Inhibition of Growth of Lactobacillus bulgaricus by Purine Deoxyribonucleotides.

Morris, George K. (University of Georgia, Athens), and William L. Williams. Inhibition of growth of Lactobacillus bulgaricus by purine deoxyribonucleotides. J. Bacteriol. 90:715-719. 1965.-An inhibition of growth of Lactobacillus bulgaricus GS was observed with deoxyadenylic acid and deoxyguanylic acid. Deoxynucleotides of cytosine, thymine, and uracil, and the deoxynucleosides of adenine, guanine, cytosine, and thymine were inactive as inhibitors. The inhibition was reversed by liver extract (a crude source of two unidentified growth factors for this organism). With suboptimal concentrations of liver extract, the inhibition was reversed by nucleotides of adenine, guanine, uracil, cytosine, and thymine. When the medium contained partially purified sources of the two growth factors rather than crude liver extract, fewer compounds reversed the inhibition. Adenylic acid and guanylic acid reversed the action of either inhibitor. Inosinic acid reversed inhibition caused by deoxyguanylic acid, but not that caused by deoxyadenylic acid. Thymidylic acid reversed inhibition caused by deoxyadenylic acid better than that caused by deoxyguanylic acid. Uridylic acid and cytidylic acid were no longer effective in reversing the inhibitions. This organism preferentially responded to monophosphorylated compounds as inhibitors and as reversers of inhibitions. Studies on the acid-soluble nucleotide pool revealed an accumulation of adenosine triphosphate, guanosine triphosphate, and an unidentified compound which resembled a nucleotide in its physical properties. These data cannot be explained by known metabolic pathways of nucleic acid biosynthesis. This organism responds differently from other related organisms to nucleic acid derivatives; therefore, it may be a new useful tool for studying nucleic acid metabolism and biosynthesis.     

Further Nutritional Requirements of Paramecium aurelia

SYNOPSIS. An acetone-insoluble yeast fraction required for axenic growth of P. aurelia , stock 51, variety 4, sensitive , after fractionation contained at least 3 essential components: (1) one soluble in perchloric acid and completely replaceable by a mixture of ribosides or ribotides; (2) one inactivated after digestion with trypsin, chymotrypsin or papain. Proteose-Peptone restored activity to this preparation, which suggests a peptide requirement; and (3) one not yet characterized.
As for the purine and pyrimidines, these combinations, in decreasing order of activity, supported growth: guanosine + cytidine, guanosine + uridine, guanylic acid + cytidylic acid, and guanylic acid + uridylic acid. Each combination was maximally effective when the molar purine: pyrimidine ratio was ∼ 0.4. On a molar basis, the minimal riboside combinations were ∼ 1.3 × more active than the ribotides.
Sparing of the purine and pyrimidine requirement was also investigated. In the presence of limiting amounts of guanylic acid, the following compounds, in decreasing order of activity, had sparing activity: deoxyguanosine, inosine, xanthosine, adenylic acid, and guanine. Adenine, adenosine, and deoxyadenosine were inhibitory under the test conditions. The requirement for cytidylic acid was spared by deoxycytidine, uridine, uridylic acid, deoxyuridine, thymidine, thymine, and uracil, in descending order.     

Phagostimulation of larval Culexpipiens L. by nucleic acid nucleotides, nucleosides and bases

ABSTRACT. The influence of nucleic acid constituents on the rate of ingestion of charcoal powder filtered from ambient water by larvae of Culex pipiens L. was examined. All nucleotides tested stimulated ingestion to some extent. Various mono-, di- and tri-phosphates of adenosine were most effective and at concentrations of 1 mM stimulated ingestion nearly as well as yeast extract, a powerful phagostimu-lant. Guanylic, thymidylic, cytidylic and uridylic acids were less stimulatory, the latter two even at 10 mM. Cyclic AMP and deoxyadenylic acid were less effective than other adenine nucleotides. The nucleosides adenosine, guanosine and uridine were almost as effective as their corresponding nucleotides (adenylic, guanylic and uridylic acids); thymidine was less effective than thymidylic acids, whereas cytidine was non-stimulatory. Adenine, guanine, uracil and cytosine, the bases of the ribose nucleotides, were non-phagostimulatory, whereas thymine, base of the deoxynucleotide, thymidylic acid, caused low but significantly increased ingestion. These findings are compared with the reported phagostimulation by nucleic acid constituents of certain plant feeding insects and with the stimulation of engorgement of the blood meal by many blood feeding insects.     

Studies on the secondary structure of nuclear ribonucleic acids

Rat liver nuclei were separated into two fractions, the nucleolus and the nucleoplasm, by a combined salt-enzymic extraction. RNA was purified from both sources and analysed on sucrose density gradients. In both fractions a prominent heterogeneous RNA class with mean sedimentation coefficient of 18S was found. This material was analysed by measuring the rate of reaction with formaldehyde, the ultraviolet absorbance-temperature profile, the spectrophotometric observation of conformational changes as a function of pH, the spectrophotometric titration of uracil and guanine residues and the effect of both temperature and ionic strength on the spectrophotometric titration of cytosine residues. Nucleoplasmic 18S RNA fraction exhibited, on heating and also by adjustment of the pH to 2.5, a hyperchromicity of about 16%, close to that observed, in control experiments, for ribosomal RNA (22%). Titration of cytosine residues in solutions containing 1mm-NaCl and 0.1m-NaCl yielded pK values equal to 4.41 and 3.84 respectively. These results suggest that this RNA fraction is composed of structurally complex polymers. The hypochromicity of the nucleolar 18S RNA fraction determined by heating or adjusting the pH to 2.5, was not greater than 6% of the initial value. The rate of reaction with formaldehyde was 88% of that observed for the hydrolysed 18S fraction which suggested only 12% hydrogen bonding. pK values for uracil and guanine residues were 10.1 and 10.05 respectively. Titration of cytosine residues yielded a pK of 4.10, which was found to be independent of temperature and ionic strength variations.     

Photochemical demonstration of stacked C.C+ base pairs in a novel DNA secondary structure

The secondary structure of the alternating polydeoxynucleotide sequence poly[d(C-T)] was studied as a function of pH by ultraviolet absorbance and circular dichroism spectroscopy and by the analysis of UV-induced photoproducts. As the pH was lowered, poly[d(C-T)] underwent a conformational transition that was characterized by changes in the long-wavelength region (280-320 nm) of the CD spectrum. These changes have previously been interpreted as evidence for the formation of a core of stacked, protonated C X C+ base pairs in a double-helical complex of poly[d(C-T)], with the thymidyl residues being looped out into the solvent [Gray, D. M., Vaughan, M., Ratliff, R. L., & Hayes, F. N. (1980) Nucleic Acids Res. 8, 3695-3707]. In the present work, poly[d(C-T)] was labeled with [U-14C]cytosine and [methyl-3H]thymine and irradiated at pH values both above and below the conformational transition point (monitored by CD spectroscopy). The distribution of radioactivity in uracil means value of uracil dimers, uracil means value of thymine dimers (the deamination products of cytosine means value of cytosine and cytosine means value of thymine dimers, respectively), and thymine-means value of thymine dimers was then determined. As the pH was decreased, we found an increase in the yield of uracil means value of uracil dimers and a decrease in the yield of uracil means value of thymine dimers, which occurred concomitantly with the change in the CD spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nutrient media for plant-parasitic nematodes. VI. Nucleic acids content and nucleotide synthesis in Aphelenchoides rutgersi

The nucleic acids content of Aphelenchoides rutgersi, Hooper and Myers, was 0.9% DNA and 2.6% RNA dry weight. The DNA contained 29.5% adenine, 29.3% thymine, 22.5% guanine, and 18.8% cytosine, while the RNA was composed of 22.8% adenine, 23.0% uracil, 31.4% guanine, and 22.9% cytosine on a molar basis.The nematodes needed folic acid for reproduction regardless of the presence or absence of nucleic acid supplements in the culture medium. This was shown by including aminopterin, a folic acid antagonist in the culture medium. A 2-hr incubation of nematodes with glycine-¹⁴C (U) and orotic-5-³H acid resulted in the incorporation of ³H-label into both DNA and RNA. Only the RNA fraction contained a significant amount of ¹⁴C-label. When this RNA was fractionated, the adenine and guanine accounted for the ¹⁴C-label, while cytidylic and uridylic acids contained the ³H-label, thereby demonstrating purine and pyrimidine synthesis by A. rutgersi. The incorporation of orotic acid into the pyrimidines was 8 times higher than that of glycine into purines.     

Cross section calculations for electron scattering from DNA and RNA bases

Differential and integral cross sections for elastic electron collisions with uracil, cytosine, guanine, adenine and thymine have been calculated using the independent atom method with a static-polarization model potential for incident energies ranging from 50 to 4000 eV. Total cross sections for single electron-impact ionization of selected DNA and RNA bases have also been calculated with the binary-encounter-Bethe model from the ionization threshold up to 5000 eV. Cross sections within the investigated energy range, can be related to the molecular symmetry, the number of target electrons and molecular size; elastic and ionization processes are most efficient for guanine and adenine molecules, while the lowest cross sections were obtained for the uracil molecule. The ionization cross sections for cytosine, thymine, adenine and guanine are compared with those recently obtained with a semi-classical and binary-encounter-Bethe formalisms. No theoretical and experimental data for elastic electron scattering from DNA and RNA bases are available, but comparisons with calculations for molecules of similar size and geometry allows the validity of the theoretical approach to be verified.     

A spectrophotometric study of the secondary structure of ribonucleic acid isolated from the smaller and larger ribosomal subparticles of rabbit reticulocytes

The spectrum of RNA from the smaller and larger subparticles of rabbit reticulocyte ribosomes was studied as a function of pH, ionic strength, urea concentration and temperature. It was inferred that both RNA species form short double-helical segments of not more than about 10 base-pairs in length. Not more than about 70% of the base residues may be located in double-helical segments. RNA from the larger subparticle is richer in guanine and cytosine residues and its secondary structure is the more stable. These conclusions are based on the use of double-helical RNA from virus-like particles and of unfractionated Escherichia coli tRNA as model systems.     

Conserved portion in bacterial ribosomal RNA

The conserved portion in bacterial ribosomal RNA was studied by the DNA-RNA hybridization method. The hybridization percentages were as follows: Bacillus subtilis DNA and B. subtilis 23S rRNA, 0.16; Escherichia coli DNA and E. coli 23S rRNA, 0.15; B. subtilis DNA and E. coli 23S rRNA, 0.03; E. coli DNA and B. subtilis 23S rRNA, 0.04. The RNA's extracted from the heterologous hybrids could be rehybridized with DNA's of B. subtilis and E. coli. The average chain lengths of the RNA's were estimated by sucrose density gradient centrifugation and Sephadex gel filtration. The results suggested that the size might be larger than 30 nucleotides. Nucleotide compositions of the RNA's in the hybrids were also studied. Both RNA's contained higher molar percentages of guanylic acid and cytidylic acid than the whole rRNA's.     

Structural forms and transitions of poly(dG-dC) with Cd(II), Ag(I) and NaNO3

Infrared spectroscopy was used to study the structures and transitions in hydrated gels of double-helical poly(dG-dC) complexed with the metal carcinogens Cd(II) and Ag(I). For one Cd(II) per ten nucleotides (r = 0.1), the B structure was stable at high and moderate hydrations with D2O and the B and Z structures coexisted at low hydrations. For poly(dG-dC) with Cd(II) at r = 0.2 to 0.35, the Z structure was stable at high hydrations (94% r.h. for r = 0.2). At a given value of hydration, H2O gave a higher content of Z structure than D2O. Cd(II) most likely binds to guanine residues at N7 in both the B and Z forms of poly(dG-dC) but binding to guanine N3 can not be excluded. It is unlikely that Cd(II) binds to cytosine residues at the r values studied and the cytosine residues did not protonate at N3 as Cd(II) bound to guanine residues. Poly(dG-dC) with Ag(I) at r = 0.2 to 0.36, existed in a B-family structure which is different from the B-family structure of the type I complex of Ag(I) and calf-thymus DNA. Poly(dG-dC) with Ag(I) did not assume the Z structure at lower hydrations even though NO3- was present in the sample. Ag(I) differs from other soft-metal acids which promote the Z structure. Ag(I) most likely binds to the guanine N7 or N3 and not to cytosine residues. Cytosine residues did not protonate at N3 as Ag(I) was bound to guanine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uracil in formic acid hydrolysates of deoxyribonucleic acid

1. When DNA is hydrolysed with formic acid for 30min. at 175 degrees and the hydrolysate is chromatographed on paper with propan-2-ol-2n-hydrochloric acid, in addition to expected ultraviolet-absorbing spots corresponding to guanine, adenine, cytosine and thymine, an ultraviolet-absorbing region with R(F) similar to that of uracil can be detected. Uracil was separated from this region and identified by its spectra in acid and alkali, and by its R(F) in several solvent systems. 2. Cytosine, deoxyribocytidine and deoxyribocytidylic acid similarly treated with formic acid all yielded uracil, as did a mixture of deoxyribonucleotides. 3. Approx. 4% of deoxyribonucleotide cytosine was converted into uracil by the formic acid treatment.     

Characterization of Labeled Residues from 5-Diazouracil-2-C Incorporated into Ribonucleic Acids of Filamentous Escherichia coli

E. coli B, filamented with 5-diazouracil (DZU)-2-¹⁴C, yielded ribonucleic acid (RNA)-(DZU-2-¹⁴C) which was converted by pancreatic ribonuclease to ¹⁴C-mono-and oligo-nucleotides. The mixed ¹⁴C-mononucleotides isolated by diethylaminoethyl-cellulose fractionation were identified as cytidylic, uridylic, and hydroxyuridylic acids, by using a combination of paper chromatography and treatment with alkaline phosphatase and cytidine deaminase. Rifampin blocked incorporation of DZU-2-¹⁴C under conditions which inhibit RNA synthesis. Division inhibition by DZU-2-¹⁴C and the incorporation into Escherichia coli B were retarded by uracil but not by other RNA bases. In a pyrimidine-requiring E. coli, DZU substituted for uracil or cytosine to an extent limited by toxic effects. Cytosine and uracil retarded these effects and retarded the incorporation of DZU-2-¹⁴C into the pyrimidineless strain. A small proportion of DZU-2-¹⁴C was converted by the latter strain into hydroxyuridylic acid, but the bulk of the incorporated label was in cytidylic and uridylic acid, as in the wild strain.     

Enzymatic properties of glutamine 32 mutants of RNase Rh from Rhizopus niveus,a trial to alter the most preferential inter-nucleotidic linkages of RNase Rh

In order to investigate the effects of mutation of Gln32, a component of a base recognition site (B2 site) of a base-nonspecific RNase from Rhizopus niveus, we prepared several enzymes mutant at this position, Q32F, Q32L, Q32V, Q32T, Q32D, Q32N, and Q32E, and their enymatic activities toward RNA and 16 dinucleoside phosphates were measured. Enzymatic activities of the mutant enzymes towards RNA were between 10-125% of the native enzyme. From the rates of hydrolysis of 16 dinucleoside phosphates by mutant enzymes, we estimated the base specificity of both B1 and B2 sites. The results indicated that mutation of Gln32 to Asp, Asn, and Glu caused the B2 site to prefer cytosine more and to a less extent, to prefer uracil (Q32N), and that Q32F made the enzyme more guanine-base preferential. The results suggested that we are able to construct an enzyme that preferentially cleaves internucleotidic linkages, at the 5'-side of cytosine residues (Q32D, Q32N, and Q32E) and guanine residues (Q32F and Q32T), thus, cleaves purine-C(Q32D, Q32N, Q32E) and GpG and ApG (Q32F, and Q32T) most easily. The results seemed to suggest converting a base-non-specific RNase to a base-specific one.     

Reactions between Singlet Oxygen and the Constituents of Nucleic Acids: Importance of Reactions in Photodynamic Processes

Bases, nucleosides, nucleotides, and polynucleotides were exposed to chemically generated singlet oxygen to determine whether the species oxidized paralleled those oxidized in photodynamic reactions. In neutral or basic aqueous solution guanine, guanosine, deoxyguanosine, guanylic acid, deoxyguanylic acid, thymine, and uracil reacted with singlet oxygen. Since these compounds are oxidized in photodynamic processes, this study provides further evidence that singlet oxygen is the active intermediate in the photodynamic oxidation of nucleic acid constituents. Dienophilic attack by singlet oxygen is considered to be a plausible mechanism in these reactions.     

The vibrational spectra and structure of poly(rA-rU)-poly(rA-rU)

Infrared and Raman spectra of aqueous poly(rA-rU)·poly(rA-rU), the double-helical complex containing strands of alternating riboadenylate and ribouridylate residues, display significant differences from one another and from corresponding spectra of poly(rA)·poly(rU), the double-helical complex of riboadenylate and ribouridylate homopolymers. Parallel studies on the copolymer and homopolymer complexes by cesium sulfate density gradient centrifugation, ultraviolet absorption spectroscopy, hydrogenion titration, 1-N oxidation of adenine residues by monoperphthalic acid and X-ray diffraction reveal, however, that the geometry of base pairing between adenine and uracil is closely similar in each complex and apparently of the Watson-Crick type. Therefore the differences observed between vibrational spectra of poly (rA-rU)·poly (rA-rU) and poly(rA)·poly(rU) are not due to different base-pairing schemes but may be attributed to differences in vibrational coupling between vertically stacked bases. Vibrational coupling may also account for the differences between infrared and Raman spectra of the same complex. Thus, the present results indicate that infrared and Raman frequencies of RNA in the region 1750–1550 cm^?1 should be dependent on the base sequence.     

The quick approximation of DNA base composition from absorbancy ratios

The approximate base composition of pure deoxyribonucleic acid (DNA) can be quickly estimated from the absorbancy ratio E₂₆₀/E₂₈₀ in 0.1n acetic acid according to the empirical relation % GC=168.6–87.4 (E₂₆₀/E₂₈₀), valid in the range 40 to 70% GC (molar per cent guanine ... cytosine). The method is only accurate to within + 3% GC. It can be used when a quick, rough estimate of DNA base composition is required, e.g., to check the correct taxonomic position of new isolates or to give an approximation of the melting point T_m or buoyant density of an unknown DNA sample. The method can not be recommended for distinguishing between two genera with closely related % GC values, or for finer distinction within one genus.     

Characterization of Two Simian Virus 40-Specific RNA Molecules from Infected BS-C-1 Cells

Two discrete simian virus 40 (SV40) RNA species sedimenting at 19 and 16S, respectively, that are present in infected BS-C-1 cells were characterized with respect to the base composition and the ribonuclease T1 fingerprints. The base composition of the 19S SV40 RNA was found to be cytidylic acid (C), 23.0; adenylic acid (A), 28.3; guanylic acid (G), 23.9; and uridylic acid (U), 24.8; that of the 16S SV40 RNA was C, 19.3; A, 34.0; G, 22.0; and U, 24.7 mol%. Analysis of the ribonuclease T1 fingerprints indicated a difference in the base sequence of the 19 and 16S SV40 RNA. The presence of long sequences of adenylic acid residues (poly A) in these viral RNAs was confirmed.     

Selective inhibition of uracil tRNA methylases of E. coli by ethionine.

L-ethionine has been found to inhibit uracil tRNA methylating enzymes in vitro under conditions where methylation of other tRNA bases is unaffected. No selective inhibitor for uracil tRNA methylases has been identified previously. 15 mM L-ethionine or 30 mM D,L-ethionine caused about 40% inhibition of tRNA methylation catalyzed by enzyme extracts from E. coli B or E. coli M3S (mixtures of methylases for uracil, guanine, cytosine, and adenine) but did not inhibit the activity of preparations from an E. coli mutant that lacks uracil tRNA methylase. Analysis of the 14CH3 bases in methyl-deficient E. coli tRNA after its in vitro methylation with E. coli B3 enzymes in the presence or absence of ethionine showed that ethionine inhibited 14CH3 transfer to uracil in tRNA, but did not diminish significantly the 14CH3 transfer to other tRNA bases. Under similar conditions 0.6 mM S-adenosylethionine and 0.2 mM ethylthioadenosine inhibited the overall tRNA base methylating activity of E. coli B preparations about 50% but neither of these ethionine metabolites preferentially inhibited uracil methylation. Ethionine was not competitive with S-adenosyl methionine. Uracil methylation was not inhibited by alanine, valine, or ethionine sulfoxide. It is suggested that the thymine deficiency that we found earlier in tRNA from ethionine-treated E. coli B cells, resulted from base specific inhibition by the amino acid, ethionine, of uracil tRNA methylation in vivo.     

Sequence selective modification of DNA with muta-carcinogenic 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole

2-Acetoxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole binds covalently to the 8 position of guanine residues in DNA. Treatment of the modified DNA with aqueous piperidine causes the liberation of the modified nucleic acid base, 2-(C8-guanyl)amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole, and cleavage of DNA at the sites of the modified guanylic acid residues. By use of 5'-end 32P-labelled DNA and sequence analysing gel electrophoresis, we discovered the base sequence specificity of DNA modification with 2-acetoxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole. The guanine residues in G-C cluster-like regions are modified more frequently.     

Determination of trace amounts of 5-methylcytosine in DNA by reverse-phase high-performance liquid chromatography

A high-performance liquid chromatographic method to separate five major bases (cytosine, thymine, guanine, adenine, and uracil) and three minor methylated bases (5-methylcytosine, N6-methyladenine, and 7-methylguanine) has been developed using a volatile mobile phase under isocratic conditions. It is extended to quantitate 5-methylcytosine in trace amounts (1 in 20,000 cytosine residues). The suitability of the method has been verified by estimating 5-methylcytosine in DNAs of phi X174 and pBR322. The method has been applied to quantitate the extent of cytosine methylation in DNA of larval silk glands of Bombyx mori. Our results confirm that the pupal DNA of Drosophila melanogaster does not contain detectable amounts of 5-methylcytosine.