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PubMed2001

Analytical characterization of the conformational transitions of polynucleotides by means of different molecular spectroscopies and multivariate curve resolution.

A general procedure for the study of conformational transitions of polynucleotides is described. The equilibria between different conformations induced by salt, ethidium bromide, and temperature of poly(dG-dC). poly(dG-dC) and induced by salt and temperature of poly(A). poly(U) are investigated using molecular absorption, circular dichroism, and fluorescence spectroscopies. Spectral data obtained from experiments are analyzed by means of a factor analysis method, namely, multivariate curve resolution, which allows possible intermediate states to be detected and the pure spectra and the concentration profiles of all species present in the system to be estimated. This work shows the application of this procedure for the analysis of data matrices obtained in individual experiments but also for the analysis of several data matrices simultaneously.

Circular Dichroism↗
PubMed1977

DNA synthesis and degradation in UV-irradiated toluene treated cells of E. coli K12: the role of polynucleotide ligase.

Toluene treated cells have been used to study the processes of DNA synthesis and DNA degradation in ultra-violet irradiated Escherichia coli K12. Synthesis and degradation are both shown to occur extensively if polynucleotide ligase is inhibited, and to occur to a much lesser extent if ligase activity is optimal. Extensive UV-induced DNA synthesis in toluene-treated cells requires ATP for the initial incision step, and DNA polymerase I. Extensive degradation also depends on the early ATP-dependent incision step, and the subsequent degradation shows a partial requirement for ATP. Curtailment of degradation by ligase requires DNA polymerase activity, but is not dependent upon DNA polymerase I. Apparently this process can be carried out with equal facility by either DNA polymerase II or polymerase III. These observations suggest that extensive DNA polymerase I-dependent repair synthesis and extensive DNA degradation are facets of two divergent pathways of excision repair, both of which depend upon the early uvrABC determined ATP-dependent incision step.

DNA Repair↗
PubMed1982

Polynucleotide replication coupled to protein synthesis: a possible mechanism for the origin of life.

A mechanism is suggested for the replication under primitive conditions of long polynucleotides by the sequential incorporation of sequences related to those of modern transfer RNAs. It is proposed that replication of such molecules became established as the result of a replicative advantage arising from the concomitant linkage together of amino acids to form polypeptides. Initially these polypeptides may have been of random sequence. Selection of primitive tRNAs in which the amino acid and anticodon stem sequences were rotationally symmetrical could have led to specific, anticodon-directed aminoacylation and fixation of the genetic code along the lines suggested by Hopfield. (Hopfield, 1978). The primitive replication-coupled system would then have been able to synthesize specific proteins containing one amino acid residue for each primitive tRNA incorporated during replication. The end result of this line of evolution is postulated to have been a nucleoprotein structure resembling the ribosome. The primitive system would then have been able to give rise directly to triplet-coded protein synthesis. Some recent RNA sequence data are discussed which are consistent with derivation of modern protein synthesis from the primitive replication-coupled mechanism.

Amino Acid Sequence↗
PubMed1996

Immunisation with DNA polynucleotides protects mice against lethal challenge with St. Louis encephalitis virus.

In vivo transfection by intramuscular injection with plasmids expressing the immunogenic proteins of microbial pathogens has considerable potential as a vaccination strategy against many pathogens of both man and animals. Here we report that weanling mice given a single intramuscular injection of 50 micrograms of a plasmid, pSLE1 expressing the St. Louis encephalitis virus (SLE) prM/E protein under the control of the cytomegalovirus immediate early protein promoter produced SLE-specific antibody and were protected against lethal challenge with the virulent virus. Polynucleotide vaccine technology provides a unique opportunity to produce vaccines against flavivirus diseases of low incidence cheaply and rapidly, and to produce multivalent vaccines such as would be required for immunisation against dengue virus disease.

Animals↗
PubMedDate not supplied

Template-directed polynucleotide synthesis on mineral surfaces.

Ferric hydroxide, a plausible prebiotic material, strongly adsorbs polynucleotides. We show that adsorption on ferric hydroxide and on several other minerals has no effect, under the conditions studied, on the template-directed oligomerization of guanylic acid on polycytidylic acid.

Biological Evolution↗
PubMed1989

Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization.

DNA was assayed in a homogeneous format using DNA probes containing hybridization-sensitive labels. The DNA probes were prepared from complementary DNA strands in which one strand was covalently labeled on the 5'-terminus with fluorescein and the complementary strand was covalently labeled on the 3'-terminus with a quencher of fluorescein emission, either pyrenebutyrate or sulforhodamine 101. Probes prepared in this manner were able to detect unlabeled target DNA by competitive hybridization producing fluorescence signals which increased with increasing target DNA concentration. A single pair of complementary probes detected target DNA at a concentration of approximately 0.1 nM in 10 min or about 10 pM in 20-30 min. Detection of a 4 pM concentration of target DNA was demonstrated in 6 h using multiple probe pairs. The major limiting factors were background fluorescence and hybridization rates. Continuous monitoring of fluorescence during competitive hybridization allowed correction for variable sample backgrounds at probe concentrations down to 20 pM; however, the time required for complete hybridization increased to greater than 1 h at probe concentrations below 0.1 nM. A promising application for this technology is the rapid detection of amplified polynucleotides. Detection of 96,000 target DNA molecules in a 50-microliters sample was demonstrated following in vitro amplification using the polymerase chain reaction technique.

Affinity Labels↗
PubMed1976

Kinetic studies on the reaction catalyzed by polynucleotide kinase from phage T4-infected Escherichia coli.

Kinetic properties of polynucleotide kinase (EC 2.7.1.78) isolated from Escherichia coli cells infected with phage T4 were investigated. The reaction depends on the concentration of MgATP, while free ATP or free Mg2+ have neither inhibitory nor accelerating effect. The initial reaction velocity was plotted against variable concentrations of ATP as the phosphate donor at various fixed concentrations of 5'-hydroxyl-DNA or -oligo(rA) as the phosphate acceptor in the presence or absence of products. The double reciprocal plot analysis of the data suggested that the reaction obeys the random sequential mechanism. Various constants were determined and the reaction mechanism was discussed.

Adenosine Triphosphate↗
PubMed1978

Inhibition of T4 polynucleotide kinase by the ATP analog, beta, gamma-imidoadenylyl 5'-triphosphate.

The inhibition of T4 polynucleotide kinase by beta,gamma-imidoadenylyl 5'-triphosphate has been investigated. It was found that the ATP analog was a competitive inhibitor with regard to ATP and a noncompetitive inhibitor with regard to DNA possessing a 5'-hydroxyl group. At pH 8.0, the Ki values were 3 and 11 mM, respectively. beta,gamma-imidoadenylyl 5'-triphosphate was not a substrate in the forward reaction, but would replace ADP and ATP in the reverse reaction. The reverse reaction was also used to make beta,gamma-imidoadenylyl 5'-tetraphosphate.

Adenosine Triphosphate↗
PubMed1978

Binding of polynucleotides to fibroblasts. Effects of complex formation with vinyl analogs of nucleic acids.

The binding of polynucleotides or of their vinyl analogs to human fibroblasts is changed when complexes are formed between these compounds. The following polymers have been studied: poly(1-vinylcytosine), poly(1-vinyluracil, poly(9-vinyladenine), polyuridylate, polyadenylate and polyinosinate. Only that complex formation (between poly(1-vinylcytosine) and polyinosinate) which is accompanied by aggregation leads to a considerable increase (30 fold) in binding to cells; all the other complex formations have only moderate effects (0.2-3 fold). Furthermore, a comparison of unordered complexes containing polyinosinate whows that enhanced binding to cells is paralleled by an increased cellular resistance to viral infection.

Cell Line↗
PubMed1978

The activity of mammalian polynucleotide ligase on x-irradiated DNAs.

Selected samples of heterogeneous DNA from calf thymus with similar number-average molecular weight, Mn, and a low incidence of single-strand breaks were exposed in aqueous solutions to a mild X-ray dose of 1500 rads. The irradiation produced on the average about 0.2 bihelical and 2.2 monohelical scissions per DNA molecule of 1708 000 Mn. The percent distribution of the chemical termini released at the radiation nicks of DNA was as follows: 64.0 OH, 9.0 PO4 and 27.0 unknowns at the 3' ends: 3.8 OH, 68.2 PO4 and 28.0 unknowns at the 5' ends. A nuclease-free polynucleotide ligase I purified about 3000-fold over the crude homogenate from calf thymus succeeded in rejoining 50% of the breaks in the X-irradiated DNA. The ability of the enzyme to close radiation nicks in DNA directly was confirmed also by experiments on synthetic poly(dA).poly([3H]dT),poly(dT)-cellulose substrates with an irradiated dT chain at either the 3' or the 5' side of the functional break. The poor discrimination of mammalian ligase versus nicked DNA containing radiation damage is of practical relevance. While rejoining altered nucleotide chains in the helices of DNA, the enzyme might contribute to the fixation of premutational lesions in the genetic material.

Animals↗
PubMed1981

A computer method for construction of secondary structure from polynucleotide sequence. Possible structure of the bacterial replication origin.

A computer method to search the possible secondary structure of a long polynucleotide was developed. As a criterion for the stabilization of a secondary structure, free energy originating from base-pairing was employed, since the structure in solution would be at the free energy minimum. The method is summarized as follows: all possible helices are collected from a given nucleotide sequence under restrictions that the length of a helix is greater than N0 bases (e.g., four bases) and the free energy of the helix calculated according to free energies of two successive sequence-dependent basepairs is lower than E0 (e.g., -5 kcal/mol). The search of secondary structures of low free energy is performed by connecting one helix to another without allowing any base-pairing between loops. For connecting single-stranded regions, destabilizing free energy of 2--3 kcal/mol is added. The method was first applied to several tRNAs and the clover-leaf structure of tRNA was obtained as a free energy minimum. Then, possible secondary structures of the replication origin regions of the Escherichia coli and Salmonella typhimurium chromosomes were examined by the method, assuming that one of the strands in the origin region takes a specific secondary structure. The lowest-energy structure for the E. coli origin was found to be approximately identical to that for the S. typhimurium origin region.

Base Sequence↗
PubMed1987

Inhibition of bovine and human brain 2':3'-cyclic nucleotide 3'-phosphodiesterase by heparin and polyribonucleotides and evidence for an associated 5'-polynucleotide kinase activity.

The present paper establishes a 5'-polynucleotide kinase activity associated with the bovine and human brain enzyme 2':3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37) in addition to known extremely high hydrolysis rates against 2':3'-cyclic nucleotides. Modulation of the enzyme activity by the addition of polyadenylate (5') and polyuridylate (5'), histone F3, myelin basic protein (MBP), and other basic molecules suggest that RNA may be the natural substrate for both enzymes. These enzymes, isolated from brain and present in very high activities in oligodendrocytes and in isolated myelin, probably have complex functions.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed1991

Specificity of guanylic RNases to polynucleotide substrates.

Kinetic parameters kcat and KM were measured for cleavage of poly I, poly A, poly U, poly C and poly I poly C by guanyl-specific RNases Sa, Pb1 and T1 and compared with that of guanyl-preferential RNase Bi. Catalytic efficiencies of the investigated enzymes to polynucleotide substrates vary considerably. The structural basis for specificity of these RNases is discussed. A hypothesis is suggested that Ser-56 plays an important role in recognition of poly A by RNase Bi.

Aspergillus oryzae↗
PubMed1976

Spectrophotometric studies on the binding with polynucleotides of 4,4'-diacetyldiphenylurea-bis(guanylhydrazone) and methylglyoxal-bis(guanylhydrazone).

4,4'-Diacetyldiphenylurea-bis(guanylhydrazone) (DDUG), an agent very effective against several animal leukemias and tumors, was found, spectrophotometrically, to interact in a biphasic manner with several natural, native and heat-denatured, and synthetic DNAs. The spectrum of DDUG was shifted towards the visible region with a hypochromic shift reaching a maximum hypochromicity at 316 mmu at a 1 : 1 molar ratio of DDUG to DNA nucleotide. Increasing molarity of DNA nucleotide resulted in a further shift towards the visible end, but with hyperchromicity rather than hypochromicity, and reaching its peak at 323 mmu. The interaction with yeast RNA was much weaker than that with DNA. 4,4'-Diacetyldiphenylurea (DDU) did not show any interaction with DNA; its monoguanylhydrazone (DDUM) showed only a hypochromic interaction. In contrast to DDUG, methylglyoxal-bis( guanylhydrazone (CH3-G), an aliphatic bisguanylhydrazone with antileukemic properties, showed only a hypochromic interaction with DNA at low ionic strength. Unlike DDUG, CH3-G was a very weak inhibitor of the DNA polymerase reaction. The hypochromic shift of the DDUG spectrum with DNA was abolished in the presence of 15 mM sodium citrate or 500 mM NaCl but not in the presence of 150 mM NaCl or 100 mM sodium acetate. The hyperchromic shift was abolished in the presence of 8 M urea. From the results obtained with different DNAs, RNA, synthetic polynucleotides and nucleotides, it appears that the total shift of the DDUG spectrum in the presence of intact DNA can not be ascribed to interaction with a single base although a greater shift occurred in the presence of G-C rich DNA.

Antineoplastic Agents↗
PubMed1987

Role of tryptophan 54 in the binding of E. coli single-stranded DNA-binding protein to single-stranded polynucleotides.

Fluorescence and optical detection of triplet state magnetic resonance spectroscopy have been employed to study the complexes formed by single-stranded polynucleotides with both E. coli single-stranded DNA-binding protein and an E. coli ssb gene product in which Trp-54 is replaced by phenylalanine using site specific oligonucleotide mutagenesis. Our results strongly suggest the involvement of Trp-54 in stabilizing the protein-nucleic acid complexes via stacking interactions of the aromatic residue with the nucleotide bases.

DNA-Binding Proteins↗
PubMed1989

Mutant rho factors with increased transcription termination activities. I. Functional correlations of the primary and secondary polynucleotide binding sites with the efficiency and site-selectivity of rho-dependent termination.

We have characterized rho proteins from mutants of Escherichia coli, rho s-81 and rho s-82, which are hyperactive in termination. The two mutant rho proteins are differentially altered both in termination activities and in RNA interactions. rho s-81 generally elicits enhanced termination on various templates such as phage T7 DNA and a DNA restriction fragment containing the trpE intracistronic rho-dependent terminators, either measured as a whole or examined for individual sites. On the other hand, rho s-82 has strikingly different preferences toward individual termination sites, exhibiting overall termination activities higher or lower than normal, depending on templates. From measurements of the rho ATPase activity with T7 RNA and various homoribopolymers as cofactors, both mutant rho proteins are shown to have broadened RNA base specificities in contrast to the stringent requirement for cytosine observed with the wild-type rho. Functional tests on the two kinds of polynucleotide binding sites known for rho have indicated that rho s-81 is mainly altered in the primary site, whereas rho s-82 is simultaneously affected in the secondary binding site as well as the primary site. Thus, we conclude that the primary and secondary sites contribute distinctly in determining the overall efficiency and site-specificity of termination, respectively. Further analysis of detailed termination points at the trpE and lambda tR1 terminators has revealed that major RNA transcripts generated by the wild-type rho and rho s-81 are notably rich in adenine and poor in cytosine for the 3'-terminal five to ten nucleotides, whereas those preferentially terminated by rho s-82 are conversely richer in cytosine than adenine. This finding suggests that rho may recognize the RNA-DNA hybrid region at the 3' end of a nascent transcript in its secondary binding reaction.

Adenosine Triphosphatases↗
PubMed1981

Functional analysis of influenza RNA polymerase activity by the use of caps, oligonucleotides and polynucleotides.

The effects of caps, dinucleotides, oligonucleotides and polynucleotides on influenza virus RNA polymerase activity have been investigated. The results show that both methyl groups in a cap are necessary for optimal stimulation of polymerase activity. Both m7G(5')ppp(5')Am and ApG stimulated the influenza RNA polymerase activity and seemed to interact at different sites. Out of the 16 homopolynucleotides tested, seven inhibited influenza RNA polymerase by 50% at 2-10 micrograms/ml. Poly(G) gave a 90% reduction of influenza virus plaque formation at 10 micrograms/ml. An oligodeoxyribonucleotide complementary to the 12 terminal nucleotides of the 3' end of influenza virus RNA was synthesized. This oligonucleotide did not selectively inhibit influenza RNA polymerase.

DNA-Directed RNA Polymerases↗