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PubMed1978

Further studies on the interaction between polynucleotides and antibodies to poly(inosinic acid). poly(cytidylic acid).

Interactions between antibodies to poly(inosinic acid).poly(cytidylic acid) [poly(I).poly(C)] (or Fab fragments) and double-stranded polynucleotides have been studied by several techniques. In quantitative precipitation tests, the amounts of precipitated antibodies depend upon salt concentration. This effect of salt is reversible. From the comparison of the association constants (deduced from fluorescence measurements) for the binding of Fab fragments to several polynucleotides, a direct interaction between atoms or groups of the bases, which can be involved in hydrogen bonds, can be excluded. The complexes between poly(I).poly(C) and Fab fragments interact with ethidium bromide. The association constants for the binding of ethidium bromide to poly(I).poly(C) and to poly(I).poly(C)-Fab fragments complexes are about the same, but Fab fragments reduce the number of base pairs available to ethidium bromide binding. At saturation of poly(I).poly(C) by Fab fragments, the number of binding sites of ethidium bromide is only reduced by a factor of two. It is concluded that all the nucleotide residues covered by the Fab fragment binding site do not interact with the amino acid residues. Circular dichroism studies of the complexes between Fab fragments and polynucleotides are in favor of some conformational change of the polynucleotides.

Antibodies↗

PubMed1975

The effects of thioketo substitution upon uracil-adenine interactions in polyribonucleotides. Synthesis and properties of the alternating polynucleotides poly(r(A-s2U)) and poly(r(A-s2s4U)).

The polynucleotides poly[r(A-s-2U)] and poly]r(A-s2s4U)] have been synthesized and characterized by nearest-neighbour analysis, sedimentation analysis as well as spectroscopic techniques. Absorption-temperature profile and absorption-pH profile of poly[r(A-s-2U)] did not reveal a structural transition between 10 and 95 degrees C even at low ionic strength, although a variety of properties indicated a helical structure of poly[r(A-s-2U)]: remarkable hyperchromicity of the absorption spectrum, circular dichroic spectrum displaying extrema of large amplitudes, resistance against hydrolysis by ribonuclease and interaction with ethidium bromide in a manner which is characteristic of helical polynucleotides. Our results show that interactions of the type A-s-2U and A-s-2s-4U do in fact exist in helical polynucleotides. The properties of poly]r(As-2U)] furthermore demonstrate the general stabilizing effect of 2-thioketopyrimidine bases in helical polynucleotides by virtue of vertical stacking interactions with neighbouring pyrimiding and purine bases.

Circular Dichroism↗

PubMed1977

Isolation and characterization of a polynucleotide phosphorylase from Bacillus amyloliquefaciens.

Bacillus amyloliquefaciens BaM-2 produces large amounts of extracellular enzymes, and the synthesis of these proteins appears to be dependent upon abnormal ribonucleic acid metabolism. A polynucleotide phosphorylase (nucleoside diphosphate:polynucleotide nucleotidyl transferase) was identified, purified, and characterized from this strain. The purification scheme involved cell disruption, phase partitioning, differential (NH4)2SO4 solubilities, agarose gel filtration, and diethylaminoethyl-Sephadex chromatography. The purified enzyme demonstrated the reactions characteristic of polynucleotide phosphorylase: polymerization, phosphorolysis, and inorganic phosphate exchange with the beta-phosphate of a nucleotide diphosphate. The enzyme was apparently primer independent and required a divalent cation. The reactions for the synthesis of the homopolyribonucleotides, (A)n and (G)n, were optimized with respect to pH and divalent cation concentration. The enzyme is sensitive to inhibition by phosphate ion and heparin and is partially inhibited by rifamycin SV and synthetic polynucleotides.

Adenosine Diphosphate↗

PubMedDate not supplied

[Interaction of gene 5 protein of phage f1 with single and double-stranded DNA and polynucleotides].

The fluorescence method was used to reveal some differences in the interaction of gene 5 protein of phage f1 with single- and double-stranded polynucleotides (DNA). The binding with the duplexes is non-cooperative and the Kapp is twice lower than that for the cooperative formation of the complex with single-stranded structures. In the complex with a double-stranded polynucleotide (DNA) the protein cover 3 nucleotide pairs. The complex dissociates with a lower concentration of salt and the contribution of the energy of nonelectrostatic interactions to the total energy of complex formation for it is lower than for the complex with single-stranded DNA. In the complex of protein with single-stranded structure the fluorescence of the tyrosine (Tyr) residues is quenched to a greater degree and their accessibility to the external quencher is lower than that of the complex with double-stranded polynucleotides (DNA). The suggestion is made that in destabilization of nucleic double helices by gene 5 protein of phage f1, a great role belongs to Tyr residues because of their high affinity to single-stranded structures and because of their different localization in the complexes with single- and double-stranded polynucleotides.

Coliphages↗

PubMed1978

Purification and properties of polynucleotide kinase of calf thymus.

Polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) has been purified approx. 1500-fold from calf thymus. This enzyme phosphorylates 5'-hydroxyl termini in DNA using ATP as phosphate donor. RNA is phosphorylated at a much lower rate than DNA. The reaction requires the presence of a divalent cation, preferably Mg2+ or Mn2+ and is sensitive to sulfhydryl antagonists. The optimum pH for enzyme activity is 5.5. Enzyme activity is inhibited by low concentrations of inorganic sulfate and by some sulfate polymers. The kinase-catalyzed incorporation of the terminal phosphate of ATP into polynucleotides is inhibited by other nucleoside and deoxynucleoside triphosphates. The enzyme molecule has a molecular weight of about 70 000 and a Stokes radius of 4.3 nm. It has a frictional ratio of 1.44 indicating an asymmetrical structure. Calf thymus tissue should provide a useful alternative source for preparation of mammalian polynucleotide kinase.

Adenosine Triphosphate↗

PubMed1978

Prevention of G:C pairing in mouse DNA by complete blocking of guanine residues with glyoxal. Availability of cytosine, adenine and thymine for hydrogen bonding with added unmodified polynucleotides.

We have developed conditions for the reaction of single-stranded DNA with glyoxal which permit blocking of essentially all guanine residues. This procedure effectively prevents base pairing involving these guanine residues, yet permits cytosine, thymine and adenine residues in the DNA to pair with added polynucleotides. Modification of DNA with glyoxal greatly reduces intrastrand helical regions, resulting in a very low binding to hydroxyapatite, as compared to unmodified DNA. Annealing of modified DNA with some synthetic polynucleotides of restricted composition (but not others) leads to a significant increase in binding, presumably because mouse DNA has sequences not containing guanine which are capable of hydrogen bonding to the added polynucleotides. This relatively simple procedure may allow isolation and further study of these guanine-free sequences in DNA.

Adenine↗

PubMed1977

Escherichia coli ribosomal protein S1 has two polynucleotide binding sites.

The interaction of Escherichia coli ribosomal protein S1 with a variety of RNA and DNA oligomers and polymers has been studied, using both a sedimentation technique and the quenching of intrinsic protein fluorescence upon nucleic acid binding to obtain equilibrium binding parameters. Two polynucleotide binding sites have been detected on S1: site I binds either single-stranded DNA or RNA and does not discriminate between adenine- and cytidine-containing polynucleotides, while the II binding is highly specific for RNA over DNA and shows a marked preference for cytidine polynucleotides over the corresponding adenine-containing species. On the basis of the binding properties of S1 to denatured DNA cellulose and poly(rC)-cellulose, it is demonstrated that every S1 molecule carries both a site I and a site II. Some possible implications of these results for mechanisms of protein synthesis and phage Qbeta replication are briefly considered.

Bacterial Proteins↗

PubMed1978

Polynucleotide kinase from a T4 mutant which lacks the 3' phosphatase activity.

Polynucleotide kinase from E. coli infected with the PseT 1 mutant of bacteriophage T4 has been isolated. The PseT 1 enzyme purifies similarly to normal polynucleotide kinase and effectively transfers the gamma phosphate of ATP to the 5' terminal hydroxyl of DNA and RNA. The PseT 1 and normal enzymes require similar magnesium ion concentrations, have the same pH optima and are both inhibited by inorganic phosphate. However, the PseT 1 enzyme is totally lacking the 3' phosphatase activity associated with normal polynucleotide kinase. The PseT 1 enzyme is a useful tool for the preparation of oligonucleotides with 3' and 5' terminal phosphates for use as susbstrates for RNA ligase.

Coliphages↗

PubMedDate not supplied

[Effect of synthetic polynucleotides and RNA on poly(C)-dependent poly(G) polymerase activity of Q beta replicase].

The effect of synthetic polynucleotides and phage RNA on poly(C)-dependent synthesis of poly (G) by Qbeta replicase is studied. It is shown that single stranded poly(U) and poly (dT) are strong inhibitors whereas structured polynucleotides poly(A), MS2 RNA as well as double stranded complexes poly(A)-poly(U) and poly(A)-poly(dT) do not affect the synthesis of poly(G). It is suggested that contact region of template with enzyme has single stranded unhelical structure and affinity of polynucleotides to Qbeta replicase is determined by degree of their secondary structure.

Kinetics↗

PubMed1975

The adjuvant activity of mycobacterial RNA preparations and synthetic polynucleotides for induction of delayed hypersensitivity to purified protein derivative in guinea pigs.

The adjuvant activity of mycobacterial RNA and synthetic polynucleotides for the induction of delayed hypersensitivity to PPD was determined. It was shown that when mycobacterial RNA or synthetic polynucleotides are injected together with purified protein derivative (PPD), delayed hypersensitivity to PPD developed as compared to no detectable delayed response when PPD was administered alone without adjuvant into guinea pigs. Four different criteria were employed to detect delayed hypersensitivity responses. These were the time, appearance, and magnitude of dermal reactions, histologic examination of dermal sections, passive transfer of sensitivity with sensitized spleen cells and the elaboration of migration inhibitory factor (MIF) by sensitized spleen cells. When synthetic polynucleotides were used as adjuvants and were injected into guinea pigs in combination with PPD, dermal reactions as well ad MIF assays gave evidence that these animals exhibited delayed-type hypersensitivity. Poly U alone exhibited adjuvant activity for induction of delayed hypersensitivity to PPD. Trypsin and pronase treatment did not affect the adjuvant activity of mycobacterial RNA whereas KOH treatment completely abolished any adjuvant effect, suggesting that ribosomal protein did not contribute to the adjuvant characteristics of mycobacterial RNA. Titration experiments indicated that the adjuvant activity of mycobacterial RNA was greater than that of poly A:U.

Adjuvants, Immunologic↗

PubMedDate not supplied

[Calculation of the conformation of stereo-regular, double-spiral polynucleotides].

By the step-by-step selection method energetically optimal conformation of double-stranded polynucleotides were calculated proceeding from the conformations of mono-and dinucleotides. These structures were found to belong to the families of DNA A and B forms. The dependence of six torsion angles of the polynucleotide chain on the glycosidic angle chi was investigated. As a result certain areas admitting of double-helix polynucleotides with sugar C3-endo with chi = -80 degrees were established.

DNA, Single-Stranded↗

PubMed1978

Use of the lipophilic tert-butyldiphenylsilyl protecting group in synthesis and rapid separation of polynucleotides.

Butyldiphenylchlorosilanes react with deoxy- and N-protected deoxymononucleotides to give, in each case, the 3'-O-butyldiphenylsilyl ether in high yield. A comparative study of n-, sec-, and tert-butyldiphenylchlorosilanes showed, as expected, a wide variation in the rate of formation and in the stability of the 3'-O-silyl ethers, the tert-butylphenylsilyl group being the one of choice in polynucleotide synthesis. The silyl group can be readily removed on treatment with fluoride ion in buffered pyridine at room temperature. This property markedly increases flexibility of the current methodology for polynucleotide synthesis. Secondly, because of its high lipophilicity, the silyl group allows rapid solvent extraction of synthetic intermediates, and thirdly, it greatly facilitates the isolation of the required product in synthetic reactions because of the selective and strong retention of the condensation product during reverse-phase high-pressure liquid chromatography. Several examples of synthetic procedures, including a stepwise synthesis of an undecanucleotide, are given which demonstrate overall simplification of polynucleotide synthesis.

Chemical Phenomena↗

PubMed1976

On the association of reverse transcriptase with polynucleotide templates during catalysis.

The association of avian myeloblastosis virus (AMV) DNA polymerase with polynucleotide templates during catalysis has been studied. During the course of polymerization, different template-primer complexes were added and the ability of the enzyme to switch from one polynucleotide template to another was determined. At 37 degrees C as well as at 4 degrees C, the polymerase is able to switch from certain template-primer complexes to others. For example, the addition of poly(A)-oligo(dT) during the course of synthesis with poly(C)-oligo(dG) results in the immediate cessation of dGMP polymerization and the start of dTMP polymerization without any lag. Early during the course of polymerization, the size of the product, as determined by alkaline sucrose gradient centrifugation, is, in part, a function of the ratio of the template-primer complex to the enzyme. These cumulative experiments indicate that catalysis on polynucleotide templates with avian myeloblastosis virus DNA polymerase under the conditions tested is not processive in a classical sense. Similar to cellular DNA polymerases the enzyme can shift from one template-primer to another. Using autoradiography after gel electrophoresis to estimate the product size, it can be calculated that the enzyme switches from one template to another within 0.25 min at 37 degrees C which corresponds to the incorporation of greater than 25 nucleotides. At 4 degrees C, switching can be calculated to occur in less than three nucleotide addition steps. Thus, with certain homopolymers, conditions can be found by which AMV DNA polymerase can switch from one template-primer complex to another, perhaps after each nucleotide addition step.

Avian Leukosis Virus↗

PubMed1979

Polynucleotides. LVII. Synthesis and properties of poly (2'-chloro-2'-deoxyinosinic acid).

Poly (2'-chloro-2'-deoxyinosinic acid) [poly(Icl)] was synthesized from Icl 5'-DP by polymerization with polynucleotide phosphorylase. UV absorption properties of poly(Icl) are very similar to those of poly(I). Poly(Icl) adopted a multi-stranded ordered form in the presence of 0.95M Na ion. The Tm value of this form was 36 degrees, which resembles that of poly(I) quadruple-stranded form at high salt. CD spectra also suggested presence of these two forms. Upon mixing with poly(C), poly-(Icl) forms a double-stranded 1 : 1 complex, which had very similar Tm-log[Na+] relationship to that of poly(I) . poly(C). Thus it was concluded that the chlorine substitution at 2'-position of the polynucleotide had the similar effect to OH on physical properties of polynucleotides.

Circular Dichroism↗

PubMed1977

Thermophilic polynucleotide phosphorylase from Thermus thermophilus. Purification and properties of an altered form of enzyme which lacks phosphorolytic activity to polynycleotide.

A thermophilic polynucleotide phosphorylase lacking polynucleotide phosphoryltic activity was purified from Thermus thermophilus HB-8 strain. The enzyme is an altered form of the native polynucleotide phosphorylase, probably attacked by the proteinase(s) of this extreme thermophile during the purification process. This modified enzyme lacks phosphorolytic activity to poly(A) while retaining weak activity to phosphorolyse tetranucleotides or hexanucleotides. The purified enzyme was shown to be homogenous by electrophoretic analysis in polyacrylamide gel. This enzyme had a molecular weight of 190 000 as calculated both from electrophoresis on polyacrylamide gel and from the Stoke's radius derived from the gel filtration pattern and the sedimentation coefficient. The enzyme was separated into three polypeptide chains by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate; their molecular weights were calculated to be 92000, 73000 and 35000. The enzyme was thermophilic and thermotolerant, exhibiting its maximal activity at 70 degrees C. The four ribonucleoside diphosphates (ADP, GDP, UDP and CDP) were polymerized to the extent of 7-S size.

Drug Stability↗

PubMed1977

Interferon induction by polynucleotides: structure-function relationship.

In view of recent developments, the structural determinants of the interferon inducing activity of polynucleotides have been (re)evaluated. To induce interferon, the polynucleotide should be sufficiently large and double-stranded, although not necessarily double-stranded over its whole length. It should be sufficiently stable to both thermal denaturation and hydrolysis by nucleases. It should also contain a particular steric conformation. This conformation is most regularly ensured by the presence of 2'-hydroxyl in the ribose moieties and intact purine-pyrimidine base pairs in the interior of the double helix. Other biologic activities of polynucleotides, such as anti-complement activity and inhibition of reverse transcriptase (RNA-directed DNA polymerase) activity, depend on structural requirements which are rather antagonistic to those governing the interferon response.

Hot Temperature↗

PubMed1976

Phosphorylation of double-stranded DNAs by T4 polynucleotide kinase.

The phosphorylation by T4 polynucleotide kinase of various double-stranded DNAs containing defined 5'-hydroxyl end group structures has been studied. Particular emphasis was placed on finding conditions that allow complete phosphorylation. The DNAs employed were homodeoxyoligonucleotides annealed on the corresponding homopolymers, DNA duplexes corresponding to parts of the genes for alanine yeast tRNA, and a suppressor tyrosine tRNA from Escherichia coli. The rate of phosphoylation of DNAs with 5'-hydroxyl groups in gaps was approximately ten times slower than for the corresponding single-stranded DNA. At low concentrations of ATP, 1 muM, incomplete phosphorylation was obtained, whereas with higher concentrations of ATP, 30 muM, complete phosphorylation was achieved. In the case of DNAs with 5'-hydroxyl groups at nicks approximately 30% phosphorylation could be detected using 30 muM ATP. A DNA containing protruding 5'-hydroxyl group ends was phosphorylated to completion using the same conditions as for single-stranded DNA, i.e., a ratio between the concentrations of ATP and 5'-hydroxyl groups of 5:1 and a concentration of ATP of approximately 1 muM. For a number of DNAs containing protruding 3'-hydroxyl group ends and one DNA containing even ends incomplete phosphorylation was found under similar conditions. For all these DNAs a plateau level was observed varying from 20 to 45% of complete phosphorylation. At 20 muM and higher ATP concentrations, the phosphorylation was complete also for these DNAs. With low concentrations of ATP a rapid production of inorganic phosphate was noted for all the latter DNAs. The apparent equilibrium constants for the forward and reverse reaction were determined for a number of different DNAs, and these data revealed that the plateau levels of phosphorylation obtained at low concentrations of ATP for DNAs with protruding 3'-hydroxyl group and even ends is not a true equilibrium resulting from the forward and reverse reaction. It is suggested that the plateau levels are due to formation of inactive enzyme-substrate and enzyme-product complexes. For all double-stranded DNAs tested, except DNAs containing protruding 5'-hydroxyl group ends, addition of KCl to the reaction mixture resulted in a drastic decrease in the rate of phosphorylation, as well as in the maximum level phosphorylated. Spermine, on the other hand, had little influence. Both of these agents have previously been shown to activate T4 polynucleotide kinase using single-stranded DNAs as substrates (Lillehaug, J.R., and Kleppe, K. (1975), Biochemistry 14, 1221). The inhibition of phosphorylation of double-stranded DNAs by salt might be the result of stabilization of the 5'-hydroxyl group regions of these DNAs.

Adenosine Triphosphate↗

PubMed1978

A rapid purification of T4 polynucleotide kinase using Blue Dextran-Sepharose chromatography.

A rapid batch procedure is described for purification of T4 polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) to near homogeneity using Blue Dextran-Sepharose chromatography. The enzyme preparation is sufficiently free of contaminating endonuclease and alkaline phosphatase activities to be suitable for radioactively labeling nucleic acids in vitro. Kinetic measurements indicate that the chromophore of Blue Dextran, Cibacron Blue F3GA, inhibits the activity of T4 polynucleotide kinase competitively with respect to single stranded DNA substrate and non-competitively with respect to the rATP substrate.

Adenosine Triphosphate↗