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PubMedDate not supplied

[Determination of the activity of a polynucleotide phosphorylase preparation from E. coli].

Thin-layer chromatography was employed to measure the activity of polynucleotide phosphorylase (PNP-ase) from E. coli during purification and in the resultant preparation. The TLC data were verified with respect to released inorganic orthophosphate and to 14C-AMP incorporation rate. It is concluded that TLC can be used to determine PNP-ase activity in crude extract, at various purification stages and in the final preparation, yielding correct results.

Chromatography, Thin Layer↗
PubMed1983

Inhibition of DNA polymerase-alpha from rat hepatoma with a series of new synthetic polynucleotides.

In continuation of efforts to correlate the antitemplate activities of chemically modified polynucleotides with their base composition and structure, four synthetic copolymers, poly(A,C), poly(C,U), poly(A,C,U), and poly(A,C,G) were modified by thiolation of 2.6-4.8% of their pyrimidine bases. The resulting 5-mercaptoheteropolynucleotides and the previously described 5-mercapto-polycytidylate (MPC) and -polyuridylate (MPU) were tested in a comparative manner as inhibitors of the DNA polymerase-alpha from rat hepatoma. A wide scale of inhibitory potencies was obtained in the following (decreasing) order: MPU greater than M-poly(A,C,U) greater than M-poly(C,U) greater than MPC greater than M-poly(A,C) greater than or equal to M-poly(A,C,G). The sensitivity of the hepatoma DNA polymerase toward these antitemplates increased upon further purification of the enzyme through the DNA-agarose step. Partially thiolated DNA-isolates from rat hepatoma and calf thymus, respectively, showed significant inhibition of the hepatoma DNA polymerase, the thiolated hepatoma DNA being the more active inhibitor.

Animals↗
PubMed1978

Polyadenylic acid-polyuridylic acid (poly A : U) and experimental murine brucellosis. I. Effect of single and double-stranded polynucleotides on Brucella abortus in vivo and in vitro.

The double stranded polynucleotide, poly A : U, when administered intraperitoneally at the same time as intravenous infection with Brucella abortus, suppressed the growth of that organism in the spleen and liver of mice. Single stranded poly A or poly U were not effective. On the other hand both the single and double stranded forms enhanced the growth of Br. abortus in broth culture. Poly A : U did not enhance the blood clearance of intravenously administered Br. abortus. Indeed its suppressive effect was not apparent until 2 days after administration. When mice given Br. abortus and poly A : U were given a second infection 2 days later, with either Br. abortus or Listeria monocytogens, the second infection was exacerbated, indicating a biphasic effect of poly A : U and antigen.

Animals↗
PubMed1978

Purification of specific DNA sequences by sulfhydryl-Sepharose chromatography of mercurated polynucleotides.

Recombinant plasmid DNA has been used to purify complementary cDNA by hybridization using a modification of sulfhydryl-Sepharose chromatography described by Dale and Ward ((1975) Biochemistry 14, 2458). Plasmid DNA containing cloned mouse globin or immunoglobulin sequences was mercurated and hybridized in solution to unpurified cDNA. The resulting hybrids were passed over a sulfhydryl-Sepharose column where mercurated polynucleotides are retained. After washing, cDNA hybridized to the mercurated plasmid DNA was melted in situ and eluted while the mercurated plasmid DNA remained bound to the column. The conditions for purification of DNA and RNA sequences are described. The purity of the cDNAs obtained by this method is analyzed by polyacrylamide gel electrophoresis and by hybridization. In addition, this nucleic acid purification procedure has been applied to two problems of general interest: (i) the sensitive titration of specific genes by saturation hybridization; (ii) the purification of DNA fragments bearing specific sequences from restriction endonuclease digests of total cellular DNA. The procedure is generally applicable to the purification by hybridization of any DNA or RNA sequence complementary to an available probe.

Base Sequence↗
PubMed1981

Specificity in the interaction of 4',6-diamidino-2-phenylindole with polynucleotides.

The binding of a fluorescent dye, 4',6-diamidino-2-phenylindole (DAPI) to polynucleotides was investigated by UV absorption and fluorescence spectroscopy. The results show that there are two types of bindings. In a higher concentration of DAPI, where UV spectroscopy is a suitable means for detecting the binding, the association of DAPI is independent of base compositions and the types of sugar moiety in spite of the spectral difference between DNA and RNA complexes with DAPI. On the other hand in a lower concentration of DAPI, where only fluorescence is measured to detect the binding, the degree of association is dependent upon the sugar types as well as base compositions.

Chemical Phenomena↗
PubMed1982

[Polynucleotide phosphorylase from rat liver: isolation and regulation of its activity by growth hormone].

Using ion-filtration chromatography on DEAE-Sephadex A-25, a homogeneous polynucleotide phosphorylase having specific activity of 350--360 u./mg and containing no admixtures of nucleases or phosphatases was obtained. Injection of 32P phosphate to hypophysectomized animals was accompanied by the label incorporation into the enzyme molecule. The data obtained indirectly indicate that the enzyme is activated by phosphorylation and is inactivated by dephosphorylation, both processes being mediated by some factor found in liver cytosol of intact animals.

Animals↗
PubMed1978

[Molecular weight distribution of hydrolysis products of polyuridylic acid by cobra venom ribonuclease. Preparation of polynucleotides of required molecular weight].

The composition of polyuridilic acid hydrolisis products with mol. weight 400.000--600.000 by endoribonuclease from cobra venom was studied. The molecular weight distribution of products formed at initial stages of hydrolysis was according to the regularities of accidental cleavage of phosphodiether linkages. The sufficient non-statistic regularities due to the high-molecular polynucleotide hydrolysis for the most part during further hydrolysis were observed out of the way of accumulation of oligonucleotides with a polymerity degree of about 15--20. As shown from the oligocytidilates hydrolysis, the velocity of process increased sufficiently with increasing substrate polymerity degree. The results obtained were used for preparation of polyuridilic fractions with a narrow range of molecular weight distribution.

Elapid Venoms↗
PubMed1981

[Polynucleotide phosphorylase from rat liver nuclei. Determination of the activity and some properties].

The activity of polynucleotide phosphorylase (PNPase) from rat liver nuclei isolated in aqueous and non-aqueous media was studied. It was shown that aqueous and non-aqueous nuclear preparations differ both in specific activity (106 and 39 units per mg of protein, respectively) and in the enzyme content (11.2 and 3.4% of homogenate activity, respectively). Nuclear PNPase reveals its maximal activity at pH 7.6-8.0 and requires Mg2+. The enzyme catalyzed polyribonucleotide phosphorelysis in the following order: poly (A) leads to poly(U) leads to poly(S) leads to RNA leads to poly(A) . poly(U). It is concluded that a comparatively low PNPase activity of the nuclei isolated in sucrose media is due to the enzyme extraction from the nuclei in the course of isolation.

Animals↗
PubMed1978

[Destruction of mRNA poly-A sequences by polynucleotide phosphorylase in rat liver microsomes].

A possible role of polynucleotide phosphorylase (PNPase) in the destruction of poly-A fragments located at the 3'-OH end of mRNA from rat liver polyribosomes was studied. Using hybridization of mRNP particles and mRNA with poly-U Sepharose 4B, it was found that polyribosomal PNPase in vitro destroys the poly-A sequences of approximately 25% of poly-A+ mRNA during the first minutes of incubation at a high rate. The destruction of the poly-A fragment of mRNA by PNPase is incomplete, since part of it is presumably protected by proteins firmly bound to the poly-A sequences of mRNA.

Animals↗
PubMed1980

[Purification and properties of a releasing factor of polynucleotide phosphorylase from sarcoma M-1 of rat].

A releasing factor (RF) of polynucleotide phosphorylase (PNPase) isolated and purified from sarcoma M-1 of the rat is a protein with molecular weight of about 8000 and the isoelectric point at 4,0--4,2. The process of PNPase release from the polyribosomes of normal rat liver does not depend on the pH of the medium within the pH range of 7,2--9,0, on temperature within the interval of 0--400 degrees and on incubation time. Inhibition of phosphorolysis by endogenous RNA from normal rat liver polyribosomes requires considerable amounts of RF--approximately 170--800 molecules of RF per molecule of poly(A)+-mRNA. The data obtained suggest that PNPase release from the polyribosomes under the action of RF is not a catalytic process and is caused by competition between RF and PNPase for the binding sites on the mRNA molecule.

Animals↗
PubMed1980

[Effect of growth hormone on polynucleotide phosphorylase activity in a primary monolayer culture of rat hepatocytes].

Growth hormone (1 microgram/ml) inoculated into the primary culture of rat hepatocytes and incubated for 3 hours at 37 degrees C decreases 2-fold the activity of polynucleotide phosphorylase (PNPase). In the culture of hepatocytes from hypophysectomized rats, the basal activity of PNPase is significantly higher as compared with that in the culture of hepatocytes from intact animals. The most pronounced inhibitory effect of growth hormone on PNPase was seen in the culture of hepatocytes from hypophysectomized animals.

Animals↗
PubMed1995

A carcinoembryonic antigen polynucleotide vaccine for human clinical use.

We have constructed a plasmid DNA encoding the full-length complementary DNA for human carcinoembryonic antigen (CEA) under transcriptional regulatory control of the cytomegalovirus early promoter/enhancer (pCEA) and demonstrated that this plasmid can function as a polynucleotide vaccine to elicit a CEA-specific immune response. This immune response protects against tumor challenge with syngeneic CEA-transduced colon carcinoma cells in mice. In the present work, the pCEA construct and purification method were modified to eliminate nonessential viral sequences, the ampicillin selectable marker, mutagens, and endotoxin to produce a reagent suitable for human clinical trials. The human use plasmid (pGT37) directs CEA expression at levels comparable with the original pCEA plasmid and can be propagated to yield large quantities of plasmid DNA based on kanamycin selection. A simple extraction technique greatly reduces contamination by endotoxin. Six weekly intramuscular injections of pGT37 elicited CEA-specific lymphoblastic transformation and antibody response in five of five mice and fully protected 10 of 10 mice against tumor challenge with syngeneic CEA-expressing colon cancer cells 42 days from the first plasmid injection. Thus, pGT37 encoding a tumor-associated antigen (CEA) has been shown to elicit cellular and humoral immune responses and mediate antitumor effects in vivo. This plasmid is suitable for human use and can be easily propagated in the laboratory.

Adenocarcinoma↗
PubMedDate not supplied

[Nucleotide composition of RNA, synthesized by polynucleotide phosphorylase, in toluene-treated cells of Escherichia coli].

On incubation of cells of E. coli B and MRE 600 (logariphmic phase of growth), treated with toluene in presence of a mixture 14C-nucleoside-5'-diphosphates, Mg2+ or Mn2+ and tris HCl buffer pH 8.0, intracellular synthesis of heteropolyribonucleotide was observed. The synthesis was catalyzed by polynucleotide phosphorylase (PNPase, E. C. 2.7.7.8). An increase in GDP concentration in the medium distinctly decreased the incorporation of other NDP into the polymer (poly-AGUC). If the ratio of ADP, UDP, CDP, GDP in the medium was 1:1:1:0.2, the composition of nitrogenous bases in the heteropolymer produced reflected completely the NDP concentrations in the incubation mixture. Addition of different amino acids (1-lysine, 1-histidine, glycine, 1-phenylalanine) and their mixtures stimulated poly-AGUC synthesis markedly and caused an appreciable alteration in the nucleotide composition of the poly-AGUC synthesized. This phenomenon resembled the effect of amino acids on the activity of partially purified PNPase and on RNA synthesis, catalized by the enzyme in vitro. These data suggest that in bacterial cell, i. e. in vivo, PNPase synthesizes specific RNA polyribonucleotide sequences, participating in protein synthesis or in its regulation.

Amino Acids↗
PubMedDate not supplied

The ability of polyamine analogues to induce Z-DNA structure in synthetic polynucleotides in vitro inversely correlates with their effects on cytotoxicity of cis-diaminedichloroplatinum (II) (CDDP) in human brain tumor cell lines.

We studied the effects of 72 h pretreatment with five polyamine analogues on the cytotoxicity of cis-diaminedichloroplatinum (II) (CDDP) in U-251 MG and SF-188 human brain tumor cells. A colony forming efficiency assay showed that the pretreatment with clinically important analogues 1,11-bis(ethylamino)-4, 8-diazaundecane (BE-3-3-3), 1,14-bis(ethylamino)-5,10-diazatetradecane (BE-4-4-4), and 1,l9-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) increased the cytotoxicity of CDDP by 1.3 to 2.3-fold; 1,19-diamino-5,10, 15-triazanonadecane (4-4-4-4) did not affect CDDP cytotoxicity, and 1,11-diamino-4,8-diazaundecane (3-3-3) protected cells from the cytotoxic effects of CDDP. An alkaline elution assay detected a small increase in DNA interstrand crosslinks accompanying the enhancement of CDDP cytotoxicity only in cells pretreated with BE-3-3-3. This study is the first to show that the Z-DNA inducing abilities of the polyamine analogues in synthetic polynucleotides in vitro correlates inversely with their effects on CDDP cytotoxicity in human tumor cells in culture.

Antineoplastic Agents↗
PubMed1976

Properties of a human liver ribonuclease. Inhibition by polynucleotides and specificity for phosphodiester bond cleavage to yield purine nucleosides at the 5' termini.

A ribonuclease, purified 2500-fold from human liver, was found to be inactive against synthetic homopolynucleotides, whereas synthetic co-polymers containing adenylic acid were rapidly degraded. The specificity of the RNase is unique in that only purine residues, in a 5:4 ratio of guanylic to adenylic acid, are found at the 5' termini of the degradation products of yeast RNA. No specificity was observed at the 3' termini of the fragments. When analyzed by DEAE-cellulose chromatography, approximately 80% of the oligonucletoides were 4 to 11 residues in length. The hydrolysis of RNA by the liver enzyme, when examined in low ionic strength buffer, could be increased severalfold over control levels by the addition of polyamines. The enzyme was found to exist as two distinct species on sucrose gradients, with molecular weights of 128,000 and 14,000. However, the addition of spermidine to the gradients resulted in the recovery of all the enzyme activity as the smaller species. The polyamines were also shown to reverse the inhibition of the enzyme by the ordered polynucleotides, polyguanylic acid and polyadenylic acid. Inhibition of enzyme activity by the polyadenylic acid segment of various mammalian mRNAs was also demonstrated.

Humans↗