SEARCH · Search PubMed
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Binding of Cu(2+) to polynucleotides has been found to occur at 1 Cu/5 polymer P and at similar molar ratios of copper to selected nucleic acid derivatives. Electron spin resonance (ESR) measurements show that the g-values and the hyperfine splitting constants vary substantially among the different complexes. Binding of Cu(2+) to the phosphates as well as to the bases is observed, and in some cases occurs simultaneously.
RNase A4 is a new RNase activity found as a contaminant in commercial polynucleotide phosphorylase. This enzyme has the ability of hydrolyzing the phosphodiester bond between pyrimidine-A in both loop and paired regions of RNA.
Two systemic antiviral resistance-inducing proteins, CIP-29 and CIP-34, isolated from Clerodendrum inerme Gaertn. leaves, were tested for ribosome-inactivating properties. It was found that CIP-29 has the characteristics of a polynucleotide:adenosine glycosidase (ribosome-inactivating protein), in that it inhibits protein synthesis both in cell-free systems and, at higher concentrations, in cells, and releases adenine from ribosomes, RNA, poly(A) and DNA. As compared with other known RIPs, CIP-29 deadenylates DNA at a high rate, and induces systemic antiviral resistance in susceptible plants.
The rpsO gene of Escherichia coli, which encodes ribosomal protein S15 is located at 69 minutes on the chromosome. It is adjacent to the pnp gene, which encodes polynucleotide phosphorylase. The two genes are separated by 249 nucleotides and are transcribed in the same direction. We report here in vivo S1 nuclease mapping and in vitro transcription experiments that demonstrate that rpsO and pnp are cotranscribed from a promoter P1, located 108 nucleotides upstream from rpsO, and that another promoter P2, located between the two genes 158 nucleotides upstream from pnp, also directs the transcription of pnp. Transcription from P1 can either terminate at the terminator t1 identified in vivo and in vitro, 18 nucleotides downstream from rpsO, or transcribe through t1 and into pnp. Comparison of the transcripts synthesized in wild-type and RNase III-deficient strains of E. coli shows that all the P1 readthrough transcripts and P2 transcripts are cleaved by RNase III. Two specific cuts are made by RNase III in a double-stranded structure about 100 nucleotides upstream rpsO. We also found that some transcripts of this operon start 47 nucleotides downstream from rpsO, in the region of t1. No promoter has been identified in this region. This mRNA is attributed to an endonucleolytic cleavage of the polycistronic transcripts and the location of the cut is named M. The order of the transcription signals and of the maturation sites in relation to rpsO and pnp can be summarized as follows: P1, rpsO, t1, M, P2, RNase III-processing sites, pnp. The possible roles of mRNA processing events in the expression of rpsO-pnp operon are discussed.
The nature of the ends of the vaccinia virus genome was determined by nucleotide sequencing. Our finding of terminal hairpins indicated that the linear double-stranded DNA molecule consists of a single continuous polynucleotide chain. The 104 nucleotide apex of the hairpin contains predominantly A and T residues and is incompletely based-paired. These loops exist in two forms, which when inverted with respect to each other are complementary in sequence. Both forms of the 104 nucleotide loop are present in nearly equimolar amounts of each end of the genome. A set of 13 tandem 70 bp repeats begins 87 bp from the proximal segment of the terminal loop, followed by a unique sequence of 325 bp, and then by a second set of 18 tandem 70 bp repeats. The sequence of the 70 bp repeats reveals a 13 bp internal redundancy. Self-priming and de novo start replication models, which involve a site-specific nick in one DNA strand proximal to the 104 nucleotide loop, account for the observed sequence inversions and incomplete base-pairing. Similar mechanisms may be involved in replication of the ends of the eucaryotic chromosome.
The interaction of the Cu(II), Ni(II) and Co(III) complexes of the following six water-soluble cationic porphyrins with calf thymus DNA, poly(dG-dC)2 and poly(dA-dT)2 was studied by UV-visible and resonance Raman spectroscopy: tetrakis(2-N-) and (3-N-methylpyridyl) porphyrin (1, 2); monophenyl-tris(4-N-methylpyridyl)porphyrin (4); cis- and trans-diphenyl-bis (4-N-methylpyridyl)porphyrin (5, 6). The binding to nucleic acids was compared with that of tetrakis(4-N-methylpyridyl)porphyrin (3). If the N(+)-CH3 group is moved from the para (3) to the meta position (2), binding of the free porphyrin as well as that of the metal complexes is only gradually modified; thus, the square-planar Cu- and Ni-2 are intercalated at the G-C site whereas Co-2 is groove-bound at A-T. Additionally, Ni-2 is probably also intercalated at the A-T site. When the N(+)-CH3 group is located at ortho position (1), the high rotation barrier of the 2-N-methylpyridyl group prevents intercalation of Cu- and Ni-1, resulting in weak outside binding. At ionic strength mu = 0.2, there is no evidence of significant interaction of Co-1 with any of the polynucleotides. When the charged N-methylpyridyl groups in 3 are subsequently replaced by phenyl groups (4, 5/6), the tendency of the Cu(II) and Ni(II) complexes to bind to the outside of the helix or to intercalate only partially increases at the expense of full intercalation. The coulombic attraction remains strong, no significant differences can be detected between 3, 4, 5, and 6. Ni-4 binds to poly(dA-dT)2 in the same complicated manner as Ni-3. The outside-binding in Co-4, -5 and -6 differs slightly from that in Co-2 and Co-3.
Administration of a single-stranded polynucleotide copolymer containing 9% cytidine residues and 91% 4-thiouridine residues [poly(C,S4U10)], 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,S4U10) was more effective than amantadine-HCl or Virazole. Polyinosinic acid in combination with poly-5-hydroxy cytidylic acid also had anti-influenza effects. Poly(C,S4U10) 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,S4U10) were apparent in the treated hamsters and ferrets, and high doses (greater than or equal to 2.86 g/kg) administered intraperitoneally to mice produced no adverse effects.
We report the polymerization of 5-mercuriuridine-5'-diphosphate (ppUHgX), in the presence of an excess of beta-mercaptoethanol, with polynucleotide phosphorylase from E. coli. A degradation of mercurated nucleotides with mercaptans was observed and about 30% of incorporation of mercury in the polymer was obtained after treatment with pancreatic RNase. The influence of ppUHgX and pUHgX with or without beta-mercaptoethanol was also studied on the polymerization of UDP. The ppUHgX did not polymerize in the absence of beta-mercaptoethanol.
Resonance Raman Spectroscopy allows a selective study of the bases of DNA and therefore of the interactions of these bases with ligands. This technique is also sensitive to structural modifications. We show here that, first, the structures of native poly(dA-dT).poly(dA-dT) and poly(dA).poly(dT) are not the same and that, secondly, it is possible to characterize the B----Z transition of poly(dG-dC).poly(dG-dC). The study of the Raman hypochromism during the thermal denaturation of the polynucleotides reveals that the stacking of the adenines in poly(dA).poly(dT) is near that observed in poly(rA) but differs of this stacking in poly(dA-dT).poly(dA-dT). The enhancement of the intensity of the guanine line at 1193 cm-1 and of the cytosine lines at 780 cm-1, 1 242 cm-1 and 1268 cm-1 as well as the shift of the guanine line at low frequency should allow to characterize a small proportion of base pairs in Z form in any DNA.
We review recent evidence on the in vivo and in vitro mRNA degradation properties of 2 3'-exonucleases, ribonuclease II and polynucleotide phosphorylase. Although secondary structures in the RNA can act as protective barriers against 3' exonucleolytic degradation, it appears that this effect depends on the stability of these structures. The fact that RNase II is more sensitive to RNA secondary structure than PNPase, could account for some differences observed in messenger degradation by the 2 enzymes in vivo. Terminator stem-loop structures are often very stable and 3' exonucleolytic degradation proceeds only after they have been eliminated by an endonucleolytic cleavage. Other secondary structures preceding terminator stem-loop seem to contribute to mRNA stability against exonucleolytic decay.
Polynucleotide phosphorylase (PNPase) has been studied in detail since its discovery in 1955 [1]. In an attempt to determine what role, if any, it has in mRNA decay in Escherichia coli, we have isolated and characterized a temperature-sensitive mutation, pnp-200, in the pnp gene. In vitro phosphorolysis, polymerization and exchange activities of the partially purified Pnp-200 enzyme are all reduced to 30-40% of wild-type activity at 50 degrees C compared to 32 degrees C. The pnp-200 mutation alone does not affect cell growth or mRNA stability. A triple mutant strain containing pnp-200 in combination with other temperature-sensitive mutations in genes known to affect mRNA metabolism (rnb-500 and ams-1) is conditionally lethal and shows increased mRNA stability after shift to the non-permissive temperature.
We review recent evidence on the in vivo and in vitro mRNA degradation properties of 2 3'-exonucleases, ribonuclease II and polynucleotide phosphorylase. Although secondary structures in the RNA can act as protective barriers against 3' exonucleolytic degradation, it appears that this effect depends on the stability of these structures. The fact that RNase II is more sensitive to RNA secondary structure than PNPase, could account for some differences observed in messenger degradation by the 2 enzymes in vivo. Terminator stem-loop structures are often very stable and 3' exonucleolytic degradation proceeds only after they have been eliminated by an endonucleolytic cleavage. Other secondary structures preceding terminator stem-loop seem to contribute to mRNA stability against exonucleolytic decay.
The PstI and BamHI fragments, containing the HIS3 (imidazoleglycerol phosphate dehydratase) gene of yeast obtained from pYehis2, and the ColE1-derived plasmid pBR322 were ligated in vitro and used to transform hisB463 strains of Escherichia coli K-12. Expression of the cloned HIS3 gene from yeast was markedly enhanced (3--5-fold) in polynucleotide phosphorylase (pnp)-deficient strains of E. coli. The levels of both HIS3 and plasmid-encoded mRNAs were increased in pnp- strains carrying the chimeric plasmids, whereas there was little difference in the levels of pBR322-specific mRNAs in pnp+ and pnp- strains. This increase in HIS3 mRNA appeared to be related to specific stabilization of the eukaryotic message due to its unique structural features, since the half-life of the HIS3 mRNA increased from 1.5 to 18.7 min, whereas no increase in the half-lives of pBR322 vehicle mRNAs was observed. A physical map of the plasmid pYehis2 was constructed using restriction endonuclease and molecular cloning techniques.
The genes encoding ribosomal protein S15 (rpsO) and polynucleotide phosphorylase (pnp) occupy adjacent positions and are oriented in the same direction on the Escherichia coli chromosomes. The nucleotide sequence of the region controlling the expression of these two genes has been determined. Two in-phase gene fusions between pnp and lacZ were constructed. The fusions define the translational reading frame of the pnp gene and indicate that the expression of pnp is independent of the upstream rpsO gene. Transcript mapping with nuclease S1 demonstrated that the two genes are transcribed from separate promoters and that the rpsO-pnp intergenic space contains a strong transcriptional terminator. The transcriptional start points have been localized.
Initiation of translation is a complicated process involving numerous accessory factors whose functions remain incompletely understood. Bacterial ribosomal protein S1 is known to contain a repeated sequence motif (S1-RM), also found in polynucleotide phosphorylase, that is thought to be involved in binding to RNA. Using the technique of profile analysis, the S1-RM can also be found in bacterial and chloroplast translation initiation factor IF-1 sequences, and in the sequences of eukaryotic translation initiation factor eIF-2 alpha chains. The significance of the similarity of the sequences is very high suggesting that the occurrence of the S1-RM in these diverse proteins represents homology. The similarity of S1 to IF-1 further suggests that S1 has evolved from an IF-1 like ancestor, and therefore that the two proteins have a similar or competitive function. The most obvious common function of the proteins containing the S1-RM seems to be RNA binding, suggesting that IF-1 and eIF-2 alpha may bind to RNA.
The interactions of tris(2,2'-bipyridyl)ruthenium(II) chloride and tris-(1,10-phenanthroline)ruthenium(II) chloride, Ru(bpy)3Cl2 and Ru(phen)3Cl2 respectively, with nucleic acids were studied by means of absorption spectroscopy and time-resolved and steady state luminescence techniques in unbuffered aqueous solution at room temperature as a function of added salt, oxygen and the [nucleotide]/[sensitizer] ratio (N/S). The hypochromicity of the visible absorption band of Ru(ligand)3(2+) and the changes in the luminescence intensity and luminescence decay kinetics are considerably larger in the presence of double-stranded calf thymus DNA (dsDNA) than in the presence of single-stranded DNA and polynucleotides. This is suggested to be the result of partial intercalation of the ruthenium complex into the dsDNA rather than just its higher charge density with respect to ssDNA. Spectral changes in the presence of dsDNA increase with increasing N/S ratio (maximum changes reached at N/S = 10-12, half-value 3-4). This is postulated to be due to a transition from mainly electrostatic binding to a binding in which partial intercalation plays an increased role. Addition of alkali or alkaline earth salts at very low concentrations stabilizes partial intercalation whereas higher salt concentrations lead to a release of the ruthenium complex from the strand. This effect of the salt cation increases in the order Cs less than Rb less than K less than Na less than Li less than Ba less than Sr less than Ca less than Mg less than Be. For Ru(bpy)3(2+) the presence of 0.5 mM Mg(ClO4)2 or 6 mM NaClO4 are sufficient to release 50% of the ruthenium complexes which are bound to the dsDNA (N/S = 10); the corresponding half-concentrations for Ru(phen)3(2+) are 0.8 mM and 40 mM respectively. The half-concentrations for release increase with increasing N/S ratio and decrease with the ionic radius of the added salt.
Polynucleotide phosphorylase (PNPase) is a phosphate-dependent 3' to 5' exonuclease widely diffused among bacteria and eukaryotes. The enzyme, a homotrimer, can also be found associated with the endonuclease RNase E and other proteins in a heteromultimeric complex, the RNA degradosome. PNPase negatively controls its own gene (pnp) expression by destabilizing pnp mRNA. A current model of autoregulation maintains that PNPase and a short duplex at the 5'-end of pnp mRNA are the only determinants of mRNA stability. During the cold acclimation phase autoregulation is transiently relieved and cellular pnp mRNA abundance increases significantly. Although PNPase has been extensively studied and widely employed in molecular biology for about 50 years, several aspects of structure-function relationships of such a complex protein are still elusive. In this work, we performed a systematic PCR mutagenesis of discrete pnp regions and screened the mutants for diverse phenotypic traits affected by PNPase. Overall our results support previous proposals that both first and second core domains are involved in the catalysis of the phosphorolytic reaction, and that both phosphorolytic activity and RNA binding are required for autogenous regulation and growth in the cold, and give new insights on PNPase structure-function relationships by implicating the alpha-helical domain in PNPase enzymatic activity.
Molecules 2-5 that include in their structure a biotin moiety as detectable unit and differently substituted phenyl diazo functions as reactive group were prepared as reagents for labeling the phosphate group in mono and polynucleotides. These molecules were shown to react selectively and quantitatively with the model nucleotide 3'-UMP. They were used successfully in the labeling step of DNA and RNA analysis using high-density DNA-chips (or microarrays) technology.