Polyadenylic Acid Research Articles

Long terminal repeat of murine retroviral DNAs: sequence analysis, host-proviral junctions, and preintegration site.

The nucleotide sequence of the long terminal repeat (LTR) of three murine retroviral DNAs has been determined. The data indicate that the U5 region (sequences originating from the 5' end of the genome) of various LTRs is more conserved than the U3 region (sequences from the 3' end of the genome). The location and sequence of the control elements such as the 5' cap, "TATA-like" sequences, "CCAAT-box," and presumptive polyadenylic acid addition signal AATAAA in the various LTRs are nearly identical. Some murine retroviral DNAs contain a duplication of sequences within the LTR ranging in size from 58 to 100 base pairs. A variant of molecularly cloned Moloney murine sarcoma virus DNA in which one of the two LTRs integrated into the viral DNA was also analyzed. A 4-base-pair duplication was generated at the site of integration of LTR in the viral DNA. The host-viral junction of two molecularly cloned AKR-murine leukemia virus DNAs (clones 623 and 614) was determined. In the case of AKR-623 DNA, a 3- or 4-base-pair direct repeat of cellular sequences flanking the viral DNA was observed. However, AKR-614 DNA contained a 5-base-pair repeat of cellular sequences. The nucleotide sequence of the preintegration site of AKR-623 DNA revealed that the cellular sequences duplicated during integration are present only once. Finally, a striking homology between the sequences flanking the preintegration site and viral LTRs was observed.

Ab initio mo study on base stacking: Adenine-adenine interaction in single-stranded polyadenylic acid (Poly A)

The interaction energy for base stacking in single-stranded poly A is calculated by an ab initio MO method. The calculated value agrees well with the observed stacking enthalpy. Dispersion and charge transfer interactions are the main factors stabilizing the polynucleotide; the contribution of the former is dominant.

Biophysical and biochemical characterization of hepatitis A virus.

Biophysical and biochemical analysis of hepatitis A virus has shown it to be a 27- to 32-nm icosahedral particle with 32 capsomers. The mature virion has a buoyant density of 1.33-1.34 g/cm3, a sedimentation coefficient of 156-160S, and is composed of four polypeptides with molecular weights of 30,000-33,000 (VP1), 24,000-27,000 (VP2), 21,000-23,000 (VP3), and 7,000-14,000 (VP4). The genome of hepatitis A virus consists of a single piece of single-stranded RNA which sediments at 32-35S and has a buoyant density of 1.64 g/cm3. The molecular weight of RNA is 2.25 x 10(6) when measured under nondenaturing conditions and 2.8 x 10(6) when measured under fully denaturing conditions. The genome contains a 40-80 nucleotide sequence of polyadenylic and is capable of infecting cell cultures. These findings, together with the observation that the virion is stable at pH 3.0 and resistant to ether and a temperature of 60 degrees for 1 h, indicate that hepatitis A virus should now be classified as an Enterovirus within the family Picornaviridae.

Characterization of a 70S polyuridylic acid polymerase isolated from foot-and-mouth disease virus-infected cells.

A polyuridylic acid polymerase complex isolated from foot-and-mouth disease virus-infected cells sedimented at 70S in a sucrose gradient and appeared in the exclusion volume of an agarose column whose molecular weight cutoff was 5 x 10(6). Phenol extraction of the complex yielded a heterogeneous band of virus-specific RNA and an apparently host cell-derived 4.5 to 5S RNA, both of which are essentially single stranded. Neither RNA served as a template in the cell-free enzyme reaction. Polyacrylamide gel analysis revealed five polypeptides with molecular weights of 50,000, 56,000, 60,000, 70,000, and 74,000 and with molar ratios of 1:2:2:1:1, respectively. Autoradiography showed P56 to be the only major virus-induced polypeptide; the other proteins are apparently of host cell origin. Electron microscopic examination suggested a cartwheel shape for the polymerase complex which was seen to dissociate as polyadenylic acid was added. Antibody previously shown to inhibit enzyme activity aggregated the 70S units.

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds

Scutellar tissues were dissected from germinating rice seeds and the incorporation of [(35)S]methionine into the alpha-amylase molecule was examined by in vivo and in vitro assay systems. Immunoprecipitation with monospecific anti-alpha-amylase immunoglobulin G raised against the purified enzyme preparation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography were used to identify alpha-amylase and its possible precursor molecule. Using freshly prepared scutellar tissues, it was demonstrated that alpha-amylase is synthesized de novo in the scutellar epithelium and secreted into endosperm. The synthesis of alpha-amylase directed by the polyadenylic acid-containing ribonucleic acid isolated from the scutellar tissues was also established using the translation system of either wheat germ extract or reticulocyte lysate. The immunoprecipitable product obtained in the in vitro translation system was smaller in molecular weight than that synthesized in vivo on the basis of mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results are discussed in relation to the processing of the nascent polypeptide precursor of the enzyme molecule and the introduction of the oligosaccharide chain to the cleaved polypeptide to make up the mature form of alpha-amylase.

Sequence heterogeneity, multiplicity, and genomic organization of alpha- and beta-tubulin genes in sea urchins.

We analyzed the multiplicity, heterogeneity, and organization of the genes encoding the alpha and beta tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. alpha- and beta-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The alpha cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of alpha tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The beta cDNA insertion contains the coding sequence for the 100-C terminal amino acids of beta tubulin and 83 pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous alpha- and beta-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of alpha-tubulin genes with beta-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered.

Sequence heterogeneity, multiplicity, and genomic organization of alpha- and beta-tubulin genes in sea urchins.

We analyzed the multiplicity, heterogeneity, and organization of the genes encoding the alpha and beta tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. alpha- and beta-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The alpha cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of alpha tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The beta cDNA insertion contains the coding sequence for the 100-C terminal amino acids of beta tubulin and 83 pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous alpha- and beta-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of alpha-tubulin genes with beta-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered.

Quasielastic light‐scattering study of polyadenylic acid solutions. II

AbstractQuasielastic light scattering is used to study saline solutions of polyadenylic acid with varying polymer concentrations and molecular masses. These experiments clearly show the existence of two relaxation times. For dilute solutions, when the chains are mutually independent, the fast mode is due to the free diffusion of the polymer chains. For concentrations above the overlap concentration C*, the fast mode is due to the propagation of collective excitations of the pseudolattice of polymer chains. The slow modes are observed when the polymer concentration is in the vicinity of the overlap concentration C*. A series of experiments shows that both their relaxation time and amplitude depend only on the polymer concentration and not on the polymer molecular mass. This result rules out any previous explanation based on individual chain motion. Furthermore, since the amplitudes depend on the time elapsed from the preparation of the solution, the slow modes are due to the diffusion of concentration inhomogeneities in the pseudolattice.

Antiviral effects of single-stranded polynucleotide inhibitors of the influenza virion-associated transcriptase against influenza virus infection of hamsters and ferrets

Administration of a single-stranded polynucleotide copolymer containing 9% cytidine residues and 91% 4-thiouridine residues [poly(C,S 4U 10)], a known potent inhibitor of the virion transcriptase of influenza viruses, suppressed the amount of virus recoverable from the nasal washes of influenza virus-infected hamsters and ferrets. The incidence of sneezing and nasal discharge in infected ferrets was also reduced. In hamsters, poly(C,S 4U 10) was more effective than amantadine-HCl given at a 5-fold higher dose; in ferrets, its effect was comparable with amantadine-HCl or Virazole. Polyinosinic acid in combination with poly-5-hydroxy cytidylic acid also had anti-influenza effects. Poly(C,S 4U 10) annealed to polyadenylic acid was not effective, nor was the double-stranded polymer (polyinosinic acid) · (polycytidylic acid), even when complexed with carboxymethylcellulose and polylysine. No toxic effects of poly(C,S 4U 10) were apparent in the treated hamsters and ferrets, and high doses (≥ 2.86 g/kg) administered intraperitoneally to mice produced no adverse effects.

Low-molecular-weight RNAs associated with tobacco ringspot virus are satellites

The low-molecular-weight RNA associated with an isolate of tobacco ringspot virus [TRSV(F)] lacks polyadenylic acid [poly(A)] sequences. Complementary DNA (cDNA) specific to this RNA was made with an oligo(dT) primer after tailing the RNA with poly(A). However, a significant amount of cDNA was made only when the RNA was treated with alkaline phosphatase prior to tailing. Hybridization of RNA in solution and by a Northern blot technique showed that there was no detectable nucleotide sequence homology between TRSV(F) RNA and two of the low-molecular-weight RNAs associated with this virus. These associated RNAs are therefore, satellites of TRSV(F). The relationship of these satellites to a previously reported satellite of TRSV is discussed.

Viral gene expression in polyoma virus-transformed rat cells and their cured revertants.

We have studied transcription of integrated viral DNA sequences in a variety of ts-a polyoma virus-transformed rat cells and cured revertants (which had undergone excision of variables amounts of integrated viral DNA) to characterize the structure of viral mRNA's produced in these lines under conditions in which integrated DNA is stable. Our results indicate that cells containing intact early region sequences, either in single-copy or tandem insertions, produce mRNA's indistinguishable from those observed early in lytic infections; sequences complementary to the polyoma late region were not transcribed from integrated viral DNA. Cured revertants no longer encoded full-length early mRNA's , but produced viral transcripts whose 3' ends mapped at an alternative early region polyadenylic acid attachment site at 99 map units or extended in to flanking host sequences. The phenotype of these revertant cells correlated with the abundance of these transcripts, suggesting that the transforming function(s) of polyoma virus controls the cellular phenotype in a dose-dependent manner. Unexpected results were obtained from studies of cells containing tandem repeats of defective viral DNA in which the polyadenylic acid attachment signal at 25.8 map units and surrounding sequences were deleted. In these cases, polyadenylated mRNA's were observed that contained sequences complementary to the early strand of the polyoma late region. These mRNA's (some larger than 8 kilobases) originated at the viral early promoter, extended into the late region, and continued into the early region of the contiguous repeat in the tandem. The multimeric mRNA's produced contained defective early regions in tandem with late region sequences. S1 analysis indicated that whereas the 5' early region sequences of readthrough transcripts were spliced in the usual manner, internal early region repeats were either unspliced or used only one of the small early region splices. When deletions in the viral readthrough transcripts were observed. This suggests that sequences nearby the AAUAAA sequence at 26 map units may control transcription termination of the polyoma early region.

Effect of polynucleotides on the dimerization of glycine.

Polynucleotides were found to suppress the dimerization reaction of aqueous glycine with trimetaphosphate as the condensing agent. Small anions (chloride, acetate, and phosphate) did not show this effect. The reaction was studied at a pH of about 11.5 and at 70 degrees C and room temperature with a 13 mM concentration of glycine and trimetaphosphate. Under these conditions, the effect of the polynucleotides was in the following order: polyguanylic acid less than polycytidylic acid less than polyadenylic acid less than polyuridylic acid. The result may have a significant implication for the understanding of processes of chemical evolution.

Purification and properties of a polyadenylate polymerase from Artemia dormant embryos

Soluble extracts from encysted dormant embryos of Artemia contain a poly(A) polymerase activity which has been partially purified and characterized. The enzyme requires manganese, an RNA primer and ATP for maximal activity. The K m for ATP is 0.04 mM and the enzyme is inhibited by concentrations higher than 0.5 mM ATP. dATP replaces ATP with a 15% efficiency. The K m for dATP is 0.06 mM. Natural RNAs, poly(A) and poly(AG) are the best RNA primers among several homopolymers and copolymers tested. The poly(A) polymerase does not show any specificity for the nucleotide in the 3′ end of the RNA primer. The product of the reaction is a polyadenylic acid chain covalently bound to the RNA primer molecule. The length of the poly(A) chain is about ten nucleotides, but this length is dependent on the incubation time and the RNA primer concentration. The molecular weight of the enzyme is 70 000 and its isoelectric point is 6.0. The existence of an active poly(A) polymerase in dormant embryos of Artemia, likely with a cytoplasmic localization, suggests a role for this enzyme in the processing and activation of the stored mRNAs after resumption of development.

Preparation of cell-size unilamellar liposomes with high captured volume and defined size distribution

Cell-size unilamellar vesicles were made by removing solvents from microscopic chloroform spherules containing smaller water droplets within. The average diameter of the vesicles in a typical preparation was 9.2 μm, comparable to that of human erythrocytes (7 μm). The standard deviation of the size distribution was 3.0 μm. The unilamellarity and bilayer unit membrane of vesicles were demonstrated by transmission electron microscopy. Materials so far succesfully incorporated into vesicles include glucose, sucrose, Arsenazo III and Ponceau S dyes, thymidine triphosphate, methotrexate, agarose, collagen, ferritin, polyadenylic acid, DNA, and whole bacteria. The captured volume per milligram of lipids (up to 144 μl/mg) was almost an order of magnitude greater than the highest value reported in the literature to date (up to 15.6 μl/mg) (Szoka, F.C. and Papahadjopoulos, D. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4194–4198).

Polycytidylate hydrolysing ribonuclease activity of human pancreatic secretions

Pure human pancreatic juice contains polycytidylate hydrolysing ribonuclease. In 0.05M sodium phosphate buffer and 0.05M sodium phosphate-borate buffer, its pH optimum is at 6.5. In 0.05M Tris-acetate buffer, its pH optimum is at 7.5. Its activity in 0.05M sodium phosphate-borate buffer of pH 6.5 and 0.05M Tris-acetate buffer of pH 7.5 respectively is 86% and 52% of that in 0.05M sodium phosphate buffer of pH 6.5. Thus, sodium phosphate buffer is preferable to other two buffers. However, the concentration of phosphate is critical. For optimal activity the phosphate concentration is 0.05M. Above this concentration, phosphate has a pronounced inhibitory effect. It is inhibited by borate. The presence of phosphate obviates to a great extent the borate inhibition. Polyadenylic and polyguanylic acids are refractory to its action. Its activity on polyuridylic acid is less than 2% of that on polycytidylic acid. Basal duodenal aspirates and those derived after secretin stimulation contain RNase with properties similar to those described above. In basal duodenal aspirates, RNase is not detectable in 7 out of 28 patients. During the first ten minute period after secretin administration, there is a rapid and dramatic increase in the RNase levels of duodenal aspirates. This rapid increase is followed by a decrease and considerable fluctuation in the RNase levels of duodenal aspirates collected during the subsequent periods.

Nucleotide sequences of the mRNA's encoding the vesicular stomatitis virus N and NS proteins.

The complete nucleotide sequences of the vesicular stomatitis virus (VSV) mRNA's encoding the N and NS proteins have been determined from the sequences of cDNA clones. The mRNA encoding the N protein is 1,326 nucleotides long, excluding polyadenylic acid. It contains an open reading frame for translation which extends from the 5'-proximal AUG codon to encode a protein of 422 amino acids. The N and mRNA is known to contain a major ribosome binding site at the 5'-proximal AUG codon and two other minor ribosome binding sites. These secondary sites have been located unambiguously at the second and third AUG codons in the N mRNA sequence. Translational initiation at these sites, if it in fact occurs, would result in synthesis of two small proteins in a second reading frame. The VSV and mrna encoding the NS protein is 815 nucleotides long, excluding polyadenylic acid, and encodes a protein of 222 amino acids. The predicted molecular weight of the NS protein (25,110) is approximately one-half of that predicted from the mobility of NS protein on sodium dodecyl sulfate-polyacrylamide gels. Deficiency of sodium dodecyl sulfate binding to a large negatively charged domain in the NS protein could explain this anomalous electrophoretic mobility.

Nucleotide sequences of the mRNA's encoding the vesicular stomatitis virus G and M proteins determined from cDNA clones containing the complete coding regions.

The complete nucleotide sequences of the vesicular stomatitis virus mRNA's encoding the glycoprotein (G) and the matrix protein (M) have been determined from cDNA clones that contain the complete coding sequences from each mRNA. The G protein mRNA is 1,665 nucleotides long, excluding polyadenylic acid, and encodes a protein of 511 amino acids including a signal peptide of 16 amino acids. G protein contains two large hydrophobic domains, one in the signal peptide and the other in the transmembrane segment near the COOH terminus. Two sites of glycosylation are predicted at amino acid residues 178 and 335. The close correspondence of the positions of these sites with the reported timing of the addition of the two oligosaccharides during synthesis of G suggests that glycosylation occurs as soon as the appropriate asparagine residues traverse the membrane of the rough endoplasmic reticulum. The mRNA encoding the vesicular stomatitis virus M protein is 831 nucleotides long, excluding polyadenylic acid, and encodes a protein of 229 amino acids. The predicted M protein sequence does not contain any long hydrophobic or nonpolar domains that might promote membrane association. The protein is rich in basic amino acids and contains a highly basic amino terminal domain. Details of construction of the nearly full-length cDNA clones are presented.

Transcription of adenovirus type 2 genes in a cell-free system: apparent heterogeneity of initiation at some promoters.

We examined the transcription of a variety of adenovirus type 2 genes in a cell-free system containing purified ribonucleic acid polymerase II and a crude extract from cultured human cells. The early EIA, EIB, EIII, and EIV genes and the intermediate polypeptide IX gene, all of which contain a recognizable TATAA sequence upstream from the cap site, were actively transcribed in vitro, albeit with apparently different efficiencies, whereas the early EII (map position 74.9) and IVa2 genes, both of which lack a TATAA sequence, were not actively transcribed. A reverse transcriptase-primer extension analysis showed that the 5' ends of the in vitro transcripts were identical to those of the corresponding in vivo ribonucleic acids and that, in those instances where initiation was heterogeneous in vivo, a similar kind of heterogeneity was observed in the cell-free system. Transcription of the polypeptide IX gene indicated that this transcript was not terminated at, or processed to, the polyadenylic acid addition site in vitro. We also failed to observe, using the in vitro system, any indication of transcriptional regulation based on the use of adenovirus type 2-infected cell extracts.

Transcription of adenovirus type 2 genes in a cell-free system: apparent heterogeneity of initiation at some promoters.

We examined the transcription of a variety of adenovirus type 2 genes in a cell-free system containing purified ribonucleic acid polymerase II and a crude extract from cultured human cells. The early EIA, EIB, EIII, and EIV genes and the intermediate polypeptide IX gene, all of which contain a recognizable TATAA sequence upstream from the cap site, were actively transcribed in vitro, albeit with apparently different efficiencies, whereas the early EII (map position 74.9) and IVa2 genes, both of which lack a TATAA sequence, were not actively transcribed. A reverse transcriptase-primer extension analysis showed that the 5' ends of the in vitro transcripts were identical to those of the corresponding in vivo ribonucleic acids and that, in those instances where initiation was heterogeneous in vivo, a similar kind of heterogeneity was observed in the cell-free system. Transcription of the polypeptide IX gene indicated that this transcript was not terminated at, or processed to, the polyadenylic acid addition site in vitro. We also failed to observe, using the in vitro system, any indication of transcriptional regulation based on the use of adenovirus type 2-infected cell extracts.

RNA and macronuclear transcription in the ciliate Stylonychia mytilus.

Nuclear and cytoplasmic RNA of Stylonychia mytilus were analyzed on denaturing polyacrylamide gels. The molecular weight of rRNA precursor molecules is within a range of 2.1 x 10(6) daltons. A comparison between the electrophoretic pattern of nuclear non-ribosomal RNA and cytoplasmic mRNA indicates that a considerable amount of nuclear RNA sequences is of higher molecular weight than cytoplasmic RNA sequences. The molecular weight distribution of cytoplasmic RNA supports the assumption that also in Stylonychia an average sized mRNA molecule contains 1,200-1,500 nucleotides according to a molecular weight of 4 x 10(5) to 5 x 10(5) daltons. The size of the polyadenylic acid fragment of poly-A+ RNA molecules is about 120 nucleotides. The total mass of cytoplasmic RNA is around 7.5/10(10)g/cell, corresponding to 1.2 x 10(7) average sized mRNA molecules per cell. RNA excess hybridization experiments show that 60% of the DNA sequences are transcribed into nuclear RNA and that the cytoplasmic mRNA sequences are homologous to about 40% of macronuclear DNA sequences. There is no indication of different frequency classes within the mRNA. The number of different mRNA species in a Stylonychia cell is 1.2-1.5 x 10(4). On the average each of them is present about 1,000 times in every cell.