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PubMed1980

Escherichia coli murein transglycosylase. Purification by affinity chromatography and interaction with polynucleotides.

Escherichia coli murein transglycosylase, a potential autolysin which splits the sugar chains of the murein sacculus, was rapidly purified from a crude cell extract by sequential chromatography on columns of blue Sepharose and poly(U)-Sepharose. In accordance with the binding to blue Sepharose and poly(U)-Sepharose, the transglycosylase is inhibited by Cibacron blue F3G-A, the affinity ligand of blue Sepharose, and also by polynucleotides, the latter, however, with varying efficiency. Among the polynucleotides tested, single-stranded DNA was found to be one of the most potent inhibitors. When bound to a blue Sepharose column, transglycosylase could be displaced from the column with single-stranded DNA. Taken together, these results point to a polynucleotide binding area on the transglycosylase molecule. Some aspects of the blue Sepharose affinity chromatography and the possible biological significance of the transglycosylase are discussed.

Chromatography, Affinity↗

PubMed1992

Study of polynucleotide conformation by resolution-enhanced ultraviolet spectroscopy poly(rC) and poly(dC).

Self-deconvolution and the fourth derivative of ultraviolet absorption spectra have been used to study stacked single-stranded and double-helix structures of different cytosine-containing polynucleotides for the first time. These compounds were studied under different solution conditions (pH and organic solvents) and at low temperatures. The red shift of the lower band (B2u band plus possibly some n-->pi* transition) of the absorption spectra in the cytosine-containing polynucleotides and the appearance of new peaks in the deconvoluted and derivative spectra in the 280-310 nm region are attributed mainly to cytosine-cytosine stacking interactions. In particular, the fourth-derivative peaks at wavelengths higher than 290 nm can be associated to coupling of electronic transitions of cytosine bases. The nature of the electronic transitions producing the absorption bands which are resolved in the aforementioned fourth-derivative peaks is discussed. It is concluded that the resolution-enhancement techniques used in this work, i.e. self-deconvolution and fourth derivative, complement each other and are useful methods to study structural changes of single-stranded and double-stranded polynucleotides allowing, at the same time, more information to be obtained about specific stacking interactions than classical absorption spectrophotometry.

Cytidine↗

PubMed1973

Studies on the toxicity and antiviral activity of various polynucleotides.

Various polynucleotides were examined for antiviral activity and toxicity in mice. Although the antiviral potency of the various interferon inducers varied, there was a concomitant variation in toxicity. This was reflected by a fivefold range in therapeutic ratio for the various compounds. In addition, no polynucleotide proved to be a more potent interferon inducer than polyinosinic.polycytidylic acid [(poly rI).(poly rC)]. Our results suggest that there may be intrinsic limitations to the development of polynucleotide interferon inducers having improved therapeutic ratios.

Animals↗

PubMed1970

Uptake of synthetic polynucleotides by competent cells of Bacillus subtilis.

A survey was made of the capacity of competent cells of Bacillus subtilis to take up synthetic polynucleotides. Polydeoxyribonucleotides but not polyribonucleotides are taken up by the cells. Both types of polynucleotide failed to compete with transforming deoxyribonucleic acid in the transformation assay whereas both stimulated amino acid incorporation. The latter phenomenon appears to be nonspecific as there is no correlation between the codon composition of the polynucleotides employed and the amino acids whose incorporation was stimulated.

Amino Acids↗

PubMed2005

Mechanism of the phosphatase component of Clostridium thermocellum polynucleotide kinase-phosphatase.

Polynucleotide kinase-phosphatase (Pnkp) from Clostridium thermocellum catalyzes ATP-dependent phosphorylation of 5'-OH termini of DNA or RNA polynucleotides and Ni(2+)/Mn(2+)-dependent dephosphorylation of 2',3' cyclic phosphate, 2'-phosphate, and 3'-phosphate ribonucleotides. CthPnkp is an 870-amino-acid polypeptide composed of three domains: an N-terminal module similar to bacteriophage T4 polynucleotide kinase, a central module that resembles the dinuclear metallo-phosphoesterase superfamily, and a C-terminal ligase-like adenylyltransferase domain. Here we conducted a mutational analysis of CthPnkp that identified 11 residues required for Ni(2+)-dependent phosphatase activity with 2'-AMP and 3'-AMP. Eight of the 11 CthPnkp side chains were also required for Ni(2+)-dependent hydrolysis of p-nitrophenyl phosphate. The ensemble of essential side chains includes the conserved counterparts (Asp187, His189, Asp233, Arg237, Asn263, His264, His323, His376, and Asp392 in CthPnkp) of all of the amino acids that form the dinuclear metal-binding site and the phosphate-binding site of bacteriophage lambda phosphatase. Three residues (Asp236, His264, and Arg237) required for activity with 2'-AMP or 3'-AMP were dispensable for Ni(2+)-dependent hydrolysis of p-nitrophenyl phosphate. Our findings, together with available structural information, provide fresh insights to the metallophosphoesterase mechanism, including the roles of His264 and Asp236 in proton donation to the leaving group. Deletion analysis defined an autonomous phosphatase domain, CthPnkp-(171-424).

Alanine↗

PubMed2006

Ultra-sensitive colorimetric method to quantitate hundreds of polynucleotide molecules by gold nanoparticles with silver enhancement.

An ultra-sensitive colorimetric method to quantitate hundreds of polynucleotide molecules by gold nanoparticles with silver enhancement has been developed. The hybridization products from the target polynucleotides with biotin-labeled probes and gold nanoparticle-functioned oligonucleotides were immobilized to microplates via avidin-biotin system, and the absorbance signals of gold nanoparticles were amplified by silver enhance solution. This sandwich colorimetric assay can detect as few as 600 molecules for single-strand oligonucleotides and as few as 6000 molecules for double-strand polynucleotides in a 50 microL reaction system.

Avidin↗

PubMed1998

Error analysis of chemically synthesized polynucleotides.

Two single-stranded polynucleotide constructs, 123 and 126 nucleotides in length, were chemically synthesized using standard phosphoramidite chemistry. Clonable, double-stranded DNA fragments about 100-bp long were prepared from the polynucleotides by primer extension with a DNA polymerase and end-trimming with two restriction endonucleases, then the fragments were ligated into separate plasmids. Errors in individual insert copies were determined by dideoxy sequencing after in vivo amplification of plasmids. Five of the ten inserts sequenced contained errors, including seven single-base-pair deletions, one four-base-pair deletion and one G-->C transversion. The origins of the latter two errors are unclear, but single-base deletions are inconsistent with errors of polymerases; thus, the most common sequence errors of chemical synthesis are deletion mutations. Deletions are most likely to result from incomplete capping or de-tritylation. The observed error rate can became a significant limiting factor in applications that depend on the correctness of a polynucleotide sequence in individual insert clones.

Cloning, Molecular↗

PubMed1975

A study of the interaction of oligonucleotides with polynucleotides by the method of equilibrium gel filtration. The effect of magnesium ions and the composition of the oligonucleotides on the stability of the complex.

The interaction of the oligonucleotides ApApA, ApApU and ApApC with polyribouridylic acid and of hexariboinosinic acid with and without phosphate near the 3'-end with polyribocytidylic acid was studied by the method of equilibrium gel filtration through Sephadexes. The free energy, the energy and entropy of the complexing were calculated from isotherms of the adsorption of the oligonucleotides on the polynucleotides in relation to the composition of the oligonucleotides and the concentration of magnesium ions in the buffer. It was shown that in the case of the interaction of ApApA and ApApC with poly(U) a perfect triple complex is formed, while in the case of the interaction of ApApU with poly(U) the noncomplementary base is partially displaced from the complex. The free energy of the interaction of neighboring oligonucleotides in a complex with polynucleotides is from --1000 to --3000 cal/mole depending on the type of the complex. It was shown that after the interaction of hexainosinic acid with poly(C) a double complex is formed. The free energy of the interaction of neighboring oligonucleotides in this complex is formed. The free energy of the interaction of neighboring oligonucleotides in this complex is about --1200 cal/mole. Magnesium ions have a different effect on the formation of triple and double complexes in the interaction of oligonucleotides with polynucleotides.

Adenine Nucleotides↗

PubMed1986

Effects of polynucleotide inhibitors on transcriptional events of influenza virions.

Polynucleotide inhibitors of the influenza virion transcriptase, which is activated in vitro by detergent, can be divided into two categories. Polyribonucleotides comprised of adenine and guanine bases or adenine and uracil bases inhibit initiation but not elongation events of transcription. In contrast, polyribonucleotides containing cytidine and uracil or inosine and uracil block both initiation and elongation. Effects on elongation are apparent on addition of polynucleotides at either 5 or 10 min post-initiation. Dose-response studies with these and other polynucleotides have shown that the concentrations causing 50% inhibition of transcription vary considerably with the most potent inhibitor being the modified polymer, poly(C,S4U10).

Orthomyxoviridae↗

PubMedDate not supplied

[Interaction of natural and synthetic polynucleotides with liposomes in the presence of divalent cations].

The formation of complexes of polynucleotides (DNA, poly A.poly U) with liposomes from egg lecithins, L-alpha-phosphatidylcholine, dimirystoyl and other lipids in the presence of divalent cations was studied by differential scanning microcalorimetry circular dichroism and turbidimetry. It was shown that the secondary structure of polynucleotides (double or triple helix) was necessary for the formation of these complexes. This structure was partially destroyed during formation of complexes. It was shown, that three main types of lipids, i.e. phosphatidylcholine, phosphatidylethanolamine and sphingomyelin participate in interactions between liposomes, polynucleotides and Mg2+.

Calorimetry, Differential Scanning↗

PubMedDate not supplied

[Effect of synthetic polynucleotides and RNA on poly(C)-dependent poly(G) polymerase activity of Q beta replicase].

The effect of synthetic polynucleotides and phage RNA on poly(C)-dependent synthesis of poly (G) by Qbeta replicase is studied. It is shown that single stranded poly(U) and poly (dT) are strong inhibitors whereas structured polynucleotides poly(A), MS2 RNA as well as double stranded complexes poly(A)-poly(U) and poly(A)-poly(dT) do not affect the synthesis of poly(G). It is suggested that contact region of template with enzyme has single stranded unhelical structure and affinity of polynucleotides to Qbeta replicase is determined by degree of their secondary structure.

Kinetics↗

PubMed1984

[Synthesis of a 33-member polynucleotide containing the "core" att site of phage lambda DNA and its cloning].

Two polynucleotides containing 33 monomeric units were synthesized by a solid-phase phosphotriester method. These polynucleotides form a duplex with protruding 5'-ends, which allows to clone the duplex in EcoRI site of a cloning vehicle. Each polynucleotide was purified by electrophoresis in polyacrylamide gel, and the duplex obtained was cloned in EcoRI site of pUR 222 plasmid DNA. The structure of the cloned duplex containing the "core" att site of phage lambda was confirmed by sequencing.

Bacteriophage lambda↗

PubMed1975

The adjuvant activity of mycobacterial RNA preparations and synthetic polynucleotides for induction of delayed hypersensitivity to purified protein derivative in guinea pigs.

The adjuvant activity of mycobacterial RNA and synthetic polynucleotides for the induction of delayed hypersensitivity to PPD was determined. It was shown that when mycobacterial RNA or synthetic polynucleotides are injected together with purified protein derivative (PPD), delayed hypersensitivity to PPD developed as compared to no detectable delayed response when PPD was administered alone without adjuvant into guinea pigs. Four different criteria were employed to detect delayed hypersensitivity responses. These were the time, appearance, and magnitude of dermal reactions, histologic examination of dermal sections, passive transfer of sensitivity with sensitized spleen cells and the elaboration of migration inhibitory factor (MIF) by sensitized spleen cells. When synthetic polynucleotides were used as adjuvants and were injected into guinea pigs in combination with PPD, dermal reactions as well ad MIF assays gave evidence that these animals exhibited delayed-type hypersensitivity. Poly U alone exhibited adjuvant activity for induction of delayed hypersensitivity to PPD. Trypsin and pronase treatment did not affect the adjuvant activity of mycobacterial RNA whereas KOH treatment completely abolished any adjuvant effect, suggesting that ribosomal protein did not contribute to the adjuvant characteristics of mycobacterial RNA. Titration experiments indicated that the adjuvant activity of mycobacterial RNA was greater than that of poly A:U.

Adjuvants, Immunologic↗

PubMed1996

Selected strategies to augment polynucleotide immunization.

We sought to amplify the immune response to polynucleotide immunization through co-delivery of complementary DNA (cDNA) encoding a cytokine or co-stimulatory molecule to enhance antigen presentation. In the context of intramuscular immunization, we examined co-delivery of cDNAs for B7-1 and human carcinoembryonic antigen (CEA) within separate plasmids or a dual plasmid with two independent expression cassettes. Intramuscular delivery of the dual expression plasmid produced anti-CEA antibody responses and antitumor effects superior to those generated by plasmid DNA encoding CEA alone. However, co-delivery of cDNAs encoding B7-1 and CEA in the form of two separate plasmids produced no augmentation. The importance of single plasmid delivery suggests the effectiveness of this strategy is contingent upon co-expression of B7-1 and CEA within the same cell. The success of cutaneous polynucleotide immunization by particle bombardment is thought to derive largely from the presence of Langerhans cells within the skin. We hypothesized that co-delivery of plasmid DNA encoding granulocyte-macrophage colony stimulating factor (GM-CSF) by particle bombardment would enhance the antigen presenting capacity of Langerhans cells at the inoculation site similar to its effects in vitro. Augmentation of CEA-specific lymphoblastic transformation and antibody response was observed when plasmid GM-CSF (pGM-CSF) was administered 3 days prior to each dose of plasmid DNA encoding CEA. These strategies for augmentation of immune response to polynucleotide immunization should be applicable to a wide variety of antigenic targets including infectious agents and other tumor-associated antigens.

Animals↗

PubMedDate not supplied

[Calculation of the conformation of stereo-regular, double-spiral polynucleotides].

By the step-by-step selection method energetically optimal conformation of double-stranded polynucleotides were calculated proceeding from the conformations of mono-and dinucleotides. These structures were found to belong to the families of DNA A and B forms. The dependence of six torsion angles of the polynucleotide chain on the glycosidic angle chi was investigated. As a result certain areas admitting of double-helix polynucleotides with sugar C3-endo with chi = -80 degrees were established.

DNA, Single-Stranded↗

PubMed1995

Characterization of the binding of YO to [poly(dA-dT)]2 and [poly(dG-dC)]2, and of the fluorescent properties of YO and YOYO complexed with the polynucleotides and double-stranded DNA.

The interaction between the fluorescent dye YO (oxazole yellow) and the alternating polynucleotides [poly(dA-dT)]2 [the duplex of alternating poly(dA-dT)] and [poly(dG-dC)]2 [the duplex of alternating poly(dG-dC)] has been studied with optical spectroscopic techniques including absorbance, flow linear dichroism, CD, and fluorescence measurements. The principal features of the spectra are very similar for the two polynucleotide solutions, showing that YO binds quite similarly to AT and GC base pairs. From a strongly negative reduced linear dichroism (LDr) in the dye absorption band, an induced negative CD, and transfer of energy from the bases to bound YO, we conclude that at low mixing ratios YO is intercalated in both [poly(dA-dT)]2 and [poly(dG-dC)]2. At higher mixing ratios an external binding mode starts to contribute, evidenced from the appearance of an exciton CD. The conclusion that YO binds in a similar way to AT and GC base pairs should be valid also for the dimer YOYO since its YO units have been found to bind to double-stranded (dsDNA) in the same way as the YO monomer. The fluorescence properties of YO and YOYO complexed with DNA or the polynucleotides have been characterized by studying the dependence of fluorescence intensity on temperature, mixing ratio, and ionic strength. The fluorescence intensity and fluorescence lifetime of YO-DNA decrease strongly with increasing mixing ratio, whereas the fluorescence intensity of YOYO-DNA shows a weaker dependence, indicating that the quantum yield depends on the distance between the YO chromophores on the DNA chain. Further, the fluorescence intensity of YO depends on the base sequence; the quantum yield and fluorescence lifetime for YO complexed with [poly(dG-dC)]2 are about twice as large as for YO complexed with [poly(dA-dT)]2. Measurements of excitation spectra at different mixing ratios and different emission wavelengths indicate that the fluorescence of the externally bound chromophores is negligible compared to the intercalated ones.

Benzoxazoles↗

PubMed1992

E.coli polynucleotide phosphorylase expression is autoregulated through an RNase III-dependent mechanism.

It has been previously shown that the pnp messenger RNAs are cleaved by RNase III at the 5' end and that these cleavages induce a rapid decay of these messengers. A translational fusion between pnp and lacZ was introduced into the chromosome of a delta lac strain to study the expression of pnp. In the presence of increased cellular concentrations of polynucleotide phosphorylase, the level of the hybrid beta-galactosidase is repressed, whereas the synthesis rate of the corresponding message is not significantly affected. In the absence of pnp, the level of the hybrid protein increases strongly. Thus, polynucleotide phosphorylase is post-transcriptionally autocontrolled. However, autocontrol is totally abolished in strains where the RNase III site on the pnp message has been deleted or in strains devoid of RNase III. These results suggest that polynucleotide phosphorylase requires RNase III cleavages to autoregulate the translation of its message. Other mutations in the ribosome binding site region support the hypothesis that this 3' to 5' processive enzyme could recognize a specific repressor binding site at the 5' end of pnp mRNA. Implications of these results on the mechanism of regulation and on messenger degradation are discussed.

Base Sequence↗

PubMed1997

Interactions of the bacteriophage T4 gene 59 protein with single-stranded polynucleotides: binding parameters and ion effects.

The gene 59 protein (gp59) of bacteriophage T4 is an important accessory protein of the phage-encoded replicative DNA helicase, gp41. The properties of this 26 kDa protein include selective binding to ssDNA, and specific interactions with both gp41 and gp32, the T4-encoded ssDNA- binding protein. gp59 stimulates many of the DNA-dependent activities of the gp41 enzyme by promoting its assembly onto gp32-ssDNA complexes. Direct interactions between gp59 and gp32-ssDNA complexes are essential for helicase assembly, and gp59-gp32 protein-protein interactions have been shown to play a central role. Presumably, the ssDNA-binding activity of gp59 is also important for helicase assembly; however, to date this activity has been poorly characterized. In this study, we present the first detailed biochemical investigation of the interactions of gp59 with single-stranded polynucleotides. Using etheno-DNA fluorescence enhancement and quantitative ssDNA-cellulose methods, we demonstrate the following: (1) gp59 binds to single-stranded polynucleotides with a binding site size of nine to ten nucleotide residues per monomer; (2) gp59 exhibits relative affinities towards four different ssDNA lattices used in this study according to the heirarchy: ssDNA (random sequence) > epsilonDNA (random sequence) > poly(dA) > poly(depsilonA); (3) gp59 exhibits two or more different polynucleotide binding modes distinguished by their cooperativities of binding, and modulated by salt and/or lattice effects; (4) gp59-ssDNA binding is characterized by a large salt effect on the association constant, consistent with multiple ionic contacts between protein and ssDNA phosphate residues and with the displacement of anions from the protein. The implications of our findings for the mechanism of action of gp59 in helicase-ssDNA assembly are discussed.

Bacteriophage M13↗