Double-stranded Polynucleotides Research Articles

Antigenic structure of double-stranded RNA analogs having varying activity in interferon induction

Antibodies were induced by immunization of rabbits with methylated bovine serum albumin complexes of: poly(I).poly(BC), an effective interferon inducer; poly(c7A).poly(rT), a noninducer that can block induction by active poly(A).poly(rT); and poly(A).poly(Um), which has neither inducing nor blocking activity. Similar complexes of f2 phage RNA or tRNA did not induce anti-nucleic acid antibodies. Each anti-polynucleotide serum contained some antibodies specific for double-stranded structure. Antibodies were immunospecifically purified from precipitates made with each serum and homologous or cross-reacting double-stranded polynucleotides. The purified antibodies distinguished among varying helices bearing base or ribose modifications. Antipoly(I).poly(BC) specificity paralleled that of the interferon induction system. Anti-poly(A).poly(Um) specificity favored the 2'-modified polymers. Anti-poly(c7A).poly(rT) antibodies were the least discriminating. Cross-reaction results indicated that some antibodies reacted with determinants that included both sugar-phosphate backbones. In far antibody excess, antigen:antibody ratios in precipitating complexes reached a minimum of 7 to 12 base pairs per bivalent IgG molecule. Single antigenic determinants may span about 4 base pairs, with primary contact sites including the phosphate groups and the furanose.

An endodeoxyribonuclease of human KB cells. Purification and properties of the enzyme.

An endodeoxyribonuclease has been purified 750-fold from human KB cells. The purified endonuclease requires Mg2+ for maximum activity: Mn2+ was less than half as active and Ca2+ inhibited the reaction. The optimum pH is 8.8 in Tris-HCl and the optimum buffer concentration is 10 mM. KCl (and NaCl), --SH-reacting reagents, and tRNA strongly inhibit the reaction. An apparent molecular weight of 54,000 was determined by sedimentation in a glycerol gradient. The purified endonuclease cleaved native, double-stranded adenovirus 2 DNA, and the reaction proceeded stepwise during the initial stage of degradation by cleavage of the DNA substrate in half, then in half again, etc. At longer digestion times, single strand scissions were detected. RNA was not a substrate for the enzyme. Poly(dG) . poly(dC) was susceptible but poly(dA) . poly(dT) was resistant to degradation. Hydrolysis of adenovirus 2 DNA yielded double-stranded polynucleotides containing 5'-phosphoryl and 3'-hydroxyl termini with short, single-stranded regions presumably at the ends. More than 50% of the product of a limit digest had a chain length greater than 35 to 40 nucleotides. Analysis of the 5' and 3' end groups of the digestion products indicated a preference for the site of the enzymatic cleavage; thymidylic acid residues were present at the 5' end and deoxyguanosine residues at the 3' end, each with a frequency of 40 to 50%.

Requirement for macrophages for interferon to be effective against encephalomyocarditis virus infection of mice

Suppression of macrophages in mice by treatments with silica or auro-thiomalate (Myocrisin) reduced production of serum interferon by polyriboinosinic acid:polyribocytidylic acid by 85 to 90%, indicating that this double-stranded polynucleotide caused interferon production primarily in macrophages. Suppression of macrophages in mice by silica or Myocrisin treatment did not significantly affect the susceptibility of mice to encephalomyocarditis virus, although at virus doses around 20 times the 50% lethal dose they died about 48 h earlier. Macrophage interferon protected mice from encephalomyocarditis virus infection at much lower doses than fibroblast interferon, and treatment of mice with silica or Myocrisin abolished the protection conferred by macrophage interferon, whereas these treatments had a much smaller effect on the protection afforded by fibroblast interferon. The requirement for macrophages for interferon to be effective in mice can explain why macrophage suppression can cause normally nonlethal viruses to kill adult mice.

Theoretical calculations of base-base interactions in nucleic acids: II. Stacking interactions in polynucleotides.

Base-base interactions were computed for single- and double stranded poly,ucleotides, for all possible base sequences. In each case, both right and left stacking arrangements are energetically possible. The preference of one over the other depends upon the base-sequence and the orientation of the bases with respect to helix-axis. Inverted stacking arrangement is also energetically possible for both single- and double-stranded polynucleotides. Finally, interacting energies of a regular duplex and the alternative structures were compared. It was found that the type II model is energetically more favourable than the rest.

The interaction of ethidium with synthetic double-stranded polynucleotides at low ionic strength.

The interaction of ethidium with synthetic DNA and RNA double-stranded polymers at 0.01 M ionic strength, pH 7.0, has been studied by fluorimetry at low drug to nucleotide ratios. Binding constants have been calculated assuming an excluded-neighbouring site model for the interaction of ethidium with double-stranded polymers. The values obtained are poly d(AT).poly d(AT), 9.5 X 10(6) M-1; poly dA.poly dT, 6.5 X 10(5) M-1; poly d(GC).poly d(GC), 9.9 X 10(6) M-1; poly dG,poly dC, 4.5 X 1-(6) M-1; poly d(AC); poly d(GT), 9.8 X 10(6) M-1; poly d(AG).poly d(CT), 1.3 X 10(6) M-1; poly rA.poly rU, 4.1 X 10(7) M-1. The displacement of ethidium from poly d(AT).poly d(AT) by 9-aminoacridine and an acridine-containing antitumor agent (NSC 156303; 4'-(9-acridinylamino)methanesulphon-m-anisidide) has also been examined.

Radiolysis of the Double-Stranded Polynucleotides Poly(A + U) and DNA in the Presence of Oxygen

MICHAELS, H. B., AND HUNT, J. W. Radiolysis of the Double-Stranded Polynucleotides Poly(A + U) and DNA in the Presence of Oxygen. Radiat. Res. 72, 32-47 (1977). The reaction of 02 with -OH adducts produced on poly(A + U) was studied by pulse radiolysis. In the presence of 0O, the absorption spectrum of the poly(A + U) - OH adduct decays to a new spectrum, with a rate constant of 5 X 107 M-1 sec-1. Solutions of poly(A + U) and DNA, in common with other polynucleotides, are sensitive to repetitive pulsing, and in the case of DNA, denaturation takes place and single-stranded regions and pieces are produced. The .OH adduct of native doublestranded DNA does not appear to react with O on a time scale from microseconds to seconds, yet steady-state radiolysis experiments and analysis for peroxides show that DNA hydroperoxides are produced at low doses. The *OH adduct of heat denatured, single-stranded DNA reacts with O as expected. The apparent lack of reaction of double-stranded DNA -OH adducts with 02 is hard to understand, since a reaction with the radiosensitizer NPPN is clearly observable under similar conditions. Possible explanations for the experimental results are discussed.

A boundary sedimentation velocity method for determining nonspecific nucleic acid-protein interaction binding parameters

An absolute method for measuring affinity constants and binding site sizes for the non-sequence-specific binding of protein to single- or double-stranded polynucleotide chains is described, based on the difference in the rate of sedimentation of free protein and the protein-nucleic acid complexes. In the form outlined, the technique can measure binding constants ranging from 10 3 to 10 6 m −1; methods for extending the range of measurable binding constants are also described. Data obtained for the noncooperative binding of ribonuclease to DNA are presented as fully worked-out examples of the method, and the extension of this approach to other noncooperative and cooperative protein-nucleic acid binding systems is discussed.

Depression of hepatic cytochrome P-450-dependent monooxygenase systems with administered interferon inducing agents

Cytochrome P-450-dependent monooxygenase activities and cytochrome P-450 levels were depressed in hepatic microsomes from rats treated with 12 interferon inducing agents of various types: small molecules (e.g. tilorone), an RNA virus (Mengo), a fungal mycophage (statolon), liver RNA, a synthetic double-stranded polynucleotide (poly rI · poly rC), a bacterial lipopolysaccharide ( E. coli endotoxin) and an attenuated bacteria ( B. pertussis vaccine). The results suggest that the depression of hepatic cytochrome P-450-dependent monooxygenase systems may be a general property of interferon inducing agents.

Induction of interferon by polyribonucleotides containing thiopyrimidines.

The influence of thioketo substitution in pyrimidine bases of double-stranded polynucleotides on interferon induction was investigated. The stabilizing effect of 2-thioketo substitution was reflected in the increased interferon inducing activity of poly(A-s2U) over that of poly(A-U). Poly(A-s2U) and poly(I)-poly(s2C) were as effective as poly(I)-Poly(C) in rabbit cells. Poly(I)-poly(C) and poly(I)-poly(s2C) were compared in several animal species. No differences in biological effects were observed in rabbits and dogs. In rodents, poly(I)-poly(s2C) was less effective and less toxic.Poly(I)-poly(s2C) was highly resistant against degradation by human serum. Further investigations seem to be justified to elucidate whether this property offers any advantages for the potential clinical utilization of poly(I)-poly(s2C).

Structure of the single-stranded polyribonucleotide polycytidylic acid

X-ray fibre diffraction studies have shown that at pH 7.0 polyribocytidylic acid (poly(C)) can adopt a single-stranded, right-handed, 6-fold helical structure with an axial rise per residue of 0.311 nm. The furanose rings have the C-3′-endo conformation. The other conformation angles of this structure are also remarkably similar to those observed in A -type double-stranded polynucleotide structures with the exception of θ[C-4′,z.sbnd;C-3′,z.sbnd;O-1,z.sbnd;P], which adopts a value very close to the monomer average. There is no hydrogen bonding between bases. The molecular structure appears to be stabilised primarily by base-stacking interactions and the crystal structure by hydrogen bonds between ribose hydroxyl groups.

A possible conformation for double-stranded polynucleotides.

A model is presented for double-stranded polynucleotides which involves side-by-side meshing of the two strands rather than double helical intertwining. The sugar-phosphate backbone has a twisted strip-like character, yet base-pairing of the Watson-Crick type is still possible. Structural features of the basic model are described and coordinates are presented for a representative example. The structure has, on the whole, reasonable sterochemical contacts, and can be shown to produce a fiber diffraction pattern with x-rays not unlike that of the B form of DNA.

A study of the interaction of TMV protein with single- and double-stranded polynucleotides

A study of the interaction of TMV protein with single- and double-stranded polynucleotides

A compact form of double-stranded RNA in solutions containing poly(ethyleneglycol).

Molecules of single-stranded ribosomal RNA and double-stranded replicative form of phage f2 RNA (dsRNA) adopt a compact form in solutions, containing sufficiently high concentrations of salt (NaCl) and polymer (PEG). However, only in the cases of native dsRNA molecules the compact particles are characterized by a regular internal structure, which accounts for the appearance of an intense positive band in CD spectra. Heating or acidification of PEG-containing solutions of dsRNA leads to the disappearance of the intense positive CD band, which results from the "destruction" of the regular internal structure of compact particles. Comparison of properties of DNA and dsRNA compact particles formed in PEG-containing water-salt solutions suggests the existence of similar mechanisms of compactization of double-stranded polynucleotides.

Effect of synthetic double-stranded polynucleotides on leukocyte migration

The effect of synthetic double-stranded polynucleotides (polyI:polyC) on migration of peripheral blood leukocytes from donors and patients with rheumatic fever was investigated. PolyI:polyC in large quantities was found not to inhibit migration of the donors' leukocytes, but it inhibited migration of leukocytes from patients with rheumatic fever, in whom antibodies against double-stranded RNA were found more often than in donors.

Hypochromism of polynucleotides in the nearest neighbour approximation

A method of calculating the hypochromism of polynucleotides in the nearest neighbour approximation is given by perturbation theory. The calculated hypochromism values of the first UV-absorption band of polynucleotides poly(dA)·poly(dT) and poly(dG)·poly(dC) are in good agreement with experiment. In this approximation the origin of the hypochromic effect is studied for the double-stranded polynucleotides; the dependence of the hypochromism upon the length of the polynucleotide is given.

Reduced Incidence of Spontaneous Mammary Tumors in C3H/He Mice After Treatment with Polyadenylate-Polyuridylate

The effect of treatment with the double-stranded polynucleotide complex polydenylate with polyuridylate [poly(A) with poly(U)] on tumor development in C3H/He mice was evaluated. Poly(A) with poly(U) was injected in newborn females, and mice were observed for 380 days. During this experimental period 42 percent of treated mice developed tumors, while the incidence in the control group was 63 percent. This difference was statistically significant.

Interferon Production and Resistance to Viral Infection Induced by Poly I· Poly C in Chick Embryo Cells. II. Lack of Enhancement by DEAE-Dextran

Japanese Journal of MicrobiologyVolume 19, Issue 1 p. 65-68 NoteFree Access Interferon Production and Resistance to Viral Infection Induced by Poly I· Poly C in Chick Embryo Cells. II. Lack of Enhancement by DEAE-Dextran Haruhiko Machida, Corresponding Author Haruhiko Machida Research Laboratory, Yamasa Shoyu Co., Ltd., Choshi, ChibaRequests for reprints should be addressed to Dr. Haruhiko Machida, Research Laboratory, Yamasa Shoyu Co., Ltd., Araoi 2–550, Choshi-shi, Chiba 288, Japan.Search for more papers by this authorAkira Kuninaka, Akira Kuninaka Research Laboratory, Yamasa Shoyu Co., Ltd., Choshi, ChibaSearch for more papers by this authorHiroshi Yoshino, Hiroshi Yoshino Research Laboratory, Yamasa Shoyu Co., Ltd., Choshi, ChibaSearch for more papers by this author Haruhiko Machida, Corresponding Author Haruhiko Machida Research Laboratory, Yamasa Shoyu Co., Ltd., Choshi, ChibaRequests for reprints should be addressed to Dr. Haruhiko Machida, Research Laboratory, Yamasa Shoyu Co., Ltd., Araoi 2–550, Choshi-shi, Chiba 288, Japan.Search for more papers by this authorAkira Kuninaka, Akira Kuninaka Research Laboratory, Yamasa Shoyu Co., Ltd., Choshi, ChibaSearch for more papers by this authorHiroshi Yoshino, Hiroshi Yoshino Research Laboratory, Yamasa Shoyu Co., Ltd., Choshi, ChibaSearch for more papers by this author First published: January 1975 https://doi.org/10.1111/j.1348-0421.1975.tb00848.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat REFERENCES 1Colby, C., and Chamberlin, M.J. 1969. The specificity of interferon induction in chick embryo cells by helical RNA. Proc. Nat. Acad. Sci. U.S. 63: 160– 167. 2Dianzani, F., Baron, S., Buckler, C.E., and Levy, H.B. 1971. Mechanisms of DEAE-dextran enhancement of polynucleotide induction of interferon. Proc. Soc. Exp. Biol. Med. 136: 1111– 1114. 3Dianzani, F., Cantagalli, P., Gagnoni, S., and Rita, G. 1968. Effect of DEAE-dextran on production of interferon induced by synthetic double-stranded RNA in L cell cultures. Proc. Soc. Exp. Biol. Med. 128: 708– 710. 4Dianzani, F., Gagnoni, S., and Cantagalli, P. 1970. Studies on the potentiating effect of DEAE-dextran on interferon production induced by double stranded synthetic polynucleotides. Ann. New York Acad. Sci. 173: 727– 735. 5Long, W.F. 1971. Effect of metabolic inhibitors on viral and polynucleotide induction of chick cell interferon. Microbios 4: 253– 259. 6Long, W.F., and Burke, D.C. 1971. Interferon production by double-stranded RNA: A comparison of induction by reovirus to that by a synthetic double-stranded polynucleotide. J. Gen. Virol. 12: 1– 11. 7Machida, H., Kuninaka, A., and Yoshino, H. 1974. Interferon production and resistance to viral infection induced by poly I· poly C in chick embryo cells. I. Effect of growth medium. Japan. J. Microbiol. 18: 427– 432. 8Nordlund, J.J., Wolff, S.M., and Levy, H.B. 1970. Inhibition of biologic activity of poly I: poly C by human plasma. Proc. Soc. Exp. Biol. Med. 133: 439– 444. 9Novokhatsky, A.S., and Ershov, F.I. 1972. The effect of diethylaminoethyl dextran on the antiviral activity of the complex of polyinosinic and polycytidylic acids in tissue culture. Vopr. Virusol. 17: 312– 317. 10Pitha, P.M., and Carter, W.A. 1971. The DEAE dextran : polyriboinosinate-polyribocytidylate complex: Physical properties and interferon induction. Virology 45: 777– 781. 11Stern, R. 1970. A nuclease from animal serum which hydrolyzes double-stranded RNA. Biochem. Biophys. Res. Commun. 41: 608– 614. 12Tilles, J.G. 1970. Diethylaminoethyl-dextran enhancement of interferon induction by a complexed polyribonucleotide. Proc. Soc. Exp. Biol. Med. 133: 1334– 1341. 13Vilček, J., Barmak, S.L., and Havell, E.A. 1972. Control of interferon synthesis: Effect of diethylaminoethyl-dextran on induction by polyinosinic-polycytidylic acid. J. Virol. 10: 614– 621. Citing Literature Volume19, Issue1January 1975Pages 65-68 ReferencesRelatedInformation

Relationship of Tm and S value of double-stranded polynucleotides prepared from various sizes of single-stranded homopolynucleotides.

Relationship of Tm and S value of double-stranded polynucleotides prepared from various sizes of single-stranded homopolynucleotides.

On the origin of the hypochromic effect in double-stranded polynucleotides

On the origin of the hypochromic effect in double-stranded polynucleotides

In vitro studies on mouse interferons induced with phage double-stranded RNA, synthetic double-stranded polynucleotides and viral inducers.

Highly purified and concentrated interferons obtained from L cells or from mouse peritoneal leukocytes (MPL) after induction with different inducers (Newcastle disease virus, double-stranded RNAs) were similar in their electrophoretic features. Incorporation of35S-labeled methionine during induction and production of interferons was similar in both cultures. However, differences were seen in electrophoretic profiles of interferons obtained from MPL cultures at different time intervals after induction.