been shown to be infectious [4,9] and no detectable virion transcriptase has been identified associated with IBV [4] or HCV [lo] particles. In this paper, studies on the characteristics of the genomes of HCV strain 229E (HCV 229E) and IBV strain Beaudette (IBV Beau) reported in [3,7] are extended, and the structure of the genome of another coronavirus, mouse hepatitis virus strain 3 (MHV 3) is analysed for the first time. A comparison of the results shows a much greater similarity between coronavirus genomes than has been previously observed.

DNA-dependent RNA and polyadenylic acid polymerase from phototrophically grown Rhodospirillum rubrum.

DNA-dependent RNA and polyadenylic acid polymerases have been purified from phototrophic Rhodospirillum rubrum. Their properties have been found to be very similar to those of the previously reported heterotrophic R. rubrum enzymes. However, several important differences do exist between the enzymes from the phototrophic and the heterotrophic cells, such as the lack of response to added polyadenylic acid for poly A synthesis and the presence of the sigma subunit in the phototrophic enzymes. Furthermore, additional purification steps were necessary for preparation of phototrophic enzyme fractions with high DNA-dependence.

Absence of detectable capping and methylating enzymes in influenza virions.

In the presence of Mg(2+) and a specific dinucleotide primer (ApG or GpG), the influenza virion transcriptase synthesizes the eight discrete segments of complementary RNA (cRNA) containing polyadenylic acid (Plotch and Krug, J. Virol. 21:24-34, 1977). Virions were examined for their ability to cap and methylate cRNA containing di- or triphosphorylated 5' termini. By using the primers ppApG, pppApG, or ppGpG, viral cRNA was synthesized in vitro with [alpha-(32)P]-GTP and S-[methyl-(3)H]adenosylmethionine as labeled precursors. DEAE-Sephadex chromatography of the RNase T2 digest of the cRNA product demonstrated no (3)H incorporation at all and the absence of a (32)P-labeled cap structure. The 5' terminus of ppApG-primed cRNA could be capped and methylated by enzymes from vaccinia virus, indicating that the two 5'-terminal phosphates derived from the primer were preserved in the product cRNA. The cap structure formed by the vaccinia enzymes and released by RNase T2 digestion as m(7)GpppA(m)pGp was radioactively labeled at its 3'-terminal phosphate only when [alpha-(32)P]CTP was used as the labeled precursor during transcription. This indicates that the 5'-terminal sequence of the cRNA is ppApGpC and that, therefore, ppApG most probably initiates transcription exactly at the 3' GpCpU(OH) terminus of the virion RNA templates. Virions were also tested for their ability to cap and methylate ppApG in the absence of transcription. No such activities were detected, whereas under the same conditions the vaccinia virus enzymes successfully capped and methylated this compound. Consequently, these experiments, together with those reported earlier, have not detected in influenza virions any capping and methylating enzymes active on the 5'-initiated termini of viral cRNA chains synthesized in vitro, whether these termini possess one, two, or three phosphates. Some mechanism for capping and methylation of viral cRNA must, however, exist, because the viral mRNA (cRNA) synthesized in the infected cell contains 5'-terminal methylated cap structures (Krug et al., J. Virol. 20:45-53, 1976). Possible mechanisms are discussed.

Inhibition by RNA of RNase H activity associated with reverse transcriptase in Rauscher murine leukemia virus cores.

We reported earlier that core preparations of Rauscher murine leukemia virus, when separated on an isopycnic sucrose gradient, did not contain detectable levels of RNase H activity, while retaining high levels of reverse transcriptase activity. We reexamined this phenomenon, and the earlier observation was found to be reproducible. However, when doubly banded preparations of viral cores were solubilized and reverse transcriptase was isolated by ion-exchange chromatography, a coincident peak of a nuclease activity with the specificity of RNase H was observed, which indicated that RNase H was selectively inhibited in the core fractions. By direct activity measurements using the purified reverse transcriptase-RNase H from cores, this endogenous inhibitor has been identified as the viral RNA. Viral 70S RNA strongly inhibited RNase H activity purified either from whole virions or from prefractionated cores. Other RNAs tested that had inhibitory effects were yeast tRNA, polyadenylic acid, and polyguanylic acid. Polyuridylic acid and polyadenylic acid were moderately inhibitory, and polycytidylic acid did not inhibit the RNase H. A rabbit anti-reverse transcriptase immunoglobulin G inhibited both the reverse transcriptase and RNase H activities of the enzyme purified from cores. These data provide a rational explanation for the failure to detect RNase H activity in core preparations of Rauscher murine leukemia virus. Furthermore, these data are consistent with the idea that the RNase H and reverse transcriptase activities purified from cores reside on the same protein molecule. Possible biological implications of the observed inhibition of RNase H by RNA is discussed.

Electrochemical oxidation of polyadenylic acid at graphite electrodes

The electrochemical oxidation and adsorption of polyriboadenylic acid (poly(A)) has been studied by differential pulse voltammetry and a.c. voltammetry at graphite electrodes. Poly(A) is adsorbed at graphite electrodes between at least 0.2 V and 1.4 V. The height of the single, well-defined differential pulse voltammetric oxidation peak of poly(A) provides a sensitive monitor of the transition of single-stranded poly(A) (ss poly(A)) into its double-stranded protonated form (ds poly(A.A) + ). Thus, ss poly(A) is relatively flexible and, when adsorbed at a graphite electrode surface, conforms to a large extent to the contours of the rough electrode surface so that many adenine residues are accessible to the electrode and a relatively large voltammetric peak current is observed. However, ds poly(A.A) + has a more rigid structure such that when adsorbed on a graphite electrode it cannot conform so readily to the contours of the electrode surface. Hence, fewer adenine residues are accessible to the electrode and a smaller voltammetric peak current is observed.

Electrochemical oxidation of polyadenylic acid at graphite electrodes

The electrochemical oxidation and adsorption of polyriboadenylic acid (poly(A)) has been studied by differential pulse voltammetry and a.c. voltammetry at graphite electrodes. Poly(A) is adsorbed at graphite electrodes between at least 0.2 V and 1.4 V. The height of the single, well-defined differential pulse voltammetric oxidation peak of poly(A) provides a sensitive monitor of the transition of single-stranded poly(A) (ss poly(A)) into its double-stranded protonated form (ds poly(A.A) +). Thus, ss poly(A) is relatively flexible and, when adsorbed at a graphite electrode surface, conforms to a large extent to the contours of the rough electrode surface so that many adenine residues are accessible to the electrode and a relatively large voltammetric peak current is observed. However, ds poly(A.A) + has a more rigid structure such that when adsorbed on a graphite electrode it cannot conform so readily to the contours of the electrode surface. Hence, fewer adenine residues are accessible to the electrode and a smaller voltammetric peak current is observed.

Multiple Roles for ATP in the Synthesis and Processing of mRNA by Vaccinia Virus: Specific Inhibitory Effects of Adenosine (β,γ-Imido) Triphosphate

Adenosine (beta,gamma-imido)triphosphate (AMP-PNP) and guanosine (beta,gamma-imido)triphosphate (GMP-PNP) are analogs of ATP and GTP with non-hydrolyzable gamma-phosphates. Although both AMP-PNP and GMP-PNP were used in place of ATP and GTP by Escherichia coli RNA polymerase to transcribe vaccinia virus DNA, only GMP-PNP was used by the transcriptase present within vaccinia virus cores. AMP-PNP specifically prevented initiation of transcription, since RNA initiated in the presence of ATP, GTP, and CTP was subsequently elongated by incubating the washed cores in the presence of AMP-PNP, GTP, CTP, and UTP. The RNA formed in this manner, however, was (i) several times longer than normal transcripts, indicating a defect in chain termination and/or cleavage of nascent RNA, (ii) was not polyadenylylated (although free polyadenylic acid formed), and (iii) was not extruded from the virus cores. Nearest neighbor analysis demonstrated that AMP-PNP was incorporated adjacent to all four nucleotides, and hybridization to restriction endonuclease fragments of vaccinia virus DNA indicated that the high-molecular-weight RNA was transcribed from representative fractions of the entire genome. The possibility of a block in processing rather than or in addition to a block in chain termination was suggested by the cleavage of the high-molecular-weight RNA within the core after replacement of AMP-PNP with ATP. Cleavage of purified high-molecular-weight RNA by a soluble endoribonuclease extracted from vaccinia virus cores, however, was not dependent upon ATP, nor was it inhibited by AMP-PNP. The latter results suggest that AMP-PNP blocks a step preceding cleavage.

Transfer of Fv-1 locus-specific resistance to murine N-tropic and B-tropic retroviruses by cytoplasmic RNA

A standardized bioassay for transfer of Fv-1 gene-specific resistance to N-tropic and B-tropic murine retroviruses was developed using X plaque reduction in SC-1 (Fv-1-) cells inoculated with virus. Testing of subcellular fractions of restrictive cells showed that the resistance transfer activity was present in the cytoplasmic (microsomal and cytosol) fractions. The activity of the cytoplasmic extract was destroyed by treatment with ribonuclease, but not with deoxyribonuclease or proteases. RNA prepared by phenol-chloroform extraction of mouse tissues, including embryos and livers of weanling mice, transferred Fv-1 locus-specific resistance into DEAE-dextran-treated SC-1 cells. The activity of isolated RNA preparations against virus of the appropriate host-range type has been demonstrated to correspond to the Fv-1 genotypes of the cell sources. The specific transfer of resistance with cellular RNA was effective within a 5- to 6-h period from 2 h before to 4 to 5 after virus infection. Sucrose gradient centrifugation of the RNA showed that the activity sedimented as a broad peak, with an apparent maximum in the 22S region. Affinity chromatography of whole-cell RNA on polyuridylic acid-Sepharose tended to separate more activity into the polyadenylic acid RNA fraction than the non-polyadenylic acid RNA fraction. Except for the reciprocal inhibitory activity for the two host-range virus types, the RNAs of Fv-1n and Fv-1b specificities showed similar properties in all aspects studied.

Herpesvirus infection modifies adenovirus RNA metabolism in adenovirus type 5-transformed cells

The effect of herpes simplex virus (HSV) infection of mRNA metabolism was examined in a system where the fate of specific RNA sequence can be assayed. Adenovirus type 5-transformed rat embryo cell line 107 synthesizes adenovirus-specific RNA (ad-RNA), which functions in the cytoplasm as mRNA. We have utilized ad-RNA as a model for mRNA metabolism, and in a preliminiary study we characterized ad-RNA in the nucleus and cytoplasm by hybridization to filter-bound adenovirus DNA. The results indicated the as-RNA accumulates in the nucleus and that cytoplasmic polyadenylic acid [poly(A)]-containing ad-RNA turns over with a half-life of a few hours. Pulse-chase experiments confirmed these observations and a half-life of about h was determined for the poly(A)-containing cytoplasmic ad-RNA. A second class of ad-RNA remains in the nucleus, where it turns over with a longer hlaf-life (about 24 h). The infection of 107 cells by HSV was restricted at 37 degree C, giving a burst size of 5 PFU per cell and allowing continued host DNA synthesis. Protein synthesis was inhibited greater than 50% by 7 h after infection, and total RNA synthesis was 50% inhibited by 4 h after infection. During the first 8 h after infection, HSV has little effect on the rate of synthesis of ad-RNA as determined by hybridization of nuclear RNA samples, but,during the same period, HSV inhibits the accumulation of poly(A)-containing ad-RNA in the cytoplasm. The degree of this inhibition increases steadily throughout this period and reaches 60% by 6.5 to 8 h after infection. Nosignificant effect was seen on the accumulation of total cellular poly(A)-containing RNA. It was concluded from these experiments that HSV infection alters the metabolism of ad-RNA so as to prevent the normal appearance of the poly(A)-containing mRNA in the cytoplasm. The result for ad-RNA may not represent the behavior of total cellular poly(A)-containing RNA under conditions where infection is restricted.

The genome of human coronavirus strain 229E.

The genomic RNA of human coronavirus strain 229E (HCV 229E) migrated on polyacrylamide gels as a single peak with a mol. wt. of 5.8 X 10(6). Denaturation of the genome with formaldehyde did not alter its electrophoretic mobility, which suggests that the HCV 229E genome is a single-stranded molecule. At least 30% of the genomic RNA was shown to contain covalently attached polyadenylic acid [poly(A)]sequences by binding the RNA to an oligo(dT)-cellulose column. These poly(A) tracts were shown to be about 70 nucleotides in length by measuring the resistance to digestion of HCV 229E RNA with pancreatic and T1 RNases. Finally, the genomic RNA was shown to terminate at or near the 3'-terminus on the basis of its susceptibility to polynucleotide phosphorylase.