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PubMedDate not supplied

[Induction of plant resistance to the tobacco mosaic virus with a double-stranded poly(G)-poly(C) complex].

Inoculation of double-stranded polyribonucleotide poly(G) . poly(C) complex in a concentration of 50-200 micrograms/mg into tobacco and thornapple leaves was found to produce resistance of the plants to subsequent infection with tobacco mosaic virus (TMV) manifested in decreased number and size of local virus lesions. The induced resistance may spread over the plant and be found in the upper untreated leaves. The level of systemic resistance, however, is much lower than that of the resistance demonstrated in the injected leaves. Actinomycin D (5 micrograms/ml) had no significant effect on the number but stimulated the growth of lesions developing in leaves injected with poly(G) . poly(C) as well as increased their number in the upper leaves of the same plants proximal to the treated ones. Development of tobacco resistance to TMV was accompanied by changes in the activity of terminal oxidases, particularly peroxidase. Possible mechanisms of formation of induced resistance are discussed.

Plant Diseases↗
PubMed2006

Reengineering CCA-adding enzymes to function as (U,G)- or dCdCdA-adding enzymes or poly(C,A) and poly(U,G) polymerases.

CCA-adding enzymes build and repair the 3'-terminal CCA sequence of tRNA. These unusual RNA polymerases use either a ribonucleoprotein template (class I) or pure protein template (class II) to form mock base pairs with the Watson-Crick edges of incoming CTP and ATP. Guided by the class II Bacillus stearothermophilus CCA-adding enzyme structure, we introduced mutations designed to reverse the polarity of hydrogen bonds between the nucleobases and protein template. We were able to transform the CCA-adding enzyme into a (U,G)-adding enzyme that incorporates UTP and GTP instead of CTP and ATP; we transformed the related Aquifex aeolicus CC- and A-adding enzymes into UU- and G-adding enzymes and Escherichia coli poly(A) polymerase into a poly(G) polymerase; and we transformed the B. stearothermophilus CCA-adding enzyme into a poly(C,A) polymerase by mutations in helix J that appear, based on the apoenzyme structure, to sterically limit addition to CCA. We also transformed the B. stearothermophilus CCA-adding enzyme into a dCdCdA-adding enzyme by mutating an arginine that interacts with the incoming ribose 2' hydroxyl. Most importantly, we found that mutations in helix J can affect the specificity of the nucleotide binding site some 20 A away, suggesting that the specificity of both class I and II enzymes may be dictated by an intricate network of hydrogen bonds involving the protein, incoming nucleotide, and 3' end of the tRNA. Collaboration between RNA and protein in the form of a ribonucleoprotein template may help to explain the evolutionary diversity of the nucleotidyltransferase family.

Binding Sites↗
PubMed2004

Xenopus embryonic poly(A) binding protein 2 (ePABP2) defines a new family of cytoplasmic poly(A) binding proteins expressed during the early stages of vertebrate development.

We describe a new RNA binding protein from Xenopus we have named ePABP2 (embryonic poly(A) binding protein type II). Based on amino acid similarity, ePABP2 is closely related to the ubiquitously expressed nuclear PABP2 protein that directs the elongation of mRNA poly(A) tails during pre-mRNA processing. However, in contrast to known PABP2 proteins, Xenopus ePABP2 is a cytoplasmic protein that is predominantly expressed during the early stages of Xenopus development and in adult ovarian tissue. Biochemical experiments indicate ePABP2 binds poly(A) with specificity and that this binding requires the RRM domain. Mouse and human ePABP2 proteins were also identified and mouse ePABP2 expression is also confined to the earliest stages of mouse development and adult ovarian tissue. We propose that Xenopus ePABP2 is the founding member of a new class of poly(A) binding proteins expressed in vertebrate embryos. Possible roles for this protein in regulating mRNA function in early vertebrate development are discussed.

Aging↗
PubMed2001

Binding of SLE autoantibodies to native poly(I), ROS-poly(I) and native DNA: a comparative study.

Reactive oxygen species (ROS) are implicated in a variety of human diseases. The formation of pathogenic anti-DNA antibodies in systemic lupus erythematosus (SLE) has been extensively investigated. ROS-modified DNA has been found to be a better antigen for anti-DNA antibodies found in SLE sera. A comparative binding of SLE autoantibodies with native poly(I), ROS-poly(I) and nDNA has been studied. Affinity-purified SLE IgG exhibited a high degree of specificity towards the ROS-modified poly(I) in comparison to native DNA and native poly(I), reiterated visually by gel retardation assay. The data suggested that hydroxyl radical-modified nucleic acids like RNA and DNA might be agent for the induction of circulating SLE anti-DNA autoantibodies.

Antibodies, Antinuclear↗
PubMed1978

Randomized trial with Poly A-Poly U as adjuvant therapy complementing surgery in patients with breast cancer: in vitro study of cellular immunity.

The immunologic reactivity of patients with initially operable breast cancer was measured by the leukocyte migration inhibition test using autologous tumor extract (T), autologous serum (S), and a combination of both (T + S). These patients formed part of a randomized clinical trial comparing, on the one hand, conventional treatment and, on the other, conventional treatment complemented by injections of poly A-poly U. A sequential study was carried out on 159 patients, testing them 7 days, 2 months, 4 months, and 1 year after the operation. Statistical comparisons revealed no significant difference in the reaction of the two groups. In addition, no significant differences were found between those with lymph node involvement and those without. Radiotherapy given to those with lymph node involvement did not significantly change their reactions. We were able to show that the percentage of patients with a positive leukocyte migration inhibition test (LMIT) increases regularly and significantly with time. This study confirmed the presence in some autologous serum of a synergistic factor (SS factor) which increased the inhibition of migration of leukocytes by autologous tumor extract. This factor was found in 18 patients, equally divided between both therapeutic groups. In the group with SS factor, the percentage with lymph node involvement appeared greater (83% compared with 68% among those patients who had no SS factor), and the incidence of metastases was also increased (44% compared with 21%). This factor seemed to indicate a bad prognosis. However, there was a difference in the results between the two therapeutic groups in patients with the synergistic factor. Of nine patients undergoing conventional treatment, six had devleoped metastases, whereas only two out of the nine patients who also poly A-poly U developed metastases. The same trend was observed in the whole trial population.

Breast Neoplasms↗
PubMed1984

Influence of the xyloadenosine analogue of 2',5'-oligoriboadenylate on poly(A)-specific, 2',5'-oligoriboadenylate degrading 2',3'-exoribonuclease and further enzymes involved in poly(A)(+)mRNA metabolism.

The homogeneous poly(A)-specific 2',3'-exoribonuclease from calf thymus gland, which cleaves both 3',5'- and 2',5'-linked oligoriboadenylates, does not degrade (xyloA2'p)2 xyloA, the xylofuranosyladenosine analogue of the 2-5A core. This oligonucleotide, which is supposed to enter intact cells rapidly, was found to possess an increased stability and an enhanced antiherpesvirus activity compared to the natural (A2'p)2A (Eppstein, D.A., Barnett, J.W., Marsh, Y.V., Gosselin, G. and Imbach, J.-L. (1983) Nature 302, 723-724). The poly(A) anabolic enzyme, poly(A) polymerase (Mn2+-dependent), from the same source, which is initiated by (A3'p)2A and its higher oligomers, does not accept 2-5A core and its xyloadenosine analogue as primer. Both oligonucleotides exert no influence on endoribonuclease IV and on the integrity of the poly(A)-ribonucleoprotein complex.

Adenine Nucleotides↗
PubMed1976

Specific excision of the inserted DNA segment from hybrid plasmids constructed by the poly(dA). poly (dT) method.

The hybrid plasmid PBETAG, consisting of plasmid PMB9 DNA with an insert of rabbit globin DNA (about 600 base pairs) flanked by poly(dA) poly(dT) regions (Maniatis, T., Kee, S.G., Efstratiadis, A. and Kafatos, F.C. (1976) Cell 8, 163-182), was cleaved into two fragments by endonuclease S1 under conditions of partial denaturation. Only the smaller fragment (575 base pairs) contained globin-specific sequences, showing that excision had occurred in the A-T-rich regions. This method of cleavage provides a useful procedure for assessing the length of inserts in hybrid plasmids prepared by the poly(dA)-POLY(DT) tail method, and allows the preparative recovery of the insert.

Animals↗
PubMed1980

Template-independent poly(A) x poly(U) synthesizing activity of different forms of Bacillus subtilis RNA polymerase.

Several, but not all, forms of bacillus subtilis RNA polymerase found in vegetative and sporulating cells can synthesize poly(A) x poly(U) in vitro. The vegetative delta-containing form of RNA polymerase (E delta) has little or no poly(A) x poly(U)-synthesizing activity, whereas RNA polymerase core (E) and sigma-containing core (E delta) both have significant activity. When purified vegetative delta factor was added to core, the core synthetic activity was reduced essentially to that of the vegetative enzyme E delta. When E sigma enzymes from vegetative and sporulating cells were compared for their salt sensitivity, it was found that the sporulation enzyme E sigma retained much more of its activity at 0.1 M KCl than the vegetative enzyme E sigma. Furthermore, when sporulation enzyme E delta 1 was compared with vegetative enzyme E sigma, it was found that the activity of the E sigma 1 form was much more resistant to high KCl concentrations than that of the vegetative E sigma form. These differences in enzyme activity, as affected by salt concentrations, suggest that the conformations of the sporulation E sigma and E delta 1 enzymes are different from that found in vegetative E sigma enzyme. These differences in conformation may be involved in selective gene expression during sporularion.

Bacillus subtilis↗
PubMed1989

A unique deletion in pBR322 DNA caused by insertion of poly(dA).poly(dT).

A unique deletion covering around 43% of the pBR322 genome was found after attempting to insert 100 or 200 bp poly(dA).poly(dT) into the EcoRV site of pBR322 DNA. This result was not observed if an equivalent size heterologous DNA or a larger poly(dA).poly(dT) fragment of 10-20,000 bp was introduced at the same site. DNA sequencing analysis at the junctions suggests that a specific intramolecular pairing may be involved in the formation of this deletion mutant.

Base Sequence↗
PubMed1983

Interferon induction by platinum(II)-poly(I).poly(C) complexes.

The effect of the interaction between poly(I).poly(C) and cis-dichloro-diammineplatinum(II) (cis-Pt), its trans analogue and chloro-diethylene-triamminoplatinum(II) (dien-Pt) on interferon induction activity was investigated. The covalent monodentate fixation of the three compounds on N7 of inosine has different effects on the structure and thermostability of poly(I). poly(C) which is well reflected by the interferon induction activity of the samples. Thus, the sandwich stabilization by dien-Pt at low binding ratios is manifested by an increased interferon induction and a high resistance towards RNAase degradation. The destabilization of the duplex by cis-Pt decreases interferon induction, accompanied by an increase in RNAase sensitivity of the complexes. In the case of trans-Pt the duplex structure is little perturbed and interferon induction is essentially maintained.

Animals↗
PubMed1992

Effect of tumor burden and route of administration on the immunotherapeutic properties of polyinosinic-polycytidylic acid stabilized with poly-L-lysine in carboxymethyl cellulose [Poly(I,C)-LC].

We examined the immunomodulatory and therapeutic activities of poly(I,C)-LC. Mice received a subcutaneous (s.c.) injection of sufficient numbers of MBL-2 lymphoma cells to produce in 1 week either a high or low tumor burden. A week after tumor cell injection, poly(I,C)-LC treatment was initiated; the agent was administered intraperitoneally (i.p.) at 5 mg/kg twice a week or at 2.5 or 0.5 mg/kg every day or as an intravenous (i.v.) injection at 0.5, 0.05, or 0.005 mg/kg three times a week. Poly(I,C)-LC treatment significantly increased antitumor effector cell functions in a variety of organs (including spleen, lungs, and peritoneum), as shown by increased killing of MBL-2 cells in vitro and increased tumor cell killing by natural killer cells and macrophages. Furthermore, prolongation of survival correlated with peritoneal macrophage tumoricidal activity when poly(I,C)-LC was given i.p. and with pulmonary effector cell function (including natural killer, cytolytic T-lymphocyte and macrophage tumoricidal activity) when the agent was administered i.v.

Animals↗
PubMed1988

Analysis of double-stranded poly(A).poly(U) molecules by reversed-phase high-performance liquid chromatography.

The behaviour of different batches of synthetic Poly(A).Poly(U) in reversed-phase high-performance liquid chromatography (HPLC) was studied. They consist of large molecules mainly in the form of a double strand. Differences in the elution patterns were correlated with properties detected by conventional methods such as electrophoresis, centrifugation, fusion analysis or enzymatic digestions. Under the present conditions, contamination by products and precursors used during synthesis was detectable, but was absent in most of the preparations. The differences in elution patterns between batches appear to be correlated with the size of the molecules synthesized. The chromatograms suggested that Poly(A).Poly(U) molecules contain single-strand portions at least transiently. The presence of such portions was confirmed by enzymatic digestion with S1 nuclease. The rapidity, reproducibility and ease of reversed-phase HPLC qualify this technique as a tool for routine analysis.

Chromatography, High Pressure Liquid↗
PubMed1983

Salt-induced conformational transition of poly[d(A-T)] X poly[d(A-T)].

Unique chiroptical properties of poly[d(A-T)] X poly[d(A-T)] observed in CsF solutions (Vorlícková et al., 1980) were specified by circular dichroism, ultraviolet light and 31P nuclear magnetic resonance spectroscopy. It was found that up to a 3 M concentration of the salt, caesium cations induced a gradual rearrangement of the polynucleotide double helix during which the phosphodiester bonds in one sequence changed the geometry and the base stacking decreased. At higher CsF concentrations poly[d(A-T)] X poly[d(A-T)] underwent a transition toward a novel conformation which had phosphodiester bonds in both sequences in considerably different non-B-DNA geometries.

Cesium↗
PubMed1980

Interaction of lac repressor with alternating poly d (A-T) and poly d (G-C). Circular dichroism studies.

The binding of lac repressor to poly d(A-T) and d(G-C) has been studied using circular dichroism. The results indicate that the binding induces the same conformational change of both polynucleotides and perturbs the same number of nucleic acid bases (28 bases). It is shown that in 0.1 M phosphate buffer the CD measurement can be used to determine the binding constant of lac repressor to poly d(A-T). Competition experiments performed at various salt concentrations show that the stronger interaction of lac repressor for poly d(A-T) than for poly d(G-C) is based on difference in the dissociation rate of the complexes whereas the association rate for both polymers are similar.

Binding Sites↗
PubMed2000

Long-range assemblies on poly(dG-dC)2 and poly(dA-dT)2: phosphonium cationic porphyrins and the importance of the charge.

The present paper describes synthesis and spectroscopic properties of novel cationic meso-tetraphenylporphyrins bearing two (trans) (P2) or three (P3) triphenylphosphonium substituents. The porphyrin aggregation in aqueous solutions is discussed in detail. Porphyrin binding to and self-organization onto long-range assemblies on poly(dA-dT)2 or poly(dG-dC)2 were probed by combination of absorption, fluorescence, circular dichroism (CD), transient and resonance light-scattering (RLS) techniques. The higher hydrophobicity of P2 is manifested by more extensive self-organization. Induced CD and intensive RLS indicate binding to the chiral environment on the nucleic acids exterior and exciton coupling between adjacent porphyrin moieties. The CD spectra of P2 on poly(dG-dC), and poly(dA-dT)2 suggest that the binding geometry is essentially independent of the base sequence. The fluorescence lifetime of about 4 ns was attributed to the long-range assembly. In the case of P3 the distinctly different CD spectra induced by GC or AT base-pair regions reveal that the number of the substituents determines how closely the porphyrin can approach the specific electronic environment on the nucleic acid exterior. The fluorescence lifetime of the P3 assembly is about 2 ns.

Circular Dichroism↗
PubMed1998

Photophysics of the cationic 5,10,15,20-tetrakis (4-N-methylpyridyl) porphyrin bound to DNA, [poly (dA-dT)]2 and [poly (dG-dC)]2: interaction with molecular oxygen studied by porphyrin triplet-triplet absorption and singlet oxygen luminescence.

Interaction between molecular oxygen and the cationic free-base 5,10,15,20-tetrakis (4-N-methylpyridyl) porphyrin (H2TMpyP4+) complexed with [poly (dA-dT)]2, [poly (dG-dC)]2 and calf thymus DNA, has been monitored in air-saturated heavy water solutions through porphyrin triplet-triplet absorption and singlet oxygen luminescence. Three different rate constants of porphyrin triplet state quenching have been found which correspond to different accessibilities of molecular oxygen to porphyrins embedded in the duplexes. The longest triplet state lifetime (30 microseconds), found for porphyrin bound to [poly (dG-dC)]2, corresponds to molecules well protected from oxygen. This supports the hypothesis of an intercalative binding mode of the porphyrin between GC base-pairs ('type A' sites). The fraction fT delta of the porphyrin triplet states quenched by molecular oxygen with singlet oxygen generation, is unity. In [poly (dA-dT)]2-porphyrin complexes, two sites ('type B' and 'C' sites of interaction) are involved, yielding very different triplet state lifetimes (5.5 microseconds and 20.5 microseconds) and efficiencies of singlet oxygen generation (fT delta = 0.50 and 0.82). The fT delta decreases can likely be explained in terms of competition between energy and electron transfer from the porphyrin excited triplet state to molecular oxygen. All three types (A, B and C) of interaction sites can be expected in porphyrin-DNA complexes.

Absorption↗
PubMed1986

Poly(dA-dT).poly(dA-dT) two-pathway proton exchange mechanism. Effect of general and specific base catalysis on deuteration rates.

The deuteration rates of the poly(dA-dT).poly(dA-dT) amino and imino protons have been measured with stopped-flow spectrophotometry as a function of general and specific base catalyst concentration. Two proton exchange classes are found with time constants differing by a factor of 10 (4 and 0.4 s-1). The slower class represents the exchange of the adenine amino protons whereas the proton of the faster class has been assigned to the thymine imino proton. The exchange rates of these two classes of protons are independent of general and specific base catalyst concentration. This very characteristic behavior demonstrates that in our experimental conditions the exchange rates of the imino and amino protons in poly(dA-dT).poly(dA-dT) are limited by two different conformational fluctuations. We present a three-state exchange mechanism accounting for our experimental results.

Deuterium↗
PubMed1976

Synthesis and properties of poly(O6-methylguanylic acid) and poly(O6-ethylguanylic acid).

The nucleotide analogues, O6-methyl- and O6-ethylguanosine diphosphate, have been synthesized and polymerized to high-molecular-weight homopolymers with polynucleotide phosphorylase. The ultraviolet spectra of these polymers show marked hypochromicity, which suggests that they possess considerable secondary structures. Graphs of optical denisty vs. temperature in 0.15 M NaC1 indicate that cooperative melting occurs for both polymers, and that the secondary structure of poly(O6-methylguanosine monophosphate) is somewhat more stable than that of poly (O6-ethyl-guanosine monophosphate). Mixing experiments show that these analogue polymers no longer form helical structures with poly(C), nor do thye form helices with poly(U). We would conclude from these data that environmental mutagens and carcinogens which react at the O6 position of guanine not only disrupt normal base-pairing relationships, but may also affect the secondary structure of nucleic acids.

Chromatography, Paper↗