In vivo radiation-induced thymine residue release from E. coli DNA

Cells of $\text{E. coli}$ C thy^?321 are examined for thymine residue release from DNA following gamma-irradiation from 5 to 15 krad. Experimental conditions are designed to inhibit enzyme activity that might promote base residue release. Enzyme action is restricted in order to assess the physicochemical action of radiation on cellular DNA, and to this end irradiation is done under O₂, N₂, and N₂O saturating conditions. Both thymine and thymidine release from bacterial DNA are detected and quantitated, and three oxygen effects are noted in comparing yields of these products. No difference in effect is observed between N₂ and N₂O gassing conditions, suggesting that the hydroxyl radical has little effect on thymine or thymidine release from irradiated DNA $\text{in vivo}$.     

Radiation chemistry of nucleic acids: identification of the major hydroperoxy thymine

The major product from γ-radiation of thymine in aerated aqueous solution is shown to be $\text{cis}$-5-hydroxy-6-hydroperoxy-5,6-dihydrothymine by considering its uv, ir, nmr spectral data, the nmr deshielding effect on the OOH proton, and its reduction to $\text{cis}$-thymine glycol. These suggest that the earlier structural assignment [Cadet and Teoule, Biochem. Biophys. Acta., $\text{238}$, 8 (1971)] was in error. Furthermore, HOOH oxidation of either $\text{cis}$- or $\text{trans}$-thymine glycol in acidic condition gives this hydro-peroxide in yields >90%. This result again directly contradicts that reported in the previous paper. Our present findings are readily explained by considering the chemistry of thymine glycols and the reaction of OH radicals with thymine. Also, contrary to the earlier notion that this peroxide should be unstable, we find that it is sufficiently stable for studying the thymine peroxide interaction with chromosomes, bacterial cells and nucleic acid components.     

The further investigation of physical and photochemical properties of quasimetacyclophane derived from thymine

The thymine derived quasimetacyclophane exist in two conformers $\text{a}$ and $\text{b}$. The absorption spectra of $\text{a}$ and $\text{b}$ were evaluated and the conformational equilibrium in different solvents /H₂O : EtOH/ were examined. The rate constant k_?1 for reaction $\text{b}\text{→}\text{a}$ was established as well as E_a.     

The absence of ribothymidine in specific eukaryotic transfer RNAs. I. Glycine and threonine tRNAs of wheat embryo

Ribothymidine, generally considered a universal nucleotide in tRNA, is completely absent in five specific wheat embryo tRNAs. These consist of two species of glycine tRNA and three species of threonine tRNA. These tRNAs, all extensively purified, are acceptable substrates for $\text{E. coli}$ - ribothymidine forming-uracil methylase, which produces one mole of ribothymidine per mole of tRNA. These five tRNAs account for about 90% of the wheat embryo tRNAs which are substrates for this methylase. Nucleotide sequence analysis of one of these tRNAs, tRNA^Gly_I, confirmed both the complete absence of ribothymidine at position 23 from the 3′end, and the presence of uridine at that site instead. In addition, it is shown that methylation with $\text{E. coli}$ uracil methylase quantitatively converts uridine at position 23 to ribothymidine, while no other uridine in the molecule is affected.Using $\text{E. coli}$ uracil methylase as an assay we have detected this class of ribothymidine lacking tRNA, in each case consisting of a few specific species, in other higher organisms, such as wheat seedling, fetal calf liver and beef liver, in addition to wheat embryo. We could not detect this class of tRNA in $\text{E. coli}$ or yeast tRNA.     

Interconversion of thymine and thymidine in a thymine requiring strain of Bacillus subtilis

In a $\text{B. subtilis}$ Thy^? strain, thymidine is rapidly converted into thymine and, at the steady state, the pool size of thymidine is very small as compared to that of thymine. Consequently when such strain is used for pulse incorporation experiments with labelled thymidine paradoxical results are obtained. A quantitative estimation of the rate of DNA synthesis can only be obtained by thymine pulses or by cumulative incorporation experiments. We also pr$\text{e}$ sent evidence that, during a short pulse, thymidine is mainly utilized for replicative DNA synthesis.     

In vitro release of thymine from DNA by neocarzinostatin.

$\text{In vitro}$ degradation of DNA to acid soluble products was induced by the combined action of neocarzinostatin and sulfhydryl agent as 2-mercaptoethanol, dithiothreitol, or reduced glutathione, but not other reducting agent as ascorbic acid or NaBH₄. From the analysis by Sephadex G-10 gel filtration, acid soluble products were found to be thymine and oligonucleotide, but not thymidylic acid and thymidine. Release of adenine or guanine from DNA was not detected.From these results, it is suggested that DNA chain breakage by the combined action of neocarzinostatin and 2-mercaptoethanol may be due to an indirect phosphodiester bond breakage with release of thymine.     

Dibutyryl cGMP: inhibitor of the effect of cholecystokinin and gastrin on the guinea pig gallbladder in vitro

N², O²-di-butyryl guanosine 3′:5′ monophosphate (Bt₂ cGMP), a known competitive and selective inhibitor of the effect of cholecystokinin on the pancreatic acinar cells $\text{in}$$\text{vitro}$ was tested for its effect on the guinea pig gallbladder $\text{in}$$\text{vitro}$. Bt₂ cGMP inhibited competitively the contractile effect of cholecystokinin octapeptide, and also inhibited the contraction induced by sulfated gastrin-17. Bt₂ cGMP failed to inhibit the contraction induced by bombesin, acetylcholine or histamine. The 8-bromo derivative of cGMP and the dibutyryl derivative of cAMP did not affect contraction stimulated by cholecystokinin octapeptide. Since it is specific for gastrincholecystokinin peptides, and not restricted to the pancreas, Bt₂ cGMP could be used to recognize the action of these peptides.     

Crystal structure of an inhibitor and a model for inhibition of replicative DNA synthesis

6-(p-Hydroxyphenylhydrazino)-uracil is an antimicrobial agent that selectively blocks replicative DNA synthesis in Bacillus subtilis by inhibiting DNA polymerase III. The drug crystallizes as a monoclinic monohydrate, space group $\text{C2}\text{c}$, with $\text{a = 23.920(6)}\text{A}\text{̊}\text{, b = 5.587(3)}\text{A}\text{̊}\text{, c = 17.466(5)}\text{A}\text{̊}\text{, β = 101.45(8) °}$, and eight hydrated molecules per cell. Three-dimensional X-ray diffraction data were collected. The structure was solved by Patterson methods and refined to an R value of 6.8% for the 1651 data. The geometry of the uracil ring is unusual. The bond distances suggest that a resonance form involving a positively charged hydrazino nitrogen and a negatively charged carbonyl oxygen, O(4), makes a large contribution to the valence bond structure of this compound. The exocyclic C(6)N bond is short (1.335 Å), the C(6)C(5) bond distance is 1.371 Å, which is longer than in uracil, and the C(5)C(4) distance (1.396 Å) is short. The uracil ring, the linked hydrazino nitrogen, and the hydrogen on this nitrogen are in the same plane. Each uracil group is hydrogen bonded to a nearly coplanar uracil across a center of symmetry. The water molecule is also near the plane of these paired bases and forms a hydrogen bond with the uracil-linked hydrazino NH group. This paired base arrangement and the restricted rotation about the exocyclic C(6)N link that constrains the hydrazino NH group to lie near the uracil plane suggest a model for the interaction of the drug with template-primer DNA. The drug acts when cytosine is the base to be copied in the template strand, and the drug is competitive with dGTP. Both cytosine and guanine can be accommodated with little distortion of the crystal structure geometry in a manner compatible with the known geometry of DNA. The structural and biochemical aspects of the model for drug action are discussed.     

A highly sensitive method for the estimation of pyrimidine dimers in DNA by high-pressure liquid cation-exchange chromatography

We have developed a rapid, highly sensitive method for the separation of thymine-dimers from thymine on a cation-exchange resin, which is capable of detecting one dimer out of 5 × 10³ to 10⁴ thymine molecules (depending on the specific activity of the used material) by the means of high-pressure liquid chromatography. The assay has been very useful in DNA-hybridization studies using $\text{(}\text{TT}\text{)}$-containing DNA-strands and there is evidence that the method will be valuable for photoreactivation studies of uv-damaged DNA and for the enzymes involved in dimer-excision studies.     

Ligation of highly modified bacteriophage DNA

After digestion by TaqI or nicking by DNAase I, five highly modified bacteriophage DNAs were tested as substrates for T4 DNA ligase. The DNAs used were from phages T4, XP12, PBS1, SP82, and SP15, which contain as a major base either glucosylated 5-hydroxymethylcytosine, 5-methylcytosine, uracil, 5-hydroxymethyluracil, or phosphoglucuronated, glucosylated 5-(4′,5′-dihydroxypentyl)uracil, respectively. The relative ability of cohesive-ended TaqI fragments of these DNAs and of normal, λ DNA to be ligated was as follows: $\text{λ}\text{DNA}\text{=}\text{XP}\text{12}\text{DNA}\text{>}\text{SP}\text{82}\text{DNA}\text{?}\text{nonglucosylated}\text{T}\text{4}\text{DNA}\text{>}\text{T}\text{4}\text{DNA}\text{=}\text{PBS}\text{1}\text{DNA}\text{?}\text{SP}\text{15}\text{DNA}$. TaqI-T4 DNA fragments were also inefficiently ligated by Escherichia coli DNA ligase. However, annealing-independent ligation of DNAase I-nicked T4, PBS1, and λ DNAs was equally efficient. We conclude that the poor ligation of TaqI fragments of T4 and PBS1 DNAs was due to the hydroxymethylation (and glucosylation) of cytosine residues at T4's cohesive ends and the substitution of uracil residues for thymine residues adjacent to PBS1's cohesive ends destabilizing the annealing of the restriction fragments. Only SP15 DNA with its negatively charged, modified base was unable to serve as a substrate for T4 DNA ligase in an annealing-independent reaction; therefore, its modification directly interfered with enzyme binding or catalysis.     

A temperature-sensitive mutant of Escherichia coli with an altered RNA polymerase beta' subunit.

$\text{Penicillium charlesii}$ extracts contain UDP-galactose:NAD⁺ 2-hexosyl oxidoreductase (1). ADP-ribose also serves as a substrate resulting in formation of NADH and an oxidized ADP-ribose derivative. Treatment of the oxidized product with NaBH₄ followed by hydrolysis at pH 2 and 100° releases xylose as well as ribose. We conclude that ADP-D-$\text{glycero}$-D-$\text{glycero}$-3-pentosulose (ADP-3-ketoribose) is the product derived from ADP-ribose.     

Solubilization and partial purification of cytochrome P-450 from rat lung microsomes

Cytochrome P-450 from rat lung microsomes has been solubilized and purified 8-fold by using affinity chromatography on an ω-amino-$\text{n}$-octyl derivative of Sepharose 4B. The purified fraction was free of cytochrome $\text{b}$₅ and NADPH-cytochrome $\text{c}$ reductase and showed spectral characteristics similar to those of lung microsomal cytochrome P-450. When combined with NADPH-cytochrome $\text{c}$ reductase partially purified from liver microsomes, the cytochrome P-450 fraction supported the hydroxylation of benzo (α)pyrene and the activity was proportional to the content of the hemoprotein. No absolute requirement for phosphatidylcholine was found.     

The acid-base properties and kinetics of dissolution of the Fe4S4 cores of Chromatin ferredoxin and high potential iron protein.

From a study of the translation of synthetic polynucleotides in the $\text{E. coli}$ and $\text{B. thuringiensis}$ cell-free systems, it is shown that the stimulation of polypeptide synthesis by spermidine depends on the uracil content of messenger ribonucleic acid. This stimulation can not be fulfilled by any amount of Mg²⁺ in the absence of polyamines.     

Induction of thymidylate synthetase activity in Tetrahymena by cyclic guanosine monophosphate.

The induction of TMP synthetase activity in Tetrahymena pyriformis depended upon growth conditions. Enzymatic activity was low in cells grown in complex medium, and was high in cells grown in, or shifted to, defined medium. TMP synthetase activity rose 5 hours after the shift from complex to defined medium using uracil as the pyrimidine source. The time of induction was decreased to $\text{3}\text{1}\text{2}$ hours using dUMP as the pyrimidine source. cGMP or its dibutyryl derivative, but not cAMP, caused the induction of TMP synthetase activity in cells grown in complex medium. Caffeine, but not theophylline, mimicked the cGMP response. cAMP decreased both the cGMP and caffeine mediated increases in TMP synthetase activity. This is the first demonstration of an effect of cGMP on induction of an enzyme of pyrimidine metabolism in any cellular system.     

Comparison of the promoting activity of pristane and n-alkanes in skin carcinogenesis with their physical effects on micellar models of biological membranes

In 1976 (Horton, A.W., Butts, C.K. and Schuff, A.R. (1976) Colloid Interface Sci. 5, 159–168) we assayed pristance (2,6,10,14-tetramethylpentadecane) in a model interfacial system that has given excellent correlation with cocarcinogenic activity among $\text{n-}\text{alkanes}$, as tested in cycloalkane diluents. It was predicted that this branched-chain derivative of the diterpenes would have higher activity than $\text{n-}\text{C}{}_{18}\text{H}{}_{38}$, one of the most cocarcinogenic of the $\text{n-}\text{alkanes}$ in such diluents. Pristane was compared with $\text{n-}\text{C}{}_{18}\text{H}{}_{38}$ and two other $\text{n-}\text{alkanes}$ for their promoting activities in cyclohexane for C3H male mice after a single application of 7,12-dimethylbenz$\text{[a]}\text{anthracene}$. The branched-chain alkane proved to be more active. 20% $\text{n-}\text{tetracosane}$ in cyclohexane was inactive, which correlated with its effects in this diluent in the physical assay system. The promoting activity of 75% $\text{n-}\text{octane}$ in cyclohexane, predicted by the physical assay, was confirmed by tests on mice. The combined by-products of the synthesis of tetracosane, including C₁₂ alkanes and alkenes, C₁₉ and C₂₀ alkylbenzenes, and C₂₄ alkenes, proved to be a very active promoter. However, a mixture in cyclohexane of purified tetracosane with this composite of potential impurities was inactive. From the alkanes behavior in physical systems involving vatious membrane phospholipids and steroids, it is hypothesized that the primary step in their biological activity is a chain-chain interaction with membrane lipids that alters the properties of liquid-crystalline phases at aqueous interfaces. Resulting changes in the microfluidity of the lipid phase and the lateral mobility of critical hormone receptors and enzyme systems, such as the nucleotidyl cyclases, would perturb control systems that maintain the normal behavior of the initiated cell. Thus, its progression to a proliferating neoplasm may be favored.     

Effect of trypsin on DNA methylation in isolated nuclei from developing sea urchin embryos

Nuclei isolated from the developing sea urchin embryo $\text{Paracentrotus lividus}$ and incubated in the presence of [³H-methyl] S-adenosylmethionine methylate their own DNA. Addition of small amounts of trypsin produces a 20-fold increase in DNA methylation. The time kinetics and the specificity of the trypsin activation of DNA methylation are described. The only products of the reaction are 5-methylcytosine and thymine. DNA adenine, guanine and cytosine are not labeled. The distribution of the counts between 5-methyl-cytosine and thymine is variable. While 5-methylcytosine originates by enzymatic methylation of DNA cytosines, the origin of the labeled thymine cannot be inferred with certainty.     

Ordered transcription of RNA tumor virus genomes.

The crystal structure of sodium adenylyl-3′,5′-uridine (ApU) hexahydrate has been determined by X-ray diffraction procedures and refined to an R factor of 0.057. ApU crystallizes with two molecules per asymmetric unit in a monoclinic unit cell, space group P2₁, with cell dimensions: $\text{a = 18.025, b = 17.501, c = 9.677}\text{A}\text{?}\text{and}\text{β = 99.45 °}$. The two independent molecules of ApU form a small segment of right-handed antiparallel double-helical RNA in the crystal, with Watson-Crick base-pairing between adenine and uracil. This is the first time that this Watson-Crick base-pair has been seen unambiguously at atomic resolution and it is also the first time that a nucleic acid fragment with double-helical symmetry has been seen at atomic resolution. The distance between the C1′ atoma of the adenine-uracil base-pair is slightly shorter than the analogous distance seen in guanine-cytosine base-pairs. The bases in each strand are heavily stacked. One sodium cation binds to the phosphates, as expected; however, the other sodium cation binds on the dyad axis in the minor groove of the double helix. It is co-ordinated directly to the two uracil carbonyl groups which protrude into the minor groove and is shielded from the nearest phosphates by a shell of water. This binding appears to be sequence-specific for ApU. One of the adenines also forms a pair of hydrogen bonds to a nearby ribose, utilizing N6 and N7. The 12 water molecules per double-helical fragment are all part of the first co-ordination shell. The ions and the symmetry of the double-helical fragment are the major organizing elements of the solvent region.     

Excision repair of uracil during replication of phiX174 DNA in vitro.

Each of the stages in the replication of ØX174 DNA $\text{in vitro}$, e. g., conversion of circular single stranded parental DNA to the duplex replicative form (SS → RF), replication of the closed circular duplex form (RF → RF), and synthesis of circular single stranded progeny DNA (RF →SS), may be affected by a reduced level of dUTPase. Thus, in enzyme preparations from mutant strains defective in dUTPase ($\text{dut}$^?), the complementary strand synthesized in the SS → RF reaction is abnormally short (7–8S vs. 14S), and the extent of RF replication is decreased 10-fold. Preferential removal of dUTPase during fractionation of enzyme preparations from wild type ($\text{dut}$⁺) cells may produce comparable effects. In particular, the single stranded circular DNA synthesized in the RF → SS reaction by a set of highly purified enzymes is rapidly degraded upon incubation with the less pure enzymes required for its conversion to RF. All of these effects are plausibly accounted for by the incorporation into DNA of uracil from dUTP, possibly present as a contaminant in one or more components of the reaction, followed by excision of the uracil and phosphodiester bond cleavage at the resulting apyrimidinic site.     

Interaction of A-nor A.19-dinor,and A-homo-5α-androstane derivatives with the androgen receptor and the epididymal androgen-binding protein

The equilibrium affinity constant for rat prostate androgen receptor and epididymal androgen binding protein (ABP) has been determined for thirty-four potential progestogens. Three A-nor-, four A,19-dinor-, and one A-homo-5α-androstane derivative bind to the androgen receptor (K_D<0.5 μM). Five of these compounds also bind to ABP with an affinity of the same order of magnitude. “Anordrin” (compound $\text{24}$) and “Dinordrins” (compounds $\text{10, 14, 15, 16, 17}$), which are potential female contraceptives, do not bind with high affinity to the androgen receptor or to ABP. The following modifications in A-nor derivatives favour binding to the receptor as compared to ABP: 19-nor substitution (compound $\text{1}$), C-18 methyl homologation (compound $\text{5}$), 2α-ethinylation (compound $\text{22}$). One 2α-allenyl A-nor derivative (compound $\text{25}$) and one A-homo derivative (compound $\text{34}$) bind almost exclusively to ABP. The interaction with either binding protein is decreased by oxidation or esterification of the hydroxyl group at C-17, and by addition of a 17 α-ethinyl group. The latter modifications are likely to increase the specificity of androstane derivatives for receptors other than androgen binding proteins, such as the progesterone receptor.     

Neocarzinostatin: Chemical characterization and partial structure of the non-protein chromophore

The molecular formula C₃₅H₃₅NO₁₂ (mol.wt. 661) is proposed for the biologically active chromophoric component of neocarzinostatin. The partial structure $\text{2}$ is proposed based on ${}^1\text{H}$ NMR and mass spectral data and consists, in part, of a 2,6-dideoxy-2-methylamino-galactose moiety and a naphthoic acid derivative. Special treatments required to obtain spectral data of the labile chromophore are described.