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PubMed1997

Purification and substrate specificity of polydeoxyribonucleotide kinases isolated from calf thymus and rat liver.

Damage to DNA can result in strand breaks with 5'-hydroxyl and 3'-phosphate termini. Before DNA polymerases and ligases can rejoin the broken strands, such termini have to be restored to 5'-phosphate and 3'-hydroxyl groups. Polydeoxynucleotide kinase is an enzyme that may fulfil this function. We have purified the kinases from calf thymus and rat liver to near homogeneity. Based on SDS-polyacrylamide gel electrophoresis and activity gels, the enzymes from both sources are approximately 60-kDa polypeptides. Both enzymes have an acidic pH optimum (5.5-6.0) for kinase activity, and similar pl values (8.5-8.6), and a specificity for DNA. The calf thymus kinase possesses a 3'-phosphatase activity, as has previously been shown for the rat liver enzyme. The minimum size of oligonucleotide that can be labelled is 7-8 nucleotides in length, but the optimal size appears to be > 18 nucleotides. Comparison of phosphorylation of oligo(dA)24 and oligo(dT)24 with oligonucleotides containing a varied nucleotide sequence indicated that the homopolymers are poorer substrates. Unlike the bacteriophage T4 polynucleotide kinase, the mammalian kinases exhibit no preference for 5'-overhanging termini when acting at DNA termini produced by restriction enzymes. With double-stranded oligonucleotide complexes designed to mode single-strand gaps and nicks, the mammalian kinases preferentially phosphorylate the 5'-terminus associated with the gap or nick, in keeping with the idea that the kinases are involved in the repair of DNA single-strand breaks.

Animals↗
PubMed1976

Template activity of synthetic deoxyribonucleotide polymers in the eukaryotic DNA-dependent RNA polymerase reaction.

Template specificities of the eukaryotic DNA-dependent RNA polymerases A and B from rat liver, pea, and cauliflower have been investigated using synthetic polydeoxyribonucleotides. Polymerases A and B from the three species exhibit different specificities for single-stranded homopolymers: polymerase A preferentially reads poly(dT) and poly (dC). and polymerase B poly (dC). This preferential reading appears to be a property of eukaryotic DNA-dependent RNA polymerases. Polymerases A and B transcribe synthetic polyribonucleotides also, but at a reduced rate. The polyribonucleotides which can be read by DNA-dependent RNA polymerases have a base sequence similar to that of the polydeoxyribonucleotides, which are effeciently transcribed, suggesting that the base sequence of the template rather than its conformation is crucial in the template specificity for synthetic polymers. Competition experiments with polydeoxyribonucleotides indicate that the enzymes have different binding specificities, which are not the same as their template specificities.

Animals↗
PubMed1971

Primer requirement and template specificity of the DNA polymerase of RNA tumor viruses.

Polyribonucleotides will act as efficient templates for the DNA polymerases found in the virions of avian myeloblastosis virus and mouse leukemia virus if a short complementary oligodeoxyribonucleotide primer is added. Synthesis of the complementary polydeoxyribonucleotide continues until an amount of polymer equal to the amount of initial template has been produced. The two viruses show slightly different specificities toward the four homoribopolymers. Polydeoxyribonucleotides are generally much poorer templates than the homologous polyribonucleotides, in most cases yielding no detectable synthesis. The DNA polymerase of RNA tumor viruses, therefore, have the same requirements for activity as do other DNA polymerases, except that they prefer polyribonucleotides over polydeoxyribonucleotides as templates.

Adenine Nucleotides↗
PubMed1981

Changes of deoxyribonucleoprotein in the spleen, thymus and liver of rats exposed to weightlessness and artificial gravity aboard the Cosmos biosatellites.

Changes of deoxyribonucleoprotein in the spleen, thymus and liver of rats exposed to weightlessness or artificial gravity on board biosatellites Cosmos 782 and Cosmos 936 after 20 days of flight were investigated. The level of polydeoxyribonucleotides in the spleen and thymus of rats exposed during the flight to weightlessness increased 4-11 hours after landing, suggesting breakdown of a part of the deoxyribonucleoprotein present. The use of artificial gravity prevented this breakdown in the thymus but not in the spleen. The breakdown was accompanied in the majority of cases by a decrease in the deoxyribonucleoprotein content. We believe the breakdown of deoxyribonucleoprotein is due to a nonspecific stress reaction to the change from the weightless state to that of terrestrial gravity during landing. The polydeoxyribonucleotide level and amount of deoxyribonucleoprotein in the majority of cases returned to normal values during the 25 days of readaptation. No substantial change of deoxyribonucleoprotein was found in the liver. The different findings in the three organs are due to the fact that breakdown of deoxyribonucleoprotein takes place in sensitive cells underlying pycnosis. These cells are found in the spleen and thymus, but not in the liver.

Animals↗
PubMedDate not supplied

Changes in chromatin and nucleic acids in rat tissues after two-week spaceflight.

The quantitative changes in nucleic acids and chromatin breakdown were followed in blood, thymus and spleen in rats after 14 day flights on board the biosatellites Cosmos-1887 and Cosmos-2044. Quantitative nucleic acid changes within 8-11 h after landing were only mild, most statistically non-significant. An analysis at 48 h after landing showed a marked decrease in a total content of DNA and RNA in spleen and thymus. Within 8-11 h after landing, the symptoms of chromatin breakdown were found as is seen in an increased concentration of its fragments-polydeoxyribonucleotides. The obtained results show that a partial adaptation to microgravity occurs up to flight day 14 in lymphoid organs. Adaptation is accompanied with a reappearing of the sensitive cells. Their chromatin breaks down, then, in a final phase of flight due to hypergravity stress manifesting itself by a temporary increase in polydeoxyribonucleotide concentration several hours after landing. The results are discussed in relation to the changes in chosen parameters after shorter or more prolonged flights.

Adaptation, Physiological↗
PubMedDate not supplied

[The inhibition by the tumor necrosis factor of the death and DNA fragmentation of lymphoid cells in irradiated rats].

The influence of a tumor necrosis factor, administered 16 h before irradiation of rats, on the radiation response of thymus and bone marrow cells has been investigated. Three and 6 h after irradiation the following indices were analyzed: the number of apoptotic cells in the thymus; the accumulation of polydeoxyribonucleotides and the appearance of single-strand breaks in DNA of bone marrow and thymus cells; and the electrophoretic properties of thymocyte DNA. The injection of a tumor necrosis factor reduced the number of polydeoxyribonucleotides, inhibited internucleosome DNA fragmentation, and did not influence the formation of single-strand breaks in DNA.

Animals↗
PubMedDate not supplied

[Computerized evaluation of reparative processes of the cervix uteri].

This study was aimed to evaluate the effect of polydeoxyribonucleotide (PDRN), as reported in relevant literature, on cervical epithelia dynamics. Particularly, the interactions taking place between columnar epithelium and the squamous one have been examined. For the purposes of the study, the following computerized techniques, already widely known, have been used: The colposcope is joined to a videocamera connected with a computer (AT compatible). The computer is equipped with a graphic card capable to record and to digit the image, i.e. to make it recognizable by the computer itself. Thereafter, many operations can be performed on the colposcopic images: reductions, enlargements, retouches, record, recall, analysis, etc. Moreover, irregular epithelial areas can be easily determined to a good approximation and, using pre-established enlargement ratios, their evolution can be evaluated. By means of this technique 12 out-patients with uterine cervix ectopias, with or without normal transformation zone (NTZ), have been examined. The monthly therapy was 12 pessaries, each containing 5 mg polydeoxyribonucleotide (POLIDES 5--Farmigea), from the 7th to the 18th day of the cycle, repeated for 3 months. Since the first month of treatment a reduction of the ectopic columnar epithelium has been noted in most patients (9 on 12), with a squamous epithelium increase (peripheral reparative process). This process has kept on increasing during the following months in the 9 patients responding to the treatment, whose ectopic areas were covered by squamous epithelium (average 55% of the area; range 33%-78%). No response to the treatment has been shown in 3 cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMedDate not supplied

[Effect of space flight conditions on deoxyribonucleoprotein and nucleic acid concentrations in rat tissues].

The spleen of rats flown for 19.5 days aboard the biosatellite Cosmos-782 showed a significant increase of polydeoxyribonucleotides and a decrease of DNP and DNA. The spleen of synchronous rats did not exhibit any differences. This suggests that the increase of polydeoxyribonucleotides in flight rats immediately post-recovery was a result of the so-called gravity stress. The liver and white blood cells did not display any significant changes in DNP or nucleic acids. The changes in the content of nucleic acids in the thymus and bone marrow were insignificant.

Animals↗
PubMedDate not supplied

[Glucocorticoid-receptor complexes of rat liver. II. Interaction with natural and synthetic polynucleotides].

The relative affinities of [3H]dexamethasone-labelled glucocorticoid-receptor complexes (GRC) from rat liver to various natural and synthetic polynucleotides differing in base composition and secondary structure have been determined. The modified competition assay procedure with use of DNA-cellulose was employed. The interaction of 150-2000 fold purified GRC with polyribo- and polydeoxyribonucleotides greatly depends on their base composition and nucleotide sequences. This interaction is hardly affected by the structure (ribo- or deoxyribo-) of the sugar-phosphate backbone or its size and is in fact independent of the secondary structure (single- or double-stranded forms) of polynucleotides. Mixtures of mononucleotides or apurinic DNA do not compete with DNA-cellulose for GRC binding. In the presence of cytosol, GRC bind double-stranded rather than single-stranded DNA, the fact possibly reflecting different effects of cytoplasmic proteins on the accessibility to GRC of polynucleotides of dissimilar secondary structure. The apparent equilibrium dissociation constants (Kd) of the ternary complexes of GRC with some synthetic polynucleotides were calculated. In case of double-stranded polydeoxyribonucleotide poly(dA) . poly(dT) Kd is equal to (1.2 +/- 0.6) x 10(-4) M; this value may characterize a nonspecific GRC--DNA interaction. It is supposed that GRC interact with high affinity (Kd approximately 10(-7)--10(-8) M) with short (5-10 nucleotides long) DNA sequences in which certain purine and pyrimidine residues are interspersed. Such DNA sequences may represent sites of primary action of GRC on the cellular genome in vivo.

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↗
PubMed1980

Isolation of a single-stranded extrachromosomal DNA from germinating wheat embryos.

Newly-synthesized cytoplasmic non-mitochondrial DNA was isolated from wheat embryos which had been germinated in the presence of [14C]-thymidine for a time period not long enough to trigger the first post-dormant round of the nuclear DNA replication. This extrachromosomal DNA fraction consisted of linear single-stranded polydeoxyribonucleotide chains, corresponding in size to approximately 1.1 X 10(6) daltons, and amounted to about 0.5% of the total cellular DNA content. It is suggested that the appearance of the newly-synthesized polydeoxyribonucleotide chains in the cytoplasm may be a physiological signal for the initiation of the nuclear DNA replication in germinating wheat embryo cells.

Centrifugation, Density Gradient↗
PubMed1981

Studies on the mode of action of single-stranded polynucleotides which are antiviral against encephalomyocarditis virus infection of mice.

Poly(dI) and poly(dC) administered separately or sequentially show no antiviral effects against EMC virus infection of mice, whereas poly(rI) and poly(rC) are antiviral in such treatment regimens without evidence of interferon induction. The antiviral effects of poly(rI) and poly(rC) appear to depend on single-strandedness because their antiviral effects are decreased by annealing to poly(dC) and poly(dI) respectively. This decrease in antiviral effect would not seem to be due to an adverse effect of polydeoxyribonucleotides on EMC virus infection because the polydeoxyribonucleotides have no effect on the antiviral activity of another single-stranded RNA, E. coli tRNA.

Animals↗
PubMed1979

Methods for limiting the action of SP3 DNAase and for the determination of the direction of hydrolysis of processive exonucleases.

The action of the exonuclease SP3 DNAase is inhibited by chemical modification of DNA with the cation N-cyclohexyl-N'-beta-(4-methylmorpholinium)-ethylcarbodiimide (CME). The limited activity of the enzyme on CMA-modified DNA makes it possible to demonstrate that the enzyme also initiates its attack on polydeoxyribonucleotides at the 5'-termini. This was determined by the analysis of the products from the digestion of CME-modified DNA containing labeled 5'-terminal phosphate groups. Such procedure can be adopted as a general approach for the determination of the direction of hydrolysis of other processive exonucleases. SP3 DNAase has been shown able to degrade oligo- and polydeoxyribonucleotides with or without 5'-terminal phosphate groups with equal efficiency (Aposhian, H.V., Friedman, N., Nichihara M., Heimer, E.P., and Nussbaum, A.L. (1970) J. Mol. Biol. 49, 367-379). The present work also shows that the enzyme can even hydrolyze oligo- and polynucleotides containing derivatized phosphate groups.

Bacillus subtilis↗