"Bridge" structures between nucleic acid molecules fixed in the structure of a liquid crystal

The binding of daunomycin and copper ions to poly(I).poly(C) molecules fixed in a particle of a liquid-crystalline dispersion was studied. A thermodynamic model of adsorption was developed, which makes it possible to describe the formation of complexes of a particular kind, "bridges" that connect adjacent nucleic acid molecules fixed in a liquid crystal. The bridges represent chelate complexes, which incorporate the molecules of the antibiotic daunomycin and copper ions. Equations describing the dependence of the concentration of these bridges in solution on the concentration of their constituents were derived. The family of dependences of experimental amplitudes of bands in CD spectra typical of "bridge" structures on the concentration of copper ions represents a set of S-shaped curves, and, as the concentration of daunomycin in solution increases, the level of saturation of these curves increases. The analysis of experimental data with the use of this model suggests that the structures of this type compete with daunomycin molecules for the binding sites on poly(I).poly(C). By using this model, the energies of formation of bridge structures were calculated.     

Protonated polynucleotides. VII. Thermal transitions between different complexes of polyinosinic acid and polycytidylic acid in an acid medium

The interaction between poly (I) and poly (C) in acid medium has been studied by potentiometric titration, mixing curves and thermal denaturation. Phase diagramms as a function of ionic strength, pH, and temperature have been established. From these data it is shown that the acid titration of the complex poly (I) · poly (C) passes through a triple-stranded intermediate poly (I) · poly (C) · poly (C⁺) to yield finally the protonated double-helical complex poly (I) · poly (C⁺). The mixing curves indicate the sole presence of the three-stranded complex in the intermediate zone. On the basis of the pK's the coexistence between the three-stranded complex with the neighboring double-stranded structure is demonstrated in a narrow rang of pH and ionic strength. The geometry of the base arrangements, their conformation and the sense of the strands are discussed in the light of the data presented. A Hoogsteen-type pairing between the bases for poly (I) · poly (C⁺) is favored, although the reverse Hoogsteen pair cannot be excluded.     

Interaction of metal ions with polynucleotides and related compounds. X. Studies on the reaction of silver (I) with the nucleosides and polynucleotides, and the effect of silver(I) on the zinc(II) degradation of polynucleotides

Potentiometric titration curves of the silver(I) complexes of cytidine, adenosine, and uridine show little uptake of base below pH 7, unlike the curve for silver(I)-guanosine, which shows extensive base uptake at neutral pH. This observation is correlated with spectrophotometric data showing little difference between the silver complex spectra of adenosine, cytidine, and uridine and the spectra of uncomplexed nucleosides, except at high pH, but showing a great difference between the silver complex spectra of guanosine and inosine and the corresponding uncomplexed nucleosides even at pH 6. Similar comparisons of the silver complexes of poly A, poly C, poly I, and poly U, both by potentiometric titration and by spectrophotometry, show that poly I behaves like guanosine and inosine as expected, differing from poly A and poly C. However, poly U behaves like poly I and thus does not resemble uridine in its complexing behavior. There is thus a dichotomy between poly A and poly C on the one hand in silver complexing phenomena, compared with poly U and poly I on the other. When silver(I) is added to systems containing zinc(II) and various polynucleotides, the same dichotomy is noted. Silver(I) inhibits the degradation by zinc(II) of all four polynucleotides, but the degradation of poly I and poly U is prevented virtually completely. Silver(I) alone has no degradative effect on RNA and inhibits, the zinc(II) degradation of RNA. Polynucleotide complexes in which silver(I) and zinc(II) are simultaneously bound to different positions on the macromolecules are postulated as intermediates in the inhibited degradation reactions.     

Vibrational dynamics and heat capacity of polyglycine I

Earlier works on polyglycine I suffer from several infirmities, such as the dynamic methylene group being replaced by a mass unit and the use of poorly resolved inelastic neutron spectra, which have resulted in wrong assignments and imprecise profile of dispersion curves. In addition, the density-of-states and heat capacity variation as a function of temperature are being reported for the first time. The heat capacity is in good agreement with the measurements reported earlier by Roles and Wunderlich within a certain range (230-350 K). Deviations set in beyond this could be due to the presence of two crystalline states (I and II) in the sample used for the heat capacity measurements.     

8-Azido double-stranded RNA photoaffinity probes. Enzymatic synthesis, characterization, and biological properties of poly(I,8-azidoI).poly(C) and poly(I,8-azidoI).poly(C12U) with 2',5'-oligoadenylate synthetase and protein kinase

The technique of photoaffinity labeling has been applied to the double-stranded RNA (dsRNA)-dependent enzyme 2',5'-oligoadenylate (2-5A) synthetase to provide a means for the examination of RNA-protein interaction(s) in the dsRNA allosteric binding domain of this enzyme. The synthesis, characterization, and biological properties of the photoaffinity probe poly[( 32P]I,8-azidoI).poly(C) and its mismatched analog poly[( 32P]I,8-azidoI).poly(C12U), which mimic the parent molecules poly(I).poly(C) and poly(I).poly(C12U), are described. The efficacy of poly[( 32P]I,8-azidoI).poly(C) and poly[( 32P]I,8-azidoI).poly(C12U) as allosteric site-directed activators is demonstrated using highly purified 2-5A synthetase from rabbit reticulocyte lysates and from extracts of interferon-treated HeLa cells. The dsRNA photoprobes activate these two 2-5A synthetases. Saturation of 2-5A synthetase is observed at 6 x 10(-4) g/ml poly[( 32P]I,8-azidoI).poly(C) following photolysis for 20 s at 0 degrees C. The photoincorporation of poly[( 32P]I,8-azidoI).poly(C) is specific, as demonstrated by the prevention of photoincorporation by native poly(I).poly(C). DNA, poly(I), and poly(C) are not competitors of poly[( 32P]I,8-azidoI).poly(C). Following UV irradiation of 2-5A synthetase with poly[( 32P]I,8-azidoI).poly(C), the reaction mixture is treated with micrococcal nuclease to hydrolyze azido dsRNA that is not cross-linked to the enzyme. A radioactive band of 110 kDa (the same as that reported for native rabbit reticulocyte lysate 2-5A synthetase) is observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The specific photolabeling of the 2-5A synthetase suggests that the azido dsRNA is intrinsic to the allosteric binding domain. The utility of poly[( 32P]I,8-azidoI).poly(C) for the detection of dsRNA-dependent binding proteins and the isolation of peptides at or near the allosteric binding site is discussed.     

Optical rotatory dispersion and circular dichroism of silver(I):Polyribonucleotide complexes

Optical rotatory dispersion (ORD) and circular dichroism (CD) spectra of single- and multistranded polyribonucleotides undergo extensive changes on binding of the silver ion. These changes are consistent with the proposition that Ag(I) binds to the heterocyclic bases and not to the phosphate groups of polynucleotides. ORD and CD of silver complexes of poly(A)·poly(U) and double-helical rice dwarf viral RNA display negative Cotton effects when there is more than one Ag(I) per two nucleotide residues in solution. These observations suggest a significant distortion of the double-helical conformation as a result of Ag(I) binding. Silver(I) binding sites of pyrimidine polynucleotides are apparently saturated when there is one Ag(I) per two nucleotide residues and those of purine polynucleotides at one Ag(I) per nucleotide in solution. These data are consistent with the supposition that some Ag(I) binding sites exist on the pyrimidine ring and additional sites on the imidazole ring of polynucleotides. The sedimentation coefficient of poly(A) increases by severalfold when one Ag(I) is present per nucleotide residue. Silver(I) may introduce intra- and interstrand cross-links (through bidentate chelates) in single-stranded polynucleotides, resulting in structures with high sedimentation coefficients. Among the polynucleotides studied, poly(U) was an exception. Silver(I) did not affect the optical properties (absorbance, ORD, and CD) of poly(U) at neutral pH.     

Metal-polynucleotide interactions. A comparison of carcinogenic and non-carcinogenic metals in vitro.

The effects of divalent cations on mixing curves of synthetic polyribonucleotides in solution are described. Manganese and cadmium chlorides at 10(-3) M induce changes suggestive of base mispairing. MnCl2 induces mispairing in complexes formed between both poly(I) and poly(C,U) and poly(I) and poly(C,A) while CdCl2 affects base pairing between poly(I) and poly(C,U) only. By contrast, the chlorides of magnesium and zinc show no mispairing effects with either polymer pair. Manganese and cadmium are both reported carcinogens in animals while magnesium and zinc are not. The possibility that direct metal-nucleic acid interaction may be involved in metal carcinogenesis is discussed.     

An improved method for purifying 2',5'-oligoadenylate synthetases

We describe a new, rapid, and convenient procedure for purifying 2',5'-oligoadenylate synthetases, employing precipitation with ammonium sulfate, fractionation by gel filtration, rapid binding to poly(I) X poly(C) cellulose, and elution with 0.35 M KCl. Unlike previously published methods, the procedure does not require sedimentation of the enzyme at 200,000 X g. Therefore, it is more general and more likely to succeed with synthetases extracted from a variety of cells or tissues, or from different subcellular fractions. We have purified the enzymes from two sources to apparent homogeneity, about 2500-fold from the cytoplasm of HeLa cells in 40% yield and more than 400,000-fold from the cytoplasm of rabbit reticulocytes in 25% yield. The specific activity of the HeLa enzyme is about 4 times higher than reported previously. The physical and functional properties of the pure enzymes are very similar to those reported by others for preparations of 2',5'-oligoadenylate synthetase from rabbit reticulocytes, mouse L cells, and human HeLa cells. A new affinity matrix was prepared by linking periodate-oxidized poly(I) X poly(C) to a hydrazide derivative of finely divided cellulose. Poly(I) X poly(C) cellulose binds about twice as much synthetase as the corresponding amount of poly(I) X poly(C) paper and activates the bound enzyme about three times better.     

2 types of temperature transitions in liquid crystals formed from poly(I).poly(C) molecules

The small-angle X-ray scattering curves, CD spectra and textures of the liquid-crystalline phase formed from poly(I).poly(C) molecules in a water-salt solutions containing poly(ethylene glycol) at different temperatures were obtained. It was found that the heating of poly(1).poly(C) liquid-crystalline phase is accompanied by two types of transitions, the first one--a "cholesteric----"compensated" structure----cholesteric", the second--a "cholesteric----isotropic state" transition. The latter transition takes place at a temperature that corresponds to that of the separation of chains of the double-stranded poly(I).poly(C) molecule.     

Anticrossing Behavior of Surface Plasmon Polariton Dispersions in Metal-Insulator-Metal Structures

The anticrossing behavior of dispersion curves of the surface plasmon polaritons supported by metal-insulator-metal structures are studied experimentally and theoretically. Samples consisting of a poly(methyl methacrylate) layer sandwiched by Ag films are prepared and their angle- and wavelength-scan attenuated total reflection spectra are measured. From an analysis of the angle-scan spectrum, the coupled-mode nature of the surface plasmon polariton modes is suggested. The dispersion relations obtained from the wavelength-scan spectra exhibit clearly the anticrossing behavior that arises from the coupling of the modes. The experimental dispersion relations are in good agreement with theoretical ones.     

Induction of Mx protein by interferon and double-stranded RNA in salmonid cells

Mx protein is one of several antiviral proteins that are induced by the type I interferons (IFN), IFNalpha and beta, in mammals. In this work induction of a 76 kDa Mx protein by double-stranded RNA (dsRNA) or type I IFN-like activity in Atlantic salmon macrophages, Atlantic salmon fibroblast cells (AS cells) and in Chinook salmon embryo cells (CHSE-214) is reported. Type I IFN-like activity was produced by the stimulation of Atlantic salmon macrophages with the synthetic dsRNA polyinosinic polycytidylic acid (poly I:C). A correlation appeared to exist between Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) induced by IFN in CHSE-214 cells. Several observations in the present work suggest that, as in mammals, the induction of Mx protein by dsRNA in fish cells primarily occurs via induction of type I IFN. First, type I IFN-like activity but not poly I:C, induced Mx protein expression in CHSE-214 cells. These cells apparently lack the ability to produce IFN in response to poly I:C. Second, the putative IFN induced maximal Mx protein expression 48 h earlier than poly I:C in AS cells. Third, the peak expression of Mx protein in macrophages induced by poly I:C occurred after 48 h whereas peak in IFN-like activity was observed by 24 h after addition of poly I:C. The present work supports the notion of using Mx protein as a molecular marker for the production of putative type I IFN in fish.     

Temperature dependence of the hydrogen ion equilibria in poly(riboadenylic acid)

Hydrogen ion titration curves have been obtained for poly (riboadenylic acid) (poly A) at temperatures of 0–40°C, and ionic strengths of 0.001, 0.01, and 0.15. Where comparable, the data are in general agreement with those previously reported by other investigators. Correlations between the titration data and thermal denaturation curves have been obtained. Formation of a two-stranded helix is catalyzed by the uptake of protons by the adenine base. Partial protonation of the base is required for formation of the two-stranded helix, but under appropriate conditions it is stable at degrees of ionization less than 0.2. The degree of ionization required for formation of the two-stranded helix increases with temperature and decreases with ionic strength.     

Epstein-Barr Virus-Specific RNA III. Mapping of DNA Encoding Viral RNA in Restringent Infection

Namalwa and Raji cells, originally obtained from a Burkitt tumor biopsy, grow as continuous cell lines in vitro and contain the Epstein-Barr virus (EBV)-related nuclear antigen EBNA (B. M. Reedman and G. Klein, Int. J. Cancer 11:499-520, 1973) and RNA homologous to at least 17 and 30% of the EBV genome, respectively (S. D. Hayward and E. Kieff, J. Virol. 18:518-525, 1976; T. Orellana and E. Kieff, J. Virol. 22:321-330, 1977). The polyribosomal and polyadenylated [poly(A)+] RNA fractions of Namalwa and Raji cells are enriched for a class of viral RNA homologous to 5 to 7% of EBV DNA (Hayward and Kieff, J. Virol. 18:518-525, 1976; Orellana and Kieff, J. Virol. 22:321-330, 1977). The objective of the experiments described in this communication was to determine the location within the map of the EBV genome (D. Given and E. Kieff, J. Virol. 28:524-542, 1978) of the DNA which encodes the viral RNA in the poly(A)+ and non-polyadenylated [poly(A)-] RNA fractions of Namalwa cells. Hybridization of labeled DNA homologous to Namalwa poly(A)+ or poly(A)- RNA to blots containing EcoRI, Hsu I, or Hsu I/EcoRI double-cut fragments of EBV (B95-8) or (W91) DNA indicated that these RNAs are encoded by DNA contained primarily in the Hsu I A/EcoRI A and Hsu I B/EcoRI A fragments and, to a lesser extent, in other fragments of the EBV genome. Hybridizations of Namalwa poly(A)+ and poly(A)- RNA in solution to denatured labeled EcoRI A or B fragments, Hsu I A, B, or D fragments, and Hsu I A/EcoRI A or Bam I S fragments and of Raji polyribosomal poly(A)+ RNA to the EcoRI A fragment indicated that (i) Namalwa poly(A)+ RNA is encoded primarily by 6 x 10(5) daltons of a 2 x 10(6)-dalton segment of DNA, Bam I S, which is tandemly reiterated, approximately 10 times, in the Hsu I A/EcoRI A fragment and is encoded to a lesser extent by DNA in the Hsu I B, EcoRI B, and Hsu I D fragments. Raji polyribosomal poly(A)+ RNA is encoded by a similar fraction of the EcoRI A fragment as that which encodes Namalwa poly(A)+ RNA. (ii) The fraction of the Bam I S fragment homologous to Namalwa poly(A)- RNA is similar to the fraction homologous to Namalwa poly(A)+ RNA. However, Namalwa poly(A)- RNA is homologous to a larger fraction of the DNA in the Hsu I B, Hsu I D, and EcoRI B fragments.     

Polynucleotides. LVI. Synthesis and properties of poly(2-deoxy-2''-fluoroinosinic acid).

Poly (2'-deoxy-2'-fluoroinosinic acid) [ poly(If)] was synthesized by polymerization of 2'-deoxy-2'-fluoroinosine 5'-diphosphate catalyzed by Escherichia coli polynucleotide phosphorylase. Although the UV absorption properties of poly(If) closely resembled those of poly(I), thermal melting curves at Na+ concentrations of 0.15M and 0.75M suggested two ordered structures for poly(If) neutral form. CD psectra taken at 0.15M Na+ concentration showed rather larger amplitudes in both a peak at 273 nm and a trough at 246 nm, suggesting rather strong vertical stacking of bases. When complexed with poly(C), poly(If) forms a double-stranded complex, poly(If).poly(C) which has Tm's higher by 10-20 degrees than those of poly(If).poly(C) measured under the same conditions. The CD spectrum of this complex resembled that of poly(I).poly(C). The effect of the fluorine atom at the 2'-position on thermal stability of polynucleotides is discussed.     

A family of cold-regulated RNA-binding protein genes in the cyanobacterium Anabaena variabilis M3.

I previously found a cold-regulated RNA-binding protein gene rbpA (now named rbpA1) in Anabaena variabilis M3 [Sato, N. (1994) Plant Mol. Biol. 24, 819-823]. I show here that this gene is a member of a gene family containing at least eight members as evidenced by Southern blot and immunoblot analyses. I have isolated three additional genes (rbpB, rbpC and rbpD) in this family. Of these, rbpB was 100% identical to the rbpB gene of Anabaena 7120 reported previously. Another gene named rbpA in Anabaena 7120 was also found to exist in A.variabilis M3 with identical sequence and named rbpA2. The amino acid sequences of these gene products were highly conserved, except that the RbpD protein lacked glycine-rich C-terminal domain present in all other known members of the gene family. RNA blot and immunoblot analyses showed that the expression of rbpA1, rbpA2, rbpB, rbpC and rbpD, as well as uncloned rbp genes was regulated by cold, though the exact time-course and extent of response to cold were different among these genes. Gel-filtration assay showed that all of the Rbp proteins have higher affinities to poly(G) and poly(U) than to poly(A) and poly(C).     

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.     

The dipolar origin of protein relaxation

1. A set of parameters is proposed to check the interpretation of the dielectric behaviour of protein solutions as a rigid-dipole relaxation of prolate ellipsoids of revolution in the frequency range between 20 kHz and 10 MHz. Besides the delta(b)-function of Scheraga, another analogous function (delta(a)) is presented to establish size and shape of globular proteins. A study of the influence of solvent viscosity on the dielectric dispersion also gives strong evidence in favour of rigid-dipole relaxation. 2. Measurements of the dielectric dispersion of monomer solutions of bovine serum albumin and transferrin are reported. Monomers of bovine serum albumin were obtained by fractionation on Sephadex G-150. Low-conductivity solutions of both proteins are obtained by passage through an ion-exchange resin. 3. Computer analysis of the experimental dispersion curves by use of a two-term Debye dispersion gives valuable information about transferrin and leads to an axial ratio 4.5 for a prolate ellipsoid of revolution. The dielectric increment of bovine serum albumin is very low and no conclusive results have yet been obtained.     

Poly(2-methylthio-7-deazainosinic acid)--hydrophobic stabilization of polynucleotide secondary structure by the 2-methylthio group.

Poly(2-methylthio-7-deazainosinic acid) [poly(ms2c7I)] was enzymatically synthesized by polymerization of 2-methylthio-7-deazainosine 5'-diphosphate with polynucleotide phosphorylase from Micrococcus luteus in high yield. The homopolymer shows much higher thermal stability than its parent polynucleotides poly(7-deazainosinic acid) [poly(c7I)] and poly(I). Its sigmoidal melting curve and pronounced hypochromicity imply a rigid, ordered structure. Poly(ms2c7I), like poly(2-methylthio-inosinic acid) [poly(ms2I)], does not form a complex with poly(C) because of the bulky 2-methylthio substituent. On the other hand, two poly(ms2c7I) strands form very rigid triple strands with poly(A). Different from poly(I) and poly(c7I) the homopolymer poly(ms2c7I) is very stable against cleavage by nuclease S1 and ribonuclease T2 as expected from its rigid secondary structure.