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D C Ward

Publications and source records attributed to D C Ward.

At least 253 records · Page 14Linked to original sources
PubMed1976

5-Iodo-5'-amino-2',5'-dideoxyuridine-5'-N'-triphosphate. Synthesis, chemical properties, and effect on Escherichia coli thymidine kinase activity.

5-Iodo-5'-amino-2',5'-dideoxyuridine-5'-N'-triphosphate (AIdUTP), a phosphoramidate analog of 5-iodo-2',5'-dideoxyuridine 5'-triphosphate (IdUTP), was synthesized and some of its chemical and biological properties were investigated. Although AIdUTP is stable in alkaline solutions, below pH 8 it undergoes degradation by a novel phosphorylysis reaction which exhibits first order kinetics. Inclusion of magnesium ion in the reaction mixture decreased the rate of degradation. Protonation of a group on AIdUTP which has a pKa of 6.10, presumably the secondary ionized oxygen on the gamma phosphate, precedes phosphorylysis. The only detectable reaction products are the nucleoside, 5-iodo-5'-amino-2',5'-dideoxyuridine (AIdUrd), and trimetaphosphate. A mechanism for the acid catalyzed phosphorylysis of AIdUTP is proposed. AIdUTP, like TTP, converts Escherichia coli thymidine kinase into an inactive dimer with a sedimentation coefficient of 5.78 S. AIdUTP is, however, 60-fold more potent as an allosteric inhibitor than is TTP at pH 7.8. Although the inhibitory effect of TTP is markedly reduced at high pH, the activity of AIdUTP is lowered only slightly. The allosteric effects of AIdUTP also differ from those of IdUTP, which is an inhibitor at low pH but a strong activator above pH 7.4. 5-Iodo-2'-deoxycytidine 5'-triphosphate, a potent enzyme activator, cannot completely reverse the AIdUTP inhibition, even when present at a 150-fold molar excess.

Escherichia coli↗
PubMed1976

Three structural polypeptides coded for by minite virus of mice, a parvovirus.

Purified full and empty virions of minute virus of mice were separated on CsCl gradients, and their polypeptides were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The empty particle contains two polypeptides, A (83,300 daltons) and B (64,300 daltons), which are 15 to 18% and 82 to 85%, respectively, of the virion mass. The full particle contains the single-stranded DNA genome, proteins A and B, and a third polypeptide, C (61,400 daltons). Again A is 15 to 18% of the protein mass, but the amounts of B and C vary inversely in different preparations of full particles. These polypeptides comprise greater than 99.6% of the protein in either virion, and their molecular weights and molar ratios are independent of the species of host cell on which the virus is propagated, They are not found in uninfected cells, and no protein component of uninfected cells copurifies with either virion under our conditions. Pulse-chase experiments show that the three proteins are synthesized only after virus infection and are therefore probably virus coded. Sequential harvesting from the nuclei of cells infected under one cycle growth conditions shows an increase in the proportion of C in full particles as infection progresses, suggesting that C is derived from B in a late maturation step.

Amino Acids↗
PubMed1976

DNA of minute virus of mice: self-priming, nonpermuted, single-stranded genome with a 5'-terminal hairpin duplex.

The genome of the nondefective parvovirus minute virus of mice (MVM) is a linear DNA molecular weight 1.48 x 10(6), which is single stranded for approximately 94% of its length. In contrast to the genomes from defective parvoviruses MVM DNA does not contain a detectable inverted terminal redundancy. A combination of enzymatic and physical techniques has shown that the molecule contains a stable hairpin duplex of approximately 130 base pairs located at the 5' terminus of the genome. MVM DNA is efficiently utilized as a template-primer by a number of DNA polymerases, including reverse transcriptases. Polymerases lacking 5' to 3' exonuclease activity yield a duplex DNA product with a molecular weight 1.96 times that of the viral genome, in which the newly synthesized complementary strand is covalently attached to the template. This duplex contains an internal "nick" that can be sealed by DNA ligase to produce a self-complementary single-strand circle. The MVM DNA duplex is cleaved twice by EcoR-RI restriction endonuclease to yield three distinct fragments in molar amounts. These results suggest that the initiation of DNA synthesis in vitro occurs at a point within 100 bases of the 3' end of the genome, using the 3' terminus of viral DNA as a primer, and that the sequence of nucleotides in the genome is not permuted.

Base Sequence↗
PubMed1976

Successful therapy of herpes hominis keratitis in rabbits by 5-iodo-5'-amino-2'5'-dideoxyuridine (AIU): a novel analog of thymidine.

The efficiency of 5-iodo-5'-amino-2'5'-dideoxyuridine (AIU) in the therapy of experimental herpes keratitis in rabbits has been examined. Virus infections were established bilaterally in 40 animals using herpes simplex, type 1 (NIH strain 11124). Twenty-four hours after infection the rabbits were divided into five matched groups of eight and each group was treated, double-blind, with topical drugs at four-hour intervals for a total of 72 hours. The solutions instilled were: (1) saline; (2) IdUrd, 1 mg. per milliliter; (3) AIU, 1 mg. per milliliter; (4) AIU, 4 mg. per milliter; and (5) AIU, 8 mg. per milliliter. Each eye was examined daily for 12 days and graded independently by two ophthalmologists. Although IdUrd and AIU (8 mg. per milliliter) were effective therapeutically, IdUrd had a greater effect. The AIU at 1 and 4 mg. per milliliter were less active, but showed more rapid healing than the saline control. Viral recovery studies are consistent with the clinical observations. A second independent experiment, similar to that described above, gave essentially identical results. Although less potent than IdUrd, AIU does provide effective therapy for herpes keratitis.

Administration, Topical↗
PubMed1975

Direct covalent mercuration of nucleotides and polynucleotides.

Nucleotides of cytosine and uracil are readily mercurated by heating at 37-50 degrees in buffered aqueous solutions (pH 5.0-8.0) containing mercuric acetate. Proton magnetic resonance, elemental, electrophoretic, and chromatographic analyses have shown the products to be 5-mercuricytosine and 5-mercuriuracil derivatives, where the mercury atom is covalently bonded. Polynucleotides can be mercurated under similar conditions. Cytosine and uracil bases are modified in RNA while only cytosine residues in DNA are substituted. There is little, if any, reaction with adenine, thymine, or guanine bases. The rate of polymer mercuration is, unlike that of mononucleotides, markedly influenced by the ionic strength of the reaction mixture: the lower the ionic strength the faster the reaction rate. Pyrimidine residues in single- and double-stranded polymers react at essentially the same rate. Although most polynucleotides can be extensively mercurated at pH 7.0 in sodium or Trisacetate buffers, tRNA undergoes only limited substitution in Tris buffers. The mild reaction conditions give minimal single-strand breakage and, unlike direct iodination procedures, do not produce pyrimidine hydrates. Mercurated polynucleotides can be exploited in a variety of ways, particularly by crystallographic and electron microscopic techniques, as tools for studying polynucleotide structure.

Chromatography↗
PubMed1975

Mercurated polynucleotides: new probes for hybridization and selective polymer fractionation.

Polynucleotides containing covalently bound mercury atoms have been prepared by chemical or enzymatic syntheses and some of their physical and biochemical properties studied. The mercury substituents do not appear to alter significantly normal polynucleotide structure. Mercurated polymers function efficiently as templates for nucleic acid polymerases, they are fully susceptible to degradation by standard nucleases, and their denaturation and reannealing properties resemble those of the corresponding nonmercurated polymers. While the Tm's of DNA duplexes are lowered by extensive mercuration, the Tm's of DNA-RNA hybrids and RNA duplexes are either unaffected or elevated. Mercuration, as would be expected, greatly increases the buoyant density of both DNA and RNA. The introduction of as few as one mercury atom per 200 bases permits the selective and quantitative retention of the mercurated polymer probe (and associated nucleotide sequences) on columns of sulfhydryl-agarose. The use of mercurated nucleotides (as polymerase substrates) and oligonucleotides (as primers) in conjunction with sulfhydryl-agarose chromatography provides a simple and efficient method for the isolation of selected polynucleotide sequences, such as specific in vitro transcription products or terminal fragments of duplex DNA. Products absorbed to the affinity resin are readily recovered for further analysis by eluting with buffers containing mercaptoethanol. Although the mercury-carbon bond is somewhat thermolabile, mercurated polynucleotides are suitable as probes in low temperature hybridization studies.

Carbon Radioisotopes↗
PubMed1975

Conversion of covalently mercurated nucleic acids to tritiated and halogenated derivatives.

Mercurated nucleic acids are converted to the corresponding tritiated, brominated, and iodinated derivatives by treatment with sodium borotritiide, N-bromosuccinimide, and elemental iodine, respectively. All three reactions occur under mild conditions in neutral aqueous solutions. Mercury-halogen conversions are essentially quantitative at both the mono- and polynucleotide levels. Tritiation reactions also proceed efficiently with mononucleotides, although polymers undergo incomplete demercuration. In spite of the latter limitation , these reactions provide novel and efficient synthetic routes to radiolabeled nucleic acid derivatives.

Borohydrides↗
PubMed1975

Selective inhibition of herpes simplex virus by 5-amino-2,5-dideoxy-5-iodouridine.

5-Amino-2,5-dideoxy-5-iodouridine, a nel thymidine analogue, is a potent inhibitor of herpes simplex virus type 1 replication. In contrast to most other nucleoside analogues which possess antiviral activity, 5-amino-2,5-dideoxy-5-iodouridine exhibits little, if any, cellular toxicity. Preliminary evidence suggests that 5-amino-2,5-dideoxy-5-iodouridine selectively inhibits viral-specific DNA synthesis.

Amines↗
PubMed1973

The synthesis and enzymatic polymerization of nucleotides containing mercury: potential tools for nucleic acid sequencing and structural analysis.

A simple acetoxymercuration reaction for introducing covalently bound mercury atoms into nucleotides is described. The 5-mercuriacetate derivatives of UTP, CTP, dUTP, and dCTP, as well as the 7-mercuriacetate derivative of 7-deazaATP, have been prepared by this procedure and tested as substrates for nucleic acid polymerases. These nucleotides, in the absence of added mercaptan, are not polymerized and in most instances are potent enzyme inhibitors. However, conversion of these mercuriacetates to mercurithio compounds in situ by the addition of one of various mercaptans, yields nucleoside triphosphates that are excellent substrates for all polymerases tested: Escherichia coli and T7 RNA polymerases, DNA polymerase I of E. coli, DNA polymerase of avian myeloblastosis virus, and calf-thymus terminal deoxynucleotidyl transferase. By varying the mercaptan used to promote syntheses it is possible to access certain structural limitations in the enzyme's nucleoside triphosphate binding site. These mercurinucleotides appear to have a diversity of potential applications: (1) as heavy-atom reagents for crystallographic and microscopic studies; (2) as affinity probes for enzymes sensitive to sulfhydryl modification; (3) as steric probes of substrate-binding sites on enzymes; and (4) as reagents for forming covalent protein-polynucleotide complexes.

Adenine Nucleotides↗