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PubMedDate not supplied

[Contribution of individual structural components to stabilization of the secondary structure of double-helical polynucleotides].

Contributions of individual structural components of the double-helical polynucleotide to the stabilization of its secondary structure have been studied. The energy of intramolecular interactions was calculated by the method of atom-atom potentials. Sections of the energy function were constructed according to the parameters determining mutual location of base pairs with the optimal conformation of ribose-phosphate backbone in the points close to A- and B-forms of DNA. A complicated nature of the contribution of different structural components of the polynucleotide to its stabilization was revealed by means of different parameters. Relationships between the change of conformational parameters corresponding to optimal values of energy for A- and B-families of nucleic acids were found.

DNA↗
PubMedDate not supplied

Complex formation of spin-labeled 9-aminoacridine with DNA and polynucleotides.

Complex formation between spin-labeled 9-aminoacridine and DNA or polynucleotides has been studied by differential spectrophotometry and ESR. The differential spectra of the strong type 9-aminoacridine-DNA complex showed characteristic absorption bands at 270 and 290 nm, and the intensity ratio of these bands varied according to the degree of DNA denaturation. The ESR spectra of this complex were characterized by slow rotation of the radical; as the macromolecule became increasingly denatured and in the polynucleotide complex, a rapid signal appeared in the ESR spectrum. The temperature at which DNA undergoes a structural transition in the premelting region could be determined from the temperature dependence of the ESR spectral form of the dye-DNA complex. The spectral characteristics of the complexes give additional information about structural disturbances in DNA.

Animals↗
PubMedDate not supplied

[Several properties of complexes of polynucleotides with aromatic amino acid amides of oligonucleotides].

Residues of Phe, Tyr and Trp in the complexes of their oligonucleotide amidates and polynucleotides of A-U of G-C nucleotide composition are most likely localized in the minor groove of the Watson--Crick part of the triple helix where they interact with bases but do not intercalate into the helix. Formation of the complexes is accompanied with a change in the relative localization of amino acids and bases. The major geometrical parameters of the triple helices of the complexes are not changed by the residues of aromatic amino acids (according to CD data). A slight violation of stacking interactions between bases is observed along with an increase of the cooperativity of melting of the complexes of A-U composition (according to UV absorption data). The effect of the residues of aromatic amino acids on the stability of triple helices is determined by the nucleotide composition of the latter, i.e. complexes of A-U composition are destabilized with the Phe, Tyr and Trp residues, whereas the Trp residue does not affect the stability of the complexes of G-C composition. The hydrophobic character of aromatic amino acids and their different affinity for bases of different structure seem to account for this difference in stability. The dependence of the thermal stability of RNH-dp(An).2poly(U)-complexes on the structure of the amide radical (residues of glycin, aromatic amino acids, alkyl- and arylalkyl amines) testifies the ability of the radical to "regulate" the interaction between the oligomer and the complementary polynucleotide. This capacity for "regulation" is not observed in the system of G-C composition.

Amides↗
PubMed1981

[Isolation and some properties of polynucleotide phosphorylase from E. coli].

A new method for isolation of polynucleotide phosphorylase from E. coli, including ion-exchange chromatography and gel-filtration has been developed. The method results in 300-fold purification of the enzyme, which being devoid of nuclease and phosphatase activities can further be utilized for oligonucleotide synthesis. It was shown that upon storage the enzyme loses the primer-independent activity and in the absence of NaCl can be used for further syntheses. An addition of NaCl stimulates the elongation of the oligonucleotide chain. Some advantages of polynucleotide phosphorylase from E. coli in comparison with the M. luteus enzyme are discussed.

Drug Stability↗
PubMed1982

Characterization of polynucleotide phosphorylase from Micrococcus luteus and isolation of the 13,000 base poly(A) product of the polymerization reaction.

A new purification procedure for polynucleotide phosphorylase from freeze-dried Micrococcus luteus cells gives approximately 20% yield of nearly homogeneous, primer-independent enzyme which is free of nucleic acid. The physicochemical properties of M. luteus polynucleotide phosphorylase are similar to those previously described for the enzyme from Escherichia coli in terms of Mr, subunit structure, and amino acid composition. The purified enzyme appears to be a trimer composed of three identical subunits (Mr 92,000), but it probably does not exist as such in the cell. Ferguson plot analyses of enzyme in cell extracts indicate that prior to purification the enzyme exists in oligomeric forms characterized by both higher charge and greater Mr. Changes in size and charge of oligomers which occur during purification are probably due to the dissociation of proteins and/or nucleic acids. Dissociation of the oligomers is achieved by dilution and electrophoresis, but reassociation does not occur after concentration. The poly(A) product of the initial polymerization stages migrates as a single band on both nondenaturing and urea-agarose gels. It is 13,000 +/- 2,000 nucleotides long, as measured by electron microscopy, and 8,000 nucleotides long by gel electrophoretic analysis. This poly(A) product remains bound to the enzyme after synthesis, yet can be easily obtained free of protein by proteinase K digestion.

Amino Acid Sequence↗
PubMed1994

Evaluation of a non-radioactive chemiluminescent method for using oligonucleotide and polynucleotide probes to identify enterotoxigenic Escherichia coli.

We compared the applicability of an enhanced chemiluminescent (ECL) method for using gene probes with that of radioactive probes to identify enterotoxigenic Escherichia coli (ETEC) in stools of Bangladeshi children with diarrhoea. Colony blots of E. coli isolates were hybridized using both [alpha-32P]-dCTP labelled and fluorescein-11-dUTP labelled polynucleotide and oligonucleotide gene probes for heat-labile enterotoxin (LT), and heat-stable enterotoxin (ST). Analysis of 1,620 isolates obtained from 540 patients gave similar results by both radioactive and chemiluminescent probes. The ECL method was faster than the radioactive method. Both polynucleotide and oligonucleotide probes could be used by the ECL method. Hybridization and detection by the ECL method appeared to be a convenient alternative to radioactive probes for screening E. coli isolates for ETEC.

Base Sequence↗
PubMed1997

Mucosal immune responses associated with polynucleotide vaccination.

A variety of gene delivery technologies can be used to express antigens within somatic tissues, resulting in systemic humoral and cellular immune responses. This observation has led to the development of polynucleotide vaccine preparations for stimulation of systemic immunity. Mucosal immune responses are functionally distinct from systemic immune responses, and are stimulated by antigen presentation within specialised mucosal-associated inductor tissues. We hypothesize that mucosal genetic vaccine will require gene transfer methods which target mucosal-associated inductor tissues such as the oropharyngeal Waldeyer's ring or intestinal Peyer's patches. We have tested this hypothesis by expressing a test antigen using a replication-defective recombinant Semliki Forest Virus (SFV) preparation. Mice treated with recombinant SFV via an intravascular or intratracheal route generated systemic immune responses against the test antigen. In contrast, intranasal inoculation resulted in the production of IgA within pulmonary fluids, one hallmark of a mucosal immune response. These results indicate that transfection of mucosal effector tissues may not be sufficient for the generation of a universal mucosal immune response. Furthermore, the results predict that techniques which target transfection or transduction to mucosal inductor tissues will enable the development of a new class of polynucleotide vaccines which exploit current concepts in mucosal immunology.

Animals↗
PubMed1997

Manipulation of immune responses via particle-mediated polynucleotide vaccines.

Polynucleotide vaccines are a new approach to immunization that promises qualitative advances in vaccine technology. These vaccines mimic infection in that they result in expression of pathogen gene products in situ, which can elicit both cell-mediated immune responses and humoral responses. This approach has been applied primarily to vaccines against viral diseases, but may be significant for vaccines directed toward bacterial pathogens. Auragen has developed a generally applicable gene transfer technology and, for vaccine applications, has focused on particle-mediated gene transfer to epidermis. Results demonstrate that Accell polynucleotide vaccines induce immune responses toward human immunodefficiency virus (HIV) antigens, influenza A virus antigens, and hepatitis B virus (HBV) antigens in rodent,s swine and primates. Cellular immune responses toward these antigens have been demonstrated in rodents. In a swine influenza a challenge model Accell vaccination provides protection equivalent to that of a commercial killed-whole-virus vaccine. Vaccination of mice by this method toward a Chlamydia pneumoniae major outer-membrane protein elicits a species-specific antibody response.

AIDS Vaccines↗
PubMed1976

Effect of deoxyribonuclease on adriamycin-polynucleotide complexes.

Recent interest in the use of adriamycin-DNA complex as an approach to improve the therapeutic effectiveness and to reduce toxicity of adriamycin for cancer chemotherapy requires an in-depth understanding of the physicochemical and biochemical properties of such complexes. The interactions of adriamycin with single-strand polydeoxyribonucleotides, double-strand DNA, and double-strand ribodeoxyribopolynucleotide hybrids were therfore investigated. Association constants (Kapp) of adriamycin and polynucleotides were obtained. These data showed that the inherent variable in such complex lies in the composition of the polynucleotides. Alternate deoxyguanylate (dG)-deoxycytidylate (dC) sequence binds 7-fold better than alternate deoxyadenylate (dA)-deoxythymidylate (dT) sequence. Comparative studies of the hydrolysis of DNA duplexes by deoxyribonucleases I and II with and without adriamycin were also carried out. The rate of hydrolysis decreased in the order poly(dA-dT) greater than calf thymus DNA greater than poly(dG-dC) greater than poly(dA)-poly(dT) greater than poly(dG)-poly(dC) for DNase I and poly(dA)-dT) greater than calf thymus DNA greater than poly(dG-dC) greater than poly(dA)-poly(dT) greater than poly(dG)-poly(dC) for DNase II. Intercalation of adriamycin to deoxyribopolynucleotide duplex resulted in inhibition of DNase II two to three times more than tat of DNase I. On the other hand, intercalation of adriamycin to homodeoxypolynucleotide duplex poly(dA)-poly(dT) and poly(dG)-poly(dC) enhanced the DNase I hydrolysis. If DNase I activity could be related to serum DNase and DNase II related to tumor lyososomal DNase as in the endocytosis mechanism proposed by Trouet et al. (Cancer Chemotherapy Rept., 59: 260, 1975), the best adriamycin carrier suggested by this investigation could be poly(dA)-poly(dT) and poly(dG-dC). It is also suggested in this study that adriamycin-RNA-DNA hybrid could be of interest as an antiviral agent by a similar release mechanism via RNase H, an enzyme associated with viral reverse transcriptase.

Animals↗
PubMed1981

A new procedure for the simultaneous large-scale purification of bacteriophage-T4-induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase.

A procedure for simultaneous large-scale purification of the bacteriophage-T4-induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase has been developed. The method involves bacterial cell disruption by sonication, fractionation of cell extract with polymin P, salt elution from the polymin pellets, ammonium sulfate precipitation, and subsequent column chromatography purification of the enzymes. To enrich the enzyme content highly in the initial source non-permissive Escherichia coli B-23 cells infected with T4 amN82 phage were used. The procedure described is rapid, reproducible, high in yield, and able to handle preparations using from 1 g to 200 g cell paste. It can be easily scaled up. The method results in large amounts of the enzymes with very high specific activities, good stability essential lacking exonuclease and endonuclease contamination. The final enzyme preparations were efficiently used in DNA sequencing and in multiple experiments on construction of various recombinant DNAs for cloning and expression in vivo.

DNA Ligases↗
PubMed1964

ALKYLATION OF SYNTHETIC POLYNUCLEOTIDES.

The synthetic polynucleotides may be used as models for the effects of alkylating agents on nucleic acid. Alkylation of polyadenylic acid with methyl methanesulfonate yields a polymer methylated in the one-position of adenine. This alteration in structure produces marked changes in physical properties and decreases the capacity to code for polylysine synthesis.

Alkylating Agents↗
PubMed1988

DNA sequence and conformation specificity of lupus autoantibodies. Preferential binding to the left-handed Z-DNA form of synthetic polynucleotides.

The binding specificity of 16 sera from systemic lupus erythematosus (SLE) patients was studied by enzyme-linked immunosorbent assay (ELISA), using 4 native DNAs of different guanine-cytosine (G-C) content and a group of synthetic polynucleotides. All the SLE sera showed increased binding to poly(dA-dC).poly(dG-dT), compared with calf thymus DNA in the right-handed B conformation. No significant differences were noted in binding of selected SLE sera to the native DNAs that differed in G-C content or superhelicity of DNA. With poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC), the majority of SLE sera showed a preferential binding to the salt-induced Z form, compared with the B form. In addition, an average twelve-fold increase was found in binding to Z-form brominated poly(dG-dC).poly(dG-dC) compared with B-form poly(dG-dC).poly(dG-dC), when the polymers were coated on the plates in 0.15M NaCl. The preferential binding of SLE sera to poly(dA-dC).poly(dG-dT) and to Z-DNA may be important in the formation of circulating immune complexes and subsequent vascular damage, or may provide a clue to the mechanism of production of anti-DNA antibodies in this disease.

Antibody Specificity↗
PubMed1985

T4 polynucleotide kinase; cloning of the gene (pseT) and amplification of its product.

The T4 gene (pseT) for polynucleotide kinase (pnk) has been cloned in lambda. Induction of a lambda E-W-S-cI857 prophage in which the pseT gene can be transcribed from the late lambda promoter, PR1, leads to greater than 100-fold amplification of pnk activity; pnk comprises approximately 7% of the total soluble cell protein. The purified enzyme, as expected, is both a 5'-kinase and a 3'-phosphatase. The amino acid sequence deduced from an open reading frame identified as the pseT gene contains a sequence which corresponds particularly well with that part of the adenine nucleotide binding site of adenylate kinase shown to form a flexible loop. A deletion mutant that lacks 5'-kinase activity, and possibly also 3'-phosphatase activity, has lost two amino acids from within the proposed loop structure. A second region of the pnk sequence shares homology with phosphoglycerate kinase, yeast inorganic pyrophosphatase and histone 2b from various organisms.

Amino Acid Sequence↗
PubMed1995

5'phosphorylation of DNA in mammalian cells: identification of a polymin P-precipitable polynucleotide kinase.

Proteins that catalyze 5' phosphorylation of an oligodeoxyribonucleotide substrate can be fractionated by polymin P treatment of whole cell extracts of calf thymus glands. Anion exchange chromatography on Q-Sepharose revealed three separable peaks of activity in the polymin P supernatant fraction, and one peak of activity in the Polymin P pellet fraction. The latter activity, Polymin P-precipitable polynucleotide kinase (PP-PNK), was further purified with a 1,500-fold increase of specific activity compared to the crude Polymin P pellet fraction. Oligonucleotides, a dephosphorylated 2.9-kb EcoRI fragment, and poly(A) were phosphorylated by the enzyme preparation, but thymidine 3' monophosphate was not a substrate. PP-PNK preparations exhibited an apparent KM of 52 microM for ATP and 8 microM for oligo dT25. The enzyme preparation displayed no detectable 3' phosphatase or cyclic 2',3' phosphohydrolase activities. The sedimentation coefficient of the PP-PNK activity was 3.8S as determined by sucrose density gradient analysis; the Stokes radius was 45 A, leading to an estimated molecular mass of 72 kDa. The enzyme had a pH optimum in the neutral to alkaline range in several buffer systems and is distinct from the DNA kinase with an acidic pH optimum previously described in calf thymus.

Animals↗
PubMed1984

A rapid procedure for visualising the inner cell mass and trophectoderm nuclei of mouse blastocysts in situ using polynucleotide-specific fluorochromes.

A rapid procedure has been devised to count the numbers of outer trophectoderm (TE) and inner cell mass (ICM) cells of mouse blastocysts by differentially labelling their nuclei in situ with polynucleotide-specific fluorochromes. The TE nuclei were labelled with propidium iodide (PI) by permeabilising the cells using selective antibody-mediated complement lysis (Solter and Knowles, '75). The blastocysts were then fixed in ethanol and the ICM nuclei labelled with bisbenzimide. These two fluorochromes have widely different fluorescent spectra. Thus, by using fluorescence microscopy with appropriate filter combinations, the PI-labelled TE nuclei appeared pink or red; the bisbenzimide-labelled ICM nuclei, blue or unlabelled. The total numbers of blastocyst nuclei and the numbers of ICM nuclei counted by differential labelling were similar to the numbers detected after spreading the nuclei of intact blastocysts or immunosurgically isolated ICMs by air-drying (Tarkowski '66). Differential labelling of TE and ICM nuclei in situ has two important advantages--that the numbers of both these cell types can be determined for individual blastocysts and that spatial relationships are partially preserved so that regional interactions can be studied.

Animals↗
PubMed1994

Ethidium and azidoethidium oligonucleotide derivatives: synthesis, complementary complex formation and sequence-specific photomodification of the single-stranded and double-stranded target oligo- and polynucleotides.

Oligodeoxyribonucleotide derivatives containing ethidium or azidoethidium residues attached to 3' and/or 5' end were prepared. These derivatives formed tight specific complexes with complementary oligodeoxyribonucleotides where each attached ethidium residue led to an increase of complex Tm by 20-30 degrees C. Tandem complexes of two oligodeoxyribonucleotides containing ethidium residues with an oligodeoxyribonucleotide having two adjacent complementary sequences for these oligonucleotides were investigated. Photoinduced reactions of a number of ethidium and azidoethidium oligodeoxyribonucleotide derivatives with target complementary single-stranded and double-stranded oligo- and polydeoxyribonucleotides were investigated. The irradiation led to direct photocleavage of the target oligo- or polynucleotide, to formation of hidden (piperidine cleavable) modifications of the target and to formation of covalent adducts between ethidium oligodeoxyribonucleotide derivative and the target. In a number of experiments, azidoethidium dyes were demonstrated to be considerably stronger photosensitizers than ethidium ones. Depending on the nature of the target (single- or double-stranded DNA) and on the irradiation conditions, the total damages to the target oligo- or polydeoxyribonucleotides ranged from 10-70% (for ethidium dyes) to 30-80% (for azidoethidium dyes).

Azides↗
PubMed1995

Effect of pH of the T4 polynucleotide kinase reaction on the 32P-postlabeling assay of DNA adducts.

Among the currently available methods for detecting bulky hydrophobic compounds bound to DNA, the 32P-postlabeling is the most sensitive and most widely practiced. One of the key reaction steps of this assay is the polynucleotide kinase (PNK)-catalyzed incorporation of 32Pi into nucleotides. To ensure high sensitivity and reproducibility of the assay, it is imperative that the PNK reaction be conducted under optimal conditions. The PNK reaction is generally conducted at pH 9.6 in the 32P assay. We have investigated pH profiles of the PNK reactions using both normal (2'-deoxyadenosine 3'-monophosphate) and adducted (benzo[ alpha]pyrene- and cigarette smoke condensate-modified) nucleotides. The optimum pH range for the PNK reaction was between 7.4 and 8.4 with both types of nucleotides. Higher pH levels such as 9.4 and 9.6 were detrimental to the PNK reaction, yielding only about one-third or less of the labeling obtained at the optimum pH. Based on these results, the PNK reaction in the 32P-postlabeling assay should be conducted in the pH range between 7.8 and 8.0 to achieve quantitative labeling of adducted nucleotides.

Animals↗
PubMed1997

Synthetic polynucleotide templates for characterizing sequence-selective small molecule/nucleic acid interactions.

A synthetic polynucleotide sequence was developed and cloned to serve as a target in footprinting and other assays designed to characterize the sequence-selective binding of drugs and other small molecules to various forms of nucleic acids. The target sequence comprehensively represents all base quartet recognition sites in a minimal length sequence. Minimal length target sequences were found to be 144 nt long. One such target sequence was divided into two parts. One strand of each part was chemically synthesized and the complementary strands were generated using a DNA polymerase. Double-stranded sequences were then cloned into pGEM-3Zf(+/-) vectors (Promega, Inc.). The cloned target sequence can be used directly in double-stranded DNA form. Alternatively, features of the plasmid vector allow expression of the target sequences as single-stranded DNA or RNA or as RNA/DNA or RNA/RNA duplexes. These cloned target sequences designed for high information content overcome limitations to the use of natural DNA sequences for footprinting and related experiments arising from the unequal representation of base quartets and the potential for secondary structure formation in single-stranded forms.

Base Sequence↗