Antisera to poly(A)-poly(U)-poly(I) contain antibody subpopulations specific for different aspects of the triple helix.

Rabbit antibodies to the triple-helical polynucleotide poly(A)-poly(U)-poly(I) were fractionated into three major antibody populations, each recognizing a different conformational feature of the triple-helical immunogen. Two distinct populations were purified from precipitates made with poly(A)-poly(U)-poly(U) and poly(A)-poly(I)-poly(I). The former reacted with double-stranded poly(A)-poly(U) or poly(I)-poly(C), and similar populations could be purified with either double-stranded form. The second population recognized the poly(A)-poly(I) region of the triple helix, and the third required all three strands for reactivity. These immunochemical studies suggest that the poly(A) and poly(U) have the same orientation in the triple-helicical poly(A)-poly(U)-poly(I) as in the double-helical poly(A)-poly(U), in which they have Watson-Crick base pairing.     

Properties of unprimed poly(A)-poly(U) synthesis by Caulobacter crescentus RNA polymerase.

Some properties of unprimed poly(A)-poly(U) synthesis by DNA-dependent RNA polymerase from Caulobacter crescentus were examined. The reaction required ATP and UTP as substrates and manganese as a divalent cation. Rifampicin completely inhibited the reaction at a concentration of 1 micron/ml, and the enzyme catalyzed the polymer synthesis well regardless of the presence of GTP, CTP or both. The chain length of the poly(A)-poly(U) synthesized was about one hundred base pairs, as estimated from a sedimentation velocity and the molar ratio of [3H]AMP to [gamma-32P]ATP incorporated into the poly(A)-poly(U). The reaction was dependent on the square of the enzyme concentration and the enzyme dimers formed complexes with poly(A)-poly(U) during the reaction.     

The binding of Mg(II) and Ni(II) to synthetic polynucleotides

Equilibria and kinetics of the interactions of Mg2+ and Ni2+ with poly(U), poly(C) and poly(I) have been investigated at 25 degrees C, an ionic strength of 0.1 M, and pH 7.0 or 6.0. Analogous studies involving poly(A) were reported earlier. All binding equilibria were studied by means of the (usually small) absorbance changes in the ultraviolet range. This technique yields apparent binding constants which are fairly large for the interaction of Ni2+ with poly(A) (K = 0.9 X 10(4) M-1) and poly(I) (K approximately equal to 2 X 10(4) M-1) but considerably lower for the corresponding Mg2+ systems, Mg2+-poly(A) (K = 2 X 10(3) M-1) and Mg2+-poly(I) (K = 280 M-1). Each of the two pyrimidine nucleotides binds both metal ions with about the same strength (K approximately equal to 65 M-1 for poly(U) and K near 600 M-1 for poly(C]. In the case of poly(C) the spectral changes deviate from those expected for a simple binding equilibrium. In addition, the binding of Ni2+ to the four polynucleotides was measured by using murexide as an indicator of the concentration of free Ni2+. The results obtained by this technique agree or are at least consistent with those derived from the ultraviolet spectra. Complications are encountered in the binding studies involving poly(I), particularly at higher metal ion concentrations, obviously due to the formation of aggregated poly(I) species. Kinetic studies of the binding processes were carried out by the temperature-jump relaxation technique. Measurable relaxation effects of time constants greater than 5 microseconds were observed only in the systems Ni2+-poly(A) and Ni2+-poly(I). Such not-too-fast reaction effects are expected for processes which include inner-sphere substitution steps at Mg2+ or Ni2+. The relaxation process in Ni2+-poly(I) is characterized by (at least) four time constants. Obviously, the complicated kinetics again include reactions of aggregated poly(I). The absence of detectable relaxation effects in all other systems (except Mg2+-poly(I), the kinetics of which was not investigated) indicates that inner-sphere coordination of the metal ions to specific sites of the polynucleotides (site binding) does not occur to a significant extent. Rather, the metal ions are bound in these systems mainly by electrostatic forces, forming a mobile cloud. The differences in binding strength which are nevertheless observed are attributed to differences in the conformation of the polynucleotides which result in different charge densities.     

Binding kinetics of mercury(II) TO POLYRIBONUCLEOTIDES.

Kinetic studies of the interaction of Hg(II) with polyribonucleotides have been used to investigate structural fluctuations of the bases in nucleic acids. The reaction of Hg(II) with poly(A)-poly(U) occurs in two phases which differ in time scale by a factor of about 100. The slow phase is first order and exhibits cooperativity or autocatalytic kinetics. The rate is found to increase as decreasing chain length of poly(U) is used to make the double helical complex. The reaction appears to initiate at the ends of poly(U) strands and may be associated with a molecular rearrangement which results in strand separation with Hg(II) being linked only to uridine. The fast reaction phase is second order ans shows little cooperative behavior. Protons are released at this stage indicating alteration of the double helix. The measured second-order rate constant is nearly three orders of magnitude smaller than that found for poly(U) alone. This rate difference suggests that the reactive sites are blocked by double helix formation, and become available for reaction with Hg(II) only through a structural fluctuation. The ratio of rate constants for the reaction of Hg(II) with poly(U) and poly(A)-poly(U) was used to place an upper limit on the equilibrium constant for the structural fluctuation of 2 times 10- minus 3 at 15 degrees and 0.5 M NaClO4. The heat of the "breathing" reaction can be estimated to be similar to 9 kcal/mol from comparison of the temperature coefficient of the reaction with poly(U) to that with poly(A)-poly(U).     

Processing of exogenous poly(A) added to virus-infected cells.

Exogenous [³H]-poly(A) was taken up by and was stable for some hours in monolayers of human embryo lung cells in culture. The poly(A) was extracted and sized by G200 Sephadex chromatography. Non-infected and virus-infected cells converted the majority of [³H]-poly(A) into a smaller poly(A) molecule which could still bind to oligo(dT)cellulose. In addition virus-infected cells only converted 11% into a poly(A) containing-molecule which was at least 4-fold greater in size than the original poly(A). This larger material could also bind to oligo(dT)cellulose and did not result from the breakdown and re-utilization of the [³H]-poly(A).     

Binding of novel 9-O–N-aryl/arylalkyl amino carbonyl methyl berberine analogs to poly(U)-poly(A)·poly(U) triplex and comparison to the duplex poly(A)-poly(U)

Interaction of the 9-O–N-aryl/arylalkyl amino carbonyl methyl substituted analogs of the anticancer isoquinoline alkaloid berberine with RNA triplex, poly(U)-poly(A)·poly(U) has been studied in comparison to the duplex poly(A)-poly(U), using multiple biophysical techniques. Spectrophotometric and spectrofluorimetric studies established the non-cooperative binding mode of all the analogs with both the duplex and the triplex. However, berberine exhibited cooperative binding with poly(A)-poly(U) and non-cooperative binding with poly(U)-poly(A)·poly(U). Analog BER1 showed the highest affinity to both the duplex and the triplex followed by BER2 and BER3. The overall binding affinity varied as BER1 > BER2 > BER3 > BER. The magnitude of the quantum efficiency values (Q > 1) revealed that energy was transferred from the bases of the triplex and the duplex to the analogs. Comparative ferrocyanide quenching and viscosity studies unambiguously established a stronger intercalative geometry of the analogs to both the triplex and the duplex in comparison to berberine. Circular dichroism studies revealed that the alkaloids perturbed the conformation of both RNA helices. The binding of all the alkaloids was found to be exothermic from isothermal titration studies. Binding of the analogs was highly entropy driven while that of berberine was enthalpy dominated. The results presented here reveal strong and specific binding of these new berberine analogs to the RNA triplex and duplex and highlight the remarkable influence of the 9-substitution on the interaction profile.     

Fluorescence studies on the complex formation between poly(rA) containing 1,N6-ethenoadenosine and poly(rU)

The interaction of the 1,N6-etheno derivatives of poly(rA) (poly(epsilon rA] with poly(rU) has been studied by absorption and fluorescence spectroscopy. The stoichiometry of the interaction is found to be 1 epsilon A:1 rU and 1 epsilon A:2 rU as well as in the case of poly(rA)-poly(rU) interaction. The fluorescence properties, including the intensity and polarization of fluorescence, respond to the conformational transition of poly(epsilon rA)-poly(rU) complexes. The introduction of epsilon A groups into poly(rA) results in a marked decrease in the melting temperature, suggesting that epsilon A may destabilize the helical structure. The three-exponential decay law obtained with poly(epsilon rA)-poly(rU) complexes indicates the existence of at least three different stacked conformational states.     

Induction of differentiation of mouse myeloid leukemia cells by poly(ADP-ribose).

The effects of poly(ADP-Rib) on the differentiation of mouse myeloid leukemia cells were studied. The myeloid leukemia cells differentiated into cells with phagocytic activity, Fc receptors, and lysozyme activity on treatment with poly(ADP-Rib). Cells with morphological characteristics of macrophages and granulocytes also appeared on incubation with poly(ADP-Rib). Dextran sulfate and polyvinylsulfate were also effective for the induction of phagocytic cells, but poly(A), poly(U), poly(C), poly(I), poly(I) · poly(C), and poly(A) · poly(U) were not. The uptake of poly(ADP-Rib) by the myeloid leukemia cells is discussed in relation to their differentiation.     

Models of triple-stranded polynucleotides with optimised stereochemistry.

Detailed models are presented for the triple-stranded polynucleotide helices of poly (U)-poly (A)-poly (U) (two forms), poly (U)-poly d (A) -poly (U), poly d(C)-poly d(I)-poly d(C), poly d(T)-polyd(A)-poly d(T) and poly (I)-poly (A)-poly (I). The models were genrated using a computerized, linked-atom procedure which preserves standard bond lengths, bond anglesand sugar ring conformations, constrains the helices to have the pitches and symmetries observed in X-ray diffraction experiments, and optimises the non-bonded interatomic contacts including hydrogen bonds. The possible biological sigificance of such complexes is discussed.     

Polyadenylation of RNA in immature oocytes and early cleavage of Urechis caupo

The poly(A) content of RNA extracted from four stages of immature oocytes, mature oocytes, and cleavage embryos through the eight-cell stage was determined by hybridization with [³H]-poly(U). During oogenesis the poly(A) content per cell gradually increases from 0.007 pg of poly(A)/cell in the 10- to 39-μm oocytes to 0.20 pg of poly(A)/cell in the 125-μm mature oocytes. After fertilization there is an additional increase to approximately 1.1 pg of poly(A)/embryo at the two-cell stage which is followed by a slight decline between the two- and eight-cell stages. Most of the increase in poly(A) after fertilization occurs in a 45-min interval coincident with the appearance of the polar bodies. The size distribution of the poly(A) in RNA from the different stages of development was determined based on the length of RNase-resistant poly(U) obtained from poly(U)-poly(A) hybrids. The size distribution of the poly(A) sequences is constant through each stage of development which indicates that the increase in the poly(A) content of the cells is the result of polyadenylation of new RNA sequences.     

Relation of the biological activity of double helical interferon inducers and their penetration into the cell and suppression of protein synthesis

The quantities of 125I-ds-inductors of interferon penetrating into the cells of transplantable cultures such as M19 (human fibroblast cells) and L-929 (mouse line) were not significant i.e. 10.5-4 per cent of the drug added. Under conditions of transfection with calcium phosphate and in complex with DEAE dextran the quantities of the inductors adhering to the cells and their contents in the cytoplasmic and nuclear fractions markedly increased. During the transfection with calcium phosphate up to 50 per cent of the applied inductor bound to the cells and its content in the cytoplasm and nuclei reached at least 10 per cent. After penetration into the cells poly I.poly C probably maintained its native structure and appeared to be firmly bound to the nuclear material. Preliminarily hydrolyzed inductors showed no such penetrating capacity. Contrary to the human fibroblast cells, in the mouse cells L-929 treated with the ds-inductors there was observed inhibition of the total protein synthesis which was probably due to activation of enzymes such as 2-5A-synthetase and proteinkinase. Increased penetration of the ds-inductors into the cells was accompanied by a marked (from 10- to 1000-fold) rise in their antiviral activity and a 2-4-fold rise in their interferon-inducing activity. It was concluded that there was immediate dependence of ds-inductor biological activity manifestation on the level of the inductor penetration into the cells.     

A premelting conformational transition in poly(dA)-Poly(dT) coupled to daunomycin binding

Circular dichroism and UV absorbance spectroscopy were used to monitor and characterize a premelting conformational transition of poly(dA)-poly(dT) from one helical form to another. The transition was found to be broad, with a midpoint of tm = 29.9 degrees C and delta HVH = +19.9 kcal mol-1. The transition renders poly(dA)-poly(dT) more susceptible to digestion by DNase I and facilitates binding of the intercalator daunomycin. Dimethyl sulfoxide was found to perturb poly(dA)-poly(dT) structure in a manner similar to temperature. These combined results suggest that disruption of bound water might be linked to the observed transition. A thermodynamic analysis of daunomycin binding to poly(dA)-poly(dT) shows that antibiotic binding is coupled to the polynucleotide conformational transition. Daunomycin binding renders poly(dA)-poly(dT) more susceptible to DNase I digestion at low binding ratios, in contrast to the normal behavior of intercalators, indicating that antibiotic binding alters the conformation of the polynucleotide. The unusual thermodynamic profiles previously observed for the binding of many antibiotics to poly(dA)-poly(dT) can be explained by our results as arising from the coupling of ligand binding to the polynucleotide conformational transition. Our data further suggest a physical basis for the temperature dependence of DNA bending.     

The metabolic behaviour of nuclear and cytoplasmic non-polyadenylated RNAs with an affinity for poly(adenylic acid) from Friend murine leukaemia cells

Using poly(A)-Sepharose and poly(U)-Sepharose affinity chromatography, various classes of nuclear RNA can be distinguished in Friend leukaemia cells. One of these contains a poly(A) tract (poly(A)+-RNA) and another lacks a poly(A) tract but has an affinity for poly(A)-Sepharose (poly(A)-u+-RNA). The stability of these two particular nuclear RNA classes was examined by using a 'pulse-chase' technique involving D-glucosamine treatment. Nuclear poly(A)-u+-RNA was found to decay as a single component with a half-life of about 12 min. In contrast, nuclear poly(A)+-RNA appears to consist of at least two distinct metabolic components with half-lives of about 22 min and 120 min. Furthermore, poly(A)-u+-RNA is transported from the nuclei much more rapidly than the poly(A)+-RNA. The 'pulse-chase' approach also allowed a quantitative estimate to be made of the conversion of nuclear poly(A)+-RNA and poly(A)-u+-RNA to cytoplasmic poly(A)-RNA and poly(A)-u+-RNA.     

Localization of host poly(A)+ mRNA in the ribosome profile of mengovirus-infected Ehrlich ascites tumor cells

The distribution of poly(A)⁺ mRNA among polysomes, monosomes, and ribosome-free supernatant fractions after mengovirus infection of Ehrlich ascites tumor (EAT) cells was investigated employing sucrose gradient centrifugation of their corresponding postnuclear supernatants. Poly(A)⁺ mRNA was isolated from sucrose gradient fractions and quantitated in a cell-free protein synthesizing system from uninfected EAT cells. It was also localized by annealing [³H]-poly(U) to the poly(A)-tracts of mRNA present in the sucrose gradient fractions. Both experiments revealed a gradual shift of host poly(A)⁺ mRNA from large to small polysomes and monosomes, respectively, with the time postinfection. The greatest part of host template RNA appears to remain ribosome-bound and only a fraction seems to be detached from the ribosomes in the course of mengovirus infection. At the end of the infectious cycle, 8 h postinfection, approximately 70% of the poly(A)⁺ mRNA detected in uninfected cells is still biologically active, but not translated in vivo, in agreement with data from the [³H] poly(U) hybridization experiment.     

Intercalation of water-soluble bis-Porphyrins into Poly(dA)-Poly(dT) double helix

The association constants (K) of nucleic acid monomers with a series of water-soluble bis-porphyrins (bisMC1, bisMC3, bisMC5, bisMC7, and bisMC11) in which two porphyrin units were linked by a methylene chain of various lengths were estimated spectrophotometrically. Among the bis-porphyrins, the K values are similar for each nucleic acid monomer, indicating that the bridging chain length does not affect the association of the bis-porphyrins with the nucleic acid monomers. The melting curves of poly(dA)-poly(dT) in the presence of bisMC3 or bisMC5 were found to be biphasic, suggesting that bisMC3 and bisMC5 are bound to poly(dA)-poly(dT) with a binding mode different from the groove binding exhibited by the corresponding porphyrin monomers. A negative-induced CD peak in the Soret region of bisMC3 and bisMC5 with poly(dA)-poly(dT) is observed and the visible spectral changes of bisMC3 and bisMC5 upon addition of poly(dA)-poly(dT) are accompanied by a large red shift of the Soret band (bisMC3: 21 nm, bisMC5: 23 nm) with substantial hypochromicity (bisMC3: 49%, bisMC5: 40%). Therefore, it is reasonable to conclude that both of the porphyrin units of bisMC3 and bisMC5 intercalate into poly(dA)-poly(dT). In contrast to poly(dA)-poly(dT), the melting curves of poly(dA·dT)₂ in the presence of the bis-porphyrins did not show such biphasic behavior. Together with the CD and visible absorption data, it is certain that these bis-porphyrins do not intercalate into poly(dA·dT)₂.     

Energy requirement and kinetics of transport of poly(A)-free histone mRNA compared to poly(A)-rich mRNA from isolated L-cell nuclei

ATP-promoted efflux of poly(A)-rich RNA from isolated nuclei of prelabeled mouse lymphoma L5178y cells has an activation energy of 51.5 kJ/mol, similar to that found for the nuclear envelope nucleoside triphosphatase (48.1 kJ/mol) assumed to be involved in mediating nucleocytoplasmic transport of at least some RNA. Here we show that efflux of two specific poly(A)-rich mRNAs (actin and beta-tubulin) from isolated L-cell nuclei is almost totally dependent on the presence of ATP, while efflux of poly(A)-free histone mRNA (H4, H2B, and H1) also occurs to a marked extent in the absence of this nucleotide. Measurements of temperature dependence of transport rate revealed an activation energy of 56.1 kJ/mol for actin mRNA, while the activation energy for histone-H4-mRNA efflux was in the same range as that found for ATP-induced release of RNA from demembranated nuclei (about 15-20 kJ/mol). Addition of nonhydrolyzable nucleotide analogs of ATP to the in vitro system used for measurement of RNA transport did not result in release of nonhistone mRNA (actin), but enhanced the efflux of H4 mRNA to approximately the same extent as ATP. Although not absolutely required, addition of ATP stimulated the rate of export of histone mRNA about twofold. Only the poly(A)-rich RNA, but not the poly(A)-free RNA, released from isolated nuclei was found to compete with poly(A) for the nuclear envelope mRNA-binding site, indicating the mechanism of transport for both RNA classes to be distinct. Export of both nonhistone and histone mRNA was found to be inhibited by a monoclonal antibody against a p60 nuclear-pore-complex antigen. This antibody had no effect on the nucleoside triphosphatase, mediating transport of poly(A)-rich mRNA.     

Protonated polynucleotide structures, 20. Interaction between poly(dG)-poly(dC) and poly(rC).1.

A study of the interaction between poly(dG)-poly(dC) and poly(rC) demonstrates that, at neutral pH and high ionic strength, there is replacement of the dC strand by poly(rC). At acid pH, formation of a triple-stranded complex which equally may involve the replacement phenomenon is observed. There is no evidence for interaction at neutral pH between poly(dG)-poly(dC) and oligo(rC), while a three-stranded complex is formed at acid pH. These data are consistent with the studies of comparative stabilities of double stranded deoxy or ribo polymers and deoxy-ribo hybrids.     

Comparison of base inclination in ribo-GC and deoxybribo-GC polymers,and synthesis of poly(rGrC)-poly(rGrC)

The inclination angle between the base normal and the helix axis, and the axes around which the bases incline, are measured for ribo-GC polymers in buffer by using flow linear dichroism (LD), and compared to measurements for deoxyribo-GC polymers in buffer and under dehydrating conditions. A new method is designed to synthesize poly(rGrC) -poly(rGrC), which is not available commercially, in large quantities. The LD of this RNA reveals inclination angles that are similar to the B-form DNA in buffer, although the axes are different. The CD of poly(dGdC)-poly(dGdC) under the dehydrating conditions is similar to poly(rGrC)-poly(rGrC), indicating it is in the A form, and the LD gives larger inclination angles than either the B form or the corresponding RNA. Poly(dG)-poly(dC) is in the A form in buffer. Comparison among poly(rG)-poly(rC) in buffer, and poly (dG)-poly(dC) in buffer and under dehydrating conditions, reveals similar inclination angles and axes, although the LD shows that the DNA has the largest inclination angles. Except for poly(rGrC)-poly(rGrC), which has a unique reduced dichroism, all the axes for G are similar, as are the axes for C. © 1995 John Wiley & Sons, Inc.