Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “POLYNUCLEOTIDES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 379 records · Page 21Linked to original sources
PubMed1986

Enhanced expression of the bacterial chloramphenicol acetyltransferase gene in mouse cells cotransfected with synthetic polynucleotides able to form Z-DNA.

Recent studies have demonstrated that the left-handed, Z-DNA conformation is favored in polymers containing alternating purine/pyrimidine sequences that can exist in vivo and may play a role in gene expression. On the basis of this assumption, we have studied the effect of various cotransfected polynucleotides on the transient expression of the chloramphenicol acetyltransferase (CAT) gene in thymidine kinase-deficient murine L cells. Cotransfections were performed by calcium phosphate coprecipitation of CAT gene plasmids with various polymers, and the CAT enzymatic activity was measured in cell lysates after 48 hr. About 2- to 10-fold stimulation of CAT gene expression was observed when the cells were cotransfected with 10 micrograms (per 10-cm culture dish) of plasmid pSV2cat, which contains simian virus 40 (SV40) promoter and enhancer sequences, and 2-10 micrograms of polymers that can form Z-DNA, such as poly(dG-m5dC) X poly(dG-m5dC) or poly(dG-dC) X poly(dG-dC), as compared to transfection with pSV2cat alone. Further, enhanced CAT gene expression was also observed when cotransfections were performed with these polymers and two other plasmid vectors, one containing the SV40 promoter but no enhancer and the other lacking any SV40 regulatory sequences. However, poly(dA-dC) X poly(dG-dT), which can form Z-DNA, did not induce any stimulation. Similarly, no or very little stimulation was observed after cotransfection of pSV2cat with either poly(dG) X poly(dC) or poly(dA-dT) X poly(dA-dT), which do not adopt the Z conformation. These results suggest that certain polynucleotides may enhance transcription of the CAT gene.

Acetyltransferases↗
PubMed1996

Strandedness discrimination in peptide-polynucleotide complexes.

Preferential binding to single- or double-stranded nucleic acids is important for the activity of many proteins that process RNA and DNA. We have investigated the mechanism of strandedness discrimination with peptides derived from the putative DNA-binding domain of the RecA protein, a bacterial recombinase that modulates its affinity for single-stranded DNA by means of ATP binding and hydrolysis. Contributions of electrostatic and non-electrostatic interactions to binding of these peptides with polynucleotides were evaluated by fluorescence spectroscopy as a function of salt concentration and peptide charge. Binding of these peptides to single- and double-stranded nucleic acids was dominated by non-electrostatic interactions. Small electrostatic contributions selectively enhanced peptide complexation with single-stranded nucleic acids. Similar results were observed in control experiments carried out with tripeptides containing charged and aromatic amino acid residues. It was possible to modify the strandedness preference of peptide-polynucleotide complexes by changing electrostatic contributions to the binding free energy. These observations suggest a mechanism whereby some proteins that interact with DNA or RNA might determine and regulate their relative affinity for single- and double-stranded nucleic acids.

Amino Acid Sequence↗
PubMed1996

Nucleotides reveal polynucleotide phosphorylase activity from conventionally purified GroEL.

GroEL, as conventionally purified, can be incubated with nucleotides to produce high molecular weight material with an absorption maximum at 260 nm. This material is most clearly demonstrated when samples are subjected to gel filtration under conditions where GroEL is monomeric. There is a time-dependent increase in the high molecular weight material that occurs on incubation with ADP or, more slowly, with ATP. This material is generated during incubation, and none is present in the initial samples. Experiments with nucleases, proteases, radiolabeled nucleotides, and chemical cleavage reagents demonstrate that the high molecular weight material is polyadenylic acid whose formation is inhibited by phosphate. These results are consistent with the GroEL samples containing polynucleotide phosphorylase activity. Nondenaturing gels stained with acridine orange, after incubation in ADP, reveal that the activity producing the poly(A) coelectrophoreses with authentic polynucleotide phosphorylase. Conditions that remove the tryptophan-like fluorescence from preparations of GroEL also remove the PNPase activity. Thus, this activity is not associated with GroEL itself. The results are consistent with reports that GroEL can associate with RNase E and with other studies showing that RNase E and PNPase can form complexes. Thus, the present experiments support suggestions that GroEL can participate in multiprotein complexes that are involved in mRNA processing and degradation.

Adenosine Diphosphate↗
PubMed1983

New wrinkles on polynucleotide duplexes.

Most fibrous polynucleotides of general sequence exhibit secondary structures that are described adequately by regular helices with a repeated motif of only one nucleotide. Such helices exploit the fact that A:T, T:A, G:C, and C:G pairs are essentially isomorphous and have dyadically-related glycosylic bonds. Polynucleotides with regularly repeated base-sequences sometimes assume secondary structures with larger repeated motifs which reflect these base-sequences. The dinucleotide units of the Z-like forms of poly d(As4T):poly d(As4T), poly d(AC):poly d(GT) and poly d(GC):poly d(GC) are dramatic instances of this phenomenon. The wrinkled B and D forms of poly d(GC):poly d(GC) and poly d(AT):poly d(AT) are just as significant but more subtle examples. It is possible also to trap more exotic secondary structures in which the molecular asymmetric unit is even larger. There is, for example, a tetragonal form of poly d(AT):poly d(AT) which has unit cell dimensions a = b = 1.71nm, c = 7.40nm, gamma = 90 degrees. The c dimension corresponds to the pitch of a molecular helix which accommodates 24 successive nucleotide pairs arranged as a 4(3) helix of hexanucleotide duplexes. The great variety of nucleotide conformations which occur in these large asymmetric units has prompted us to describe them as pleiomeric, a term used in botany to describe whorls having more than the usual number of structures. Pleiomeric DNAs need not contain nucleotide conformations that are very different from one another. On the other hand, DNAs carrying nucleotides of very different conformation must be pleiomeric. This is because 4 nucleotides of different conformation are needed to join patches of secondary structure which are as different as A or B or Z. Differences in nucleotide structures may occur also between chains rather than within chains. In poly d(A):poly d(T), the purine nucleotides all contain C3'-endo furanose rings and the pyrimidine nucleotides C2'-endo rings. Analogous heteronomous structures may exist in DNA-RNA hybrids although these duplexes are also found to have symmetrical A-type conformations.

DNA↗
PubMed1983

A proposal for a specific double-helical structure in which the polynucleotide strands intercalate instead of forming base-pairs.

Double helices, since the discovery of the DNA structure by Watson and Crick, represent the single most important secondary structural form of nucleic acids. The secondary structures of a variety of polynucleotide helices have now been well characterised with hydrogen-bonded base-pairs as building blocks. We wish to propose here the possibility, in a specific case, of a double stranded helical structure without any base-pair, but having a repeat unit of two nucleotides with their bases stacked through intercalation. The proposal comes from the initial models we have built for poly(dC) using the stacking patterns found in the crystal structures of 5'-dCMPNa2 which crystallises in two forms depending on the degree of hydration. These structures have pairs of nucleotides with the cytosine rings partially overlapping and separated by 3.3A. Using these as repeat units one could generate a model for poly(dC) with parallel strands, having a turn angle of 30 degrees and a base separation of 6.6A along each strand. Both right and left handed models with these parameters can be built in a smooth fashion without any obviously unreasonable stereochemical contacts. The helix diameter is about 13.5A, much smaller than that of normal helices with base-pair repeats. The changes in the sugar-phosphate backbone conformation in the present models compared to normal duplexes only reflect the torsional flexibility available for extension of polynucleotide chains as manifested by the crystal structures of drug-inserted oligonucleotide complexes. Intercalation proposed here could have some structural relevance elsewhere, for instance to the base-mismatched regions on the double helix and the packing of noncomplementary single strands as found in the filamentous bacteriophage Pf1.

DNA↗
PubMed1991

Kinetics of radiation-induced strand break formation in single-stranded pyrimidine polynucleotides in the presence and absence of oxygen; a time-resolved light-scattering study.

Time-resolved reductions in the light-scattering intensity (LSI) of aqueous oxic and anoxic solutions of poly-C and poly-U at pH 7.8, following pulse-irradiation, have been studied as indices of strand break formation. With doses of 3-24 Gy per pulse, a number of kinetically distinct strand breakage components have been detected. A comparison of the LSI responses obtained from irradiations conducted under N2O with those conducted under air or O2 show no marked difference in the overall extent of LSI change. However, the immediate and fast (t 1/2 less than or equal to 50 microseconds) reduction in LSI, accounting for about 18-19% of the pyrimidine polynucleotide's total LSI response in oxic solution, is reduced in the absence of oxygen, to about 12% of the total LSI response found with poly-C and to about 9% for poly-U. For poly-C there is a five-fold enhancement in the rate of major strand breakage under anoxia [k1(N2O) = 7.9s-1] whereas for poly-U a more modest enhancement (about two-fold) is observed. These enhanced rates are mirrored by those for the losses of the principal optical anoxic absorptions (observed pulse radiolytically) that are assigned to the pyrimidine 6-yl base radicals. Such findings support a proposal that the rate-limiting step of major strand breakage for pyrimidine polynucleotides is a base radical mediated hydrogen atom abstraction reaction (Lemaire et al. 1987, Hildenbrand and Schulte-Frohlinde 1989). Irradiation of poly-C and poly-U in N2O/O2 (4:1, v/v) saturated solutions yields LSI changes much larger than those noted under N2O and air (or O2), which are in turn approximately double the responses observed under N2. This indicates that the major strand breaking species of water radiolysis is the OH-radical and that there is an oxygen enhancement of single strand breakage of about 1.9 for poly-C and 1.6 for poly-U.

DNA Damage↗
PubMed1973

Specific murine B-cell activation by synthetic single-and double-stranded polynucleotides.

The synthetic single- and double-stranded polynucleotides, poly I, poly C, and poly I.C, were shown to induce thymidine incorporation in six inbred strains of murine spleen cells. This stimulation was shown to be secondary to B-cell activation and not due to contamination of the polynucleotides with bacterial lipopolysaccharide (LPS). The ability of poly I.C to act as a B-cell mitogen, in addition to its behavior as a thymic-independent antigen, suggested that these two phenomena may be related. The similarity of the molecular structure of poly I.C to LPS, a material which also acts as a thymic-independent antigen and a B-cell mitogen, supports the hypothesis that the polyvalent nature of these materials accounts for their functional interaction with murine B cells.

Animals↗
PubMed1990

The binding of the 69- and 100-kD forms of 2',5'-oligoadenylate synthetase to different polynucleotides.

Three major forms of 2',5' oligoadenylate (2-5A) synthetase are induced in interferon (IFN)-treated human cells: 40-46, 69, and 100 kD. Here we studied the binding and activation of the 69- and 100-kD forms to single-stranded (ss) or double-stranded (ds) RNAs. The 69- and 100-kD form enzymes purified by immunoaffinity chromatography, were shown to be activated by synthetic dsRNAs poly(I).poly(C) or poly(A).poly(U) whereas ssRNAs poly(I), poly(C), poly(A), poly(U), and poly(G) had no effect. Both enzymes were also partially purified by binding to dsRNA or ssRNA-Sepharose. The synthetases bound to dsRNA Sepharose were partially activated but required the addition of soluble poly(I).poly(C) for maximal activity. The synthetases bound to ssRNA-Sepharose manifested no activity but became activated in the presence of soluble dsRNA, poly(I).poly(C). However, activation of such ssRNA-bound enzymes by dsRNA did not result in their dissociation from the ssRNA-Sepharose. These results indicate the presence of different polynucleotide binding sites on the 69- and 100-kD forms of 2-5A synthetase: a specific dsRNA binding site essential for activation and another polynucleotide binding site or sites which, although not specific, might be important for the optimal conformation of these proteins in cells.

2',5'-Oligoadenylate Synthetase↗
PubMed1974

Fingerprinting nonradioactive ribonucleic acid with the aid of polynucleotide phosphorylase.

We describe a method for obtaining radioactive fingerprints from nonradioactive ribonucleic acid. Fragments derived by T1 ribonuclease digestion of RNA are dephosphorylated with bacterial alkaline phosphatase. When these fragments are used as primers for the reaction of primer dependent polynucleotide phosphorylase with [alpha-(32)P]GDP in the presence of T1 ribonuclease the 3'-hydroxyl group of each fragment becomes phosphorylated. The degree of phosphorylation is reasonably uniform. The method has been applied to T1 ribonuclease digests of Escherichia coli tRNA(Met) (f); the oligonucleotides were further analyzed by spleen phosphodiesterase digestion. In a similar manner fingerprints of pancreatic ribonuclease digests of RNA can be obtained, when [alpha-(32)P]UDP, polynucleotide phosphorylase and pancreatic ribonuclease are used.

Alkaline Phosphatase↗
PubMed1974

'Single addition' and 'transnucleotidation' reactions catalyzed by polynucleotide phosphorylase. Effect of enzymatic removal of inorganic phosphate during reaction.

The reaction of the tetranucleotide, pA-A(2)-A, with 2'(3')-0-(alpha-methoxyethyl)uridine 5'-diphosphate, Mg(2+) ions, and M. luteus polynucleotide phosphorylase followed by mild acid treatment to remove the blocking groups results in a 49% yield of the desired single addition product, pA-A(3)-U, together with smaller amounts of pA-A-U, pA-A-A, pA-A(2)-U, pA-A(2)-A, pA-A(3)-A, pA-A(4)-U, and pA-A(4)-A. The side products are thought to arise from the phosphorolysis of the acceptor molecule by the inorganic phosphate formed in the reaction mixture and from subsequent additions to the various oligonucleotide species by the resulting adenosine 5'-diphosphate. A system developed for the removal of inorganic phosphate as it is formed in the synthesis involves the addition to the reaction mixture of calf spleen nucleoside phosphorylase and nicotinamide riboside and, under these conditions, pA-A(3)-U can be prepared in 90% yield with essentially no side products. Under similar conditions, pA-A(3)-A, pA-A(3)-G, and pA-A(3)-C may be prepared from pA-A(2)-A and the appropriate blocked nucleoside diphosphate in yields of 85-94%. The incubation of pA-A(2)-A alone with polynucleotide phosphorylase exhibits the phenomenon of "transnucleotidation" in that the molecule is partially converted to oligonucleotides of smaller and larger chain lengths. In the presence of the phosphate removal system, however, the tetranucleotide is not attacked by the enzyme, and thus, "transnucleotidation" appears to be simply a combination of phosphorolytic and addition reactions catalyzed by trace amounts of inorganic phosphate contaminating the enzyme and/or the substrate.

Adenine Nucleotides↗
PubMed1983

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.

Circular Dichroism↗
PubMed1983

Intercalative and nonintercalative binding of large cationic porphyrin ligands to polynucleotides.

The interactions of two positional isomers and one analogue of meso-tetra (4-N-methylpyridyl) porphine, with the synthetic polynucleotides poly[d(A-T)] . poly[d(A-T)] and poly[d(G-C)] . poly[d(G-C)] have been investigated by circular dichroism. All four porphyrins were found to bind to the polynucleotides as shown by the induction of circular dichroism in their Soret bands. Furthermore, the sign of the induced ellipticity reflects selective occupation of binding sites by the porphyrin ligands. The conformational lability of poly[d(A-T)] X poly[d(A-T)] was found to be appreciable as micromolar amounts of meso-substituted 4-N-methylpyridyl, 3-N-methylpyridyl, and p-N-trimethylanilinium porphines induced a CD spectrum similar but not identical to that of DNA in the Z-form, i.e. a negative band at 280 nm and a positive band at 259 nm. The effect of porphyrin binding to poly[d(G-C)] X poly[d(G-C)] was less pronounced and dissimilar to that seen in the AT polymer.

Chemical Phenomena↗
PubMed1984

Two rotameric forms of open ring 7-methylguanine are present in alkylated polynucleotides.

High performance liquid chromatography analysis of imidazole open ring 7-methylguanine, 2-6 diamino-4-hydroxy-5N-methyl-formamidopyrimidine (rom7G), showed two well-separated peaks (fI and fII) of the same magnitude. Rechromatography of each isolated component indicated that they are slowly interconverted to give a 1:1 mixture. NMR analysis demonstrated that the two species observed on reversed phase HPLC are rotational isomers. Thermodynamic measurements strongly suggested that the equilibrium can be assigned to rotation around the N-methyl formamido bond. The two species, fI and fII, separated by HPLC were identified as rotamers E and Z, respectively. The structures of fI and fII were also determined. A polynucleotide containing rom7G was obtained by alkaline treatment of poly (dGC) containing 7-methylguanine. In order to study its structure within the polynucleotide, rom7G was enzymatically excized by E.coli rom7G-DNA glycosylase. The analysis of the products released by the enzyme showed a 1:4 mixture of the two rotamers favoring the Z form (fII).

Alkylation↗
PubMed1987

A stopped-flow H-D exchange kinetic study of spermine-polynucleotide interactions.

The rates of H-D exchange for imino and amino protons in adenosine, calf thymus DNA, poly (dA-dT), poly(dG-dC), and poly (dG-me5dC) were determined using stopped flow kinetic methods in the presence of various concentrations of Tris, imidazole, Mg2+, and spermine in citrate buffer (pH 7, 25 degrees C). CD spectroscopic studies showed that all polynucleotides always remain in the B-form under these conditions. An increase in the concentration of Tris and imidazole from 5 mu M to 20 mM caused an increase in the rates of exchange of both fast-exchanging imino and slow-exchanging amino protons. The limiting rates of exchange at infinite concentrations of catalysts were found to be different for fast (31-57 sec-1) and slow (1-2 sec-1) exchanging protons. These results indicate that imino and amino protons of B-DNA exchange asymmetrically from two different open states as observed for Z-DNA. An increase in the concentration of spermine from a ratio of 1:50 to 1:2 of positive charge/phosphate decreased the rate of exchange of imino protons of calf-thymus DNA, poly(dG-dC), and poly(dG-me5dC), but increased the rate of exchange of the imino protons of poly(dA-dT) without affecting the exchange rate of the amino protons of any of the polynucleotides. These results are interpreted in terms of possible spermine-induced change of conformations of oligonucleotides of specific sequence that has been suggested by theoretical model building studies.

Circular Dichroism↗
PubMed1989

Direct evidence for the presence of left-handed conformation in a supramolecular assembly of polynucleotides.

Hexammine cobalt(III) chloride (Co(NH3)6(3+) provokes a B-DNA----Z-DNA----psi-DNA conformational transition in poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC). The circular dichroism spectrum of psi-DNA is characterized by a manyfold increase of positive ellipticity in the range of 300-225 nm and the complete absence of a negative peak. In order to ascertain the helical handedness of psi-DNA, we used a recently developed enzyme immunoassay technique. This method consisted of treating the polynucleotides with Co(NH3)6(3+) to convert them to the Z- or psi-DNA forms and immobilizing these conformations on a microtiter plate. The plates were subsequently treated with a monoclonal anti-Z-DNA antibody Z22, alkaline phosphatase conjugated, affinity purified immunoglobulins, and the phosphatase substrate. The enzyme-substrate reaction was monitored by reading the absorbance at 405 nm with a microplate autoreader. The monoclonal anti-Z-DNA antibody had no reactivity to the B-DNA form, but bound strongly to both the Z- and psi-DNA forms, showing that Co(NH3)6(3+)-induced psi-DNA form of the polynucleotides exists in the left-handed Z-DNA conformation.

Antibodies↗
PubMed1995

A Sindbis virus mRNA polynucleotide vector achieves prolonged and high level heterologous gene expression in vivo.

The direct intramuscular delivery of naked plasmid DNA has been demonstrated to allow expression of encoded heterologous genes in the target myocytes. The method has been employed to elicit immunization based upon delivery of antigen encoding plasmid DNA. For application in the context of achieving anti-tumor immunization against antigenic transforming oncoproteins, delivery of plasmid DNAs encoding these molecules would create significant potential safety hazards. As an alternative to DNA polynucleotide vectors, we explored the utility of mRNA vehicles for inducing foreign gene expression in muscle cells in vivo. Synthetic reporter-gene encoding mRNA transcripts were derived for this analysis. The Sindbis virus vector was also used to derive luciferase mRNA transcripts which possessed self-replication capacity. In these studies, it could be shown that the replicative vector was capable of directing significantly elevated levels of reporter gene expression in myocytes compared to a non-replicative mRNA species. In addition, the replicative species was capable of achieving significantly prolonged levels of in vivo gene expression compared to non-replicative mRNA. Both of these characteristics will make replicative mRNA vectors of utility for polynucleotide-based immunization protocols.

Animals↗
PubMed1976

A deoxyadenylate kinase activity associated with polynucleotide phosphorylase from Micrococcus luteus.

We report here the presence of two enzymatic activities associated with highly purified preparations of polynucleotide phosphorylase from Micrococcus luteus. The first, a nuclease activity, which is not separated from the phosphorylase on hydroxylapatite, may be due to substitution of H2O for phosphate in the phosphorolysis reaction. The second activity, a deoxyadenylate kinase, the bulk of which is not resolved from the phosphorylase using gel filtration, sucrose density gradient centrifugation, DEAE-Sephadex, or hydroxylapatite chromatography, may represent a new activity of polynucleotide phosphorylase or be due to an enzyme which is tightly bound to the phosphorylase. Several properties of the kinase are described and its possible significance with respect to the overall enzyme mechanism is discussed.

Adenosine Monophosphate↗
PubMed1976

Electrostatic potential modulations on polynucleotides as a function of ionic content: potentiometric determinations.

pK values of ionisation of cytidine residues, in poly C and poly I. poly C, have been determined by potentiometric titration as a function of either ionic strength (Na+) or Mg2+ concentration. The results are interpreted as a consequence of the existence of a local pH, prevailing in the vicinity of the polynucleotides, smaller than that of the bulk solution. This local pH is dependent upon the electrostatic potential psi due to the negatively charged phosphate groups of the polynucleotide. Moreover psi can be modulated through variation of either ionic strength or Mg2+ concentration. At a given pH Mg2+ addition leads to the release of protons which, in the case of complex systems of limited stability, might be detected by the use of the pH-stat method. Possible functional implications for genetic translation systems are examined.

Binding Sites↗