Effect of uracil on colonial morphology ofNeisseria gonorrhoeae

The production of cells ofNeisseria gonorrhoeae that form type-T4 colonies in cultures started with cells that originally formed only type-T2 colonies was inhibited by calf thymus RNA. Guanosine and uracil were the only nucleic acid constituents that significantly reduced the T2 to T4 shift. Uracil gave the best results in degree of inhibition. It was found that some tritiated uracil was incorporated into the RNA of growing cells ofN. gonorrhoeae but that much more was incorporated into DNA probably after conversion to guanine and adenine. The data show that the shift from T2 to T4 can be progressively inhibited by increasing the concentration of uracil in the growth medium.     

Intranuclear incorporation of thymic low molecular weight RNA by murine bone marrow immunoblasts and inhibition of plasma cell formation by a derivative of rifampicin

An in vitro culture system for the proliferation of IgG-forming plasma cells from mouse bone marrow cultures has previously been described. The present study attempts to elucidate the mode of action of thymic RNA in these cultures. Autoradiography after using radiolabeled thymic RNA showed that radioactive material was mainly incorporated into the nuclei of IgG-forming plasma cells. No radiolabeled thymic RNA was incorporated into the cells except immunoblasts. The incorporated thymic RNA was acid insoluble and digested by RNase, but resistant to DNase and pronase. Radioactivity in the nucleotide pool after the cells were cultured with radiolabeled thymic RNA was negligible, indicating that reutilization of degraded RNA did not occur in the nuclei of the plasma cells. Moreover, the incorporation of radiolabeled thymic RNA by the cells was not prevented by excess unlabeled nucleosides. Escherichia coli transfer RNA, L-cell RNA and synthetic polynucleotide poly(A-U) were incorporated but were distributed in a different manner in the cells. A derivative of rifampicin, 2'5'-dimethyl N(4') benzyl-N(4')[desmethyl]rifampicin (AF/ABDMP), a possible inhibitor of RNA-dependent DNA polymerases, suppressed both the incorporation of thymic RNA and the differentiation of immunoblasts. AF/ABDMP suppressed DNA synthesis by bone marrow cultures to the same level as those pretreated with anti-mouse B-cell antibodies and complement. DNA dependent RNA polymerase activity was observed in the supernatant of bone marrow cultures stimulated by normal syngeneic thymic RNA and human gammaglobulin as antigen. These results imply a possible relationship between B-cell differentiation and RNA-dependent DNA polymerases.     

Incorporation of zebularine from its 2'-deoxyribonucleoside triphosphate derivative and activity as a template-coding nucleobase

Zebularine (1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) was studied as both a 2 '-deoxyribosyl 5 '-triphosphate derivative and as a template incorporated into an oligonucleotide. Using a novel pyrosequencing assay, zebularine acted as cytosine analog and was incorporated into DNA with a template pairing profile most similar to cytosine, pairing with greatest efficiency opposite guanine in the template strand. Guanine was incorporated with greater affinity than adenine opposite a zebularine in the template strand. Computer modeling of base-pairing structures supported a better fit of zebularine opposite guanine than adenine. Zebularine acts as a cytosine analog, which supports its activity as an inhibitor of cytosine methyltransferase.     

Incorporation of Zebularine from its 2′-Deoxyribonucleoside Triphosphate Derivative and Activity as a Template-Coding Nucleobase

Zebularine (1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) was studied as both a 2 ′-deoxyribosyl 5 ′-triphosphate derivative and as a template incorporated into an oligonucleotide. Using a novel pyrosequencing assay, zebularine acted as cytosine analog and was incorporated into DNA with a template pairing profile most similar to cytosine, pairing with greatest efficiency opposite guanine in the template strand. Guanine was incorporated with greater affinity than adenine opposite a zebularine in the template strand. Computer modeling of base-pairing structures supported a better fit of zebularine opposite guanine than adenine. Zebularine acts as a cytosine analog, which supports its activity as an inhibitor of cytosine methyltransferase.     

Simultaneous Rates of Ribonucleic Acid and Deoxyribonucleic Acid Syntheses for Estimating Growth and Cell Division of Aquatic Microbial Communities

A method for measuring rates of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) syntheses using a single radioactive precursor has been devised and tested using bacterial cultures and natural assemblages of marine and freshwater microorganisms. The procedure is based upon the uptake and incorporation of exogenous [³H]adenine into cellular adenosine triphosphate and deoxyadenosine triphosphate pools which serve as the immediate precursors for the adenine incorporated into RNA and DNA, respectively. It is proposed that the DNA/RNA rate ratio is correlated with the specific growth rate of microorganisms and can be used as an index for estimating and comparing the productivities of microbial assemblages in nature. This technique can also be used to detect discontinuous growth and cell division processes which frequently occur in surface plankton populations. The DNA/RNA rate ratios measured in a variety of aquatic ecosystems ranged from 3.3 to 31.8% without significant correlation to total microbial biomass.     

Purine metabolism in Toxoplasma gondii

We have studied the incorporation and interconversion of purines into nucleotides by freshly isolated Toxoplasma gondii. They did not synthesize nucleotides from formate, glycine, or serine. The purine bases hypoxanthine, xanthine, guanine, and adenine were incorporated at 9.2, 6.2, 5.1, and 4.3 pmol/10(7) cells/h, respectively. The purine nucleosides adenosine, inosine, guanosine, and xanthosine were incorporated at 110, 9.0, 2.7, and 0.3 pmol/10(7) cells/h, respectively. Guanine, xanthine, and their respective nucleosides labeled only guanine nucleotides. Inosine, hypoxanthine, and adenine labeled both adenine and guanine nucleotide pools at nearly equal ratios. Adenosine kinase was greater than 10-fold more active than the next most active enzyme in vitro. This is consistent with the metabolic data in vivo. No other nucleoside kinase or phosphotransferase activities were found. Phosphorylase activities were detected for guanosine and inosine; no other cleavage activities were detected. Deaminases were found for adenine and guanine. Phosphoribosyltransferase activities were detected for all four purine nucleobases. Interconversion occurs only in the direction of adenine to guanine nucleotides.     

The formation of acetylaminofluorene adducts in poly(dC-dG) and poly(dA-dT) on reaction with N-acetoxy-2-acetylaminofluorene and the effect of such modification upon the polymers as templates for DNA polymerases

N-Acetoxy-2-acetylaminofluorene (AcO-AAF) reacts with the alternating DNA-like polynucleotides poly(dC-dG) and poly(dA-dT) in vitro to give adducts of the guanine and adenine bases similar to those reported to be formed in DNA. A previously unobserved guanine adduct was detected in the poly(dC-dG). Using a double-labelled [U-14C-dG, 8-3H-G]-poly(dC-dG) we show that this adduct does not involve the 7- or 8-positions of the guanine. Similarly a thymine adduct of unknown structure was observed in poly(dA-dT). Modification of the polymers with AcO-AAF inhibits their capacity to act as templates for Escherichia coli DNA polymerase I and mammalian DNA polymerase alpha although the binding of the polymerases to the polynucleotides is unaffected. Such modification also leads to an increase in the levels of non-complementary nucleotides incorporated into newly synthesised DNA.     

Adenine and guanine 8CH exchange in nucleic acids: resolution and measurement by Raman optical multichannel analysis

Deuterium exchange of 8C protons of adenine and guanine in nucleic acids is conveniently monitored by laser Raman spectrophotometry, and the average exchange rate so determined [kA + kG] can be exploited as a dynamic probe of the secondary structure of DNA or RNA [J. M. Benevides and G. J. Thomas, Jr. (1985) Biopolymers 24, 667-682]. The present work describes a rapid Raman procedure, based upon optical multichannel analysis, which permits discrimination of the different 8CH exchange rates, kA of adenine and kG of guanine, in a single experimental protocol. For this procedure, simultaneous measurements are made of the intensity decay or frequency shift in separately resolved Raman bands of adenine and guanine, each of which is sensitive only to 8C deuteration of its respective purine. Resolution of the rates kA and kG is demonstrated for the mononucleotide mixtures, 5'-rAMP + 5'-rGMP and 5'-dAMP + 5'-dGMP, for the polynucleotides poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC), for calf thymus DNA, and for the 17 base-pair operator OR3. We show that the different exchange rates of adenine and guanine, in nucleotide mixtures and in DNA, may also be calculated independently from intensity decay of the composite 1481-cm-1 band, comprising overlapped adenine and guanine components, over a time domain that encompasses two distinct regimes: (1) a relatively more rapid exchange of guanine, and (2) a concurrent slower exchange of adenine. Both methods developed here yield consistent results. We find, first, that exchange of guanine is approximately twofold more rapid than that of adenine when both purines are present in the same structure and solvent environment, presumably a consequence of the greater basicity of the 7N site of guanine. Second, we find that adenine suffers greater retardation of exchange than guanine when both purines are incorporated into a "classical" B-DNA secondary structure, such as that of calf thymus DNA. This finding suggests different microenvironments at the 7N-8C loci of adenine and guanine in aqueous B-DNA. We also confirm that adenine residues of B-form poly(dA-dT).poly(dA-dT) exchange much more slowly than those of other B-DNA sequences, implying a secondary structure for the alternating-AT sequence with unusual stereochemistry in the major groove. The greater resistance of adenine than guanine to 8CH exchange in the B-DNA secondary structure is more evident in high molecular weight calf thymus DNA and in the alternating AT and GC copolymer duplexes than in the smaller 17 base-pair operator OR3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purine metabolism in mesophyll protoplasts of tobacco (Nicotiana tabacum) leaves.

The overall metabolism of purines was studied in tobacco (Nicotiana tabacum) mesophyll protoplasts. Metabolic pathways were studied by measuring the conversion of radioactive adenine, adenosine, hypoxanthine and guanine into purine ribonucleotides, ribonucleosides, bases and nucleic acid constituents. Adenine was extensively deaminated to hypoxanthine, whereupon it was also converted into AMP and incorporated into nucleic acids. Adenosine was mainly hydrolysed to adenine. Inosinate formed from hypoxanthine was converted into AMP and GMP, which were then catabolized to adenine and guanosine respectively. Guanine was mainly deaminated to xanthine and also incorporated into nucleic acids via GTP. Increased RNA synthesis in the protoplasts resulted in enhanced incorporation of adenine and guanine, but not of hypoxanthine and adenosine, into the nucleic acid fraction. The overall pattern of purine-nucleotide metabolic pathways in protoplasts of tobacco leaf mesophyll is proposed.     

Human mitochondrial DNA polymerase γ exhibits potential for bypass and mutagenesis at UV-induced cyclobutane thymine dimers

Cyclobutane thymine dimers (T-T) comprise the majority of DNA damage caused by short wavelength ultraviolet radiation. These lesions generally block replicative DNA polymerases and are repaired by nucleotide excision repair or bypassed by translesion polymerases in the nucleus. Mitochondria lack nucleotide excision repair, and therefore, it is important to understand how the sole mitochondrial DNA polymerase, pol γ, interacts with irreparable lesions such as T-T. We performed in vitro DNA polymerization assays to measure the kinetics of incorporation opposite the lesion and bypass of the lesion by pol γ with a dimer-containing template. Exonuclease-deficient pol γ bypassed thymine dimers with low relative efficiency; bypass was attenuated but still detectable when using exonuclease-proficient pol γ. When bypass did occur, pol γ misincorporated a guanine residue opposite the 3'-thymine of the dimer only 4-fold less efficiently than it incorporated an adenine. Surprisingly, the pol γ exonuclease-proficient enzyme excised the incorrectly incorporated guanine at similar rates irrespective of the nature of the thymines in the template. In the presence of all four dNTPs, pol γ extended the primer after incorporation of two adenines opposite the lesion with relatively higher efficiency compared with extension past either an adenine or a guanine incorporated opposite the 3'-thymine of the T-T. Our results suggest that T-T usually stalls mitochondrial DNA replication but also suggest a mechanism for the introduction of point mutations and deletions in the mitochondrial genomes of chronically UV-exposed cells.     

Proofreading of a mutagenic nucleotide, N4-aminodeoxycytidylic acid, by Escherichia coli DNA polymerase I

N4-Aminodeoxycytidine triphosphate, a putative metabolite of N4-aminocytidine which is a potent mutagen, is incorporated, in vitro, into polynucleotides in place of dCTP and at a much lesser extent, but significantly, in place of dTTP by E. coli DNA polymerase I large fragment. The activity of the polymerase to proofread this unnatural nucleotide has now been investigated. The results indicate that the 3'-5' exonuclease in the polymerase recognizes N4-aminocytosine as an incorrect base when N4-aminocytosine is incorporated opposite adenine but the enzyme cannot distinguish N4-aminocytosine from cytosine when it is incorporated opposite guanine.     

Incorporation of label from root-applied N6[8-14C]furfiuryIadenine into the guanine nucleotide fraction of pea bud ribonucleic acid

The pattern of incorporation of label into the nucleotides of axillary bud ribonucleic acid was investigated in Pisum sativum L. cv. Meteor following the application of N ⁶[8-^I4C]furfuryladenine or of [8-¹⁴C]adenine to the root system of decapitated plants and to cultured excised buds. When N ⁶[8-¹⁴C]furifaryladenine was applied to the root system label was confined to the guanine nucleotide moiety of the axillary bud ribonucleic acid; label from [8-¹⁴C]adenine was incorporated preferentially into adenine nucleotide in the molar ratio adenine nucleotide/guanine nucleotide = 3.23. When isolated buds were incubated in media containing [8-¹⁴C]adenine or N ⁶[8-¹⁴C]furfuryladenine, label was incorporated into both purine moieties of the ribonucleic acid. However, the relative incorporation into the guanine nucleotide fraction was considerably greater for N ⁶[8-^I4C]furfuryladenine (adenine nucleotide/guanine nucleotide = 2.23) than for [8-¹⁴C]adenine (ratio = 4.67).
It was concluded that the pattern of metabolism of adenine to guanine and its incorporation into the guanine nucleotide moiety of pea axillary bud ribonucleic acid, is influenced by the presence of a substitution in the N ⁶ position of the adenine base.     

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.     

Purine deoxynucleoside salvage in Giardia lamblia

Giardia lamblia is dependent on the salvage of preformed purines and pyrimidines, including deoxythymidine. Dependence on deoxynucleoside salvage is extremely unusual among eucaryotic cells (Moore, E. C., and Hurlbert, R. B. (1985) Pharmacol & Ther. 27, 167-196). The present study investigates the possibility that giardia lacks ribonucleotide reductase and depends entirely on deoxynucleoside salvage. A ribonucleotide reductase inhibitor, hydroxyurea, at concentrations up to 2 mM had no effect on the growth of giardia. This is 15-20 times the ED50 of hydroxyurea for the protozoans Trypanosoma cruzi, Trypanosoma gambiense, and Leishmania donovani. A lysate of giardia had no detectable ribonucleotide reductase. Although radiolabeled adenine, adenosine, guanine, and guanosine were readily incorporated into RNA by cultured cells, no adenine or adenosine and only trace amounts of guanine and guanosine were detectable in DNA. This is in contrast to deoxynucleosides, where 58% of deoxyadenosine and 10% of deoxyguanosine incorporated into nucleic acid were found in DNA. Phosphorylation of both deoxyadenosine and deoxyguanosine was catalyzed by a cell lysate of giardia when nucleoside kinase co-substrates were included in the assay but not when phosphotransferase co-substrates were present. The absence of detectable ribonucleotide reductase, the failure to incorporate purine nucleobases and nucleosides into DNA to any significant extent, the ready incorporation of deoxynucleosides into DNA, and the demonstration of a purine deoxynucleoside kinase suggest that giardia are dependent on the salvage of exogenous deoxynucleosides.     

Induction of sporulation by inhibitory purines and related compounds.

Sporulation of Bacillus subtilis can be induced, in the presence of excess ammonia, glucose and phosphate, by many purine derivatives under conditions of partial growth inhibition. Some of the compounds are known inhibitors of purine nucleotide synthesis. For most compounds the effect is counteracted by adenine and guanine. Partial growth inhibition by amethopterin (methotrexate) causes sporulation in the absence of purines but not in their presence. Unable to induce sporulation at any concentration are inhibitors of DNA, RNA, and protein synthesis as well as base or amino acid analogs that are incorporated into these polymers.     

Why are there four letters in the genetic alphabet?

We list, without thinking, the four base types that make up DNA as adenine, guanine, cytosine and thymine. But why are there four? This question is now all the more relevant as organic chemists have synthesized new base pairs that can be incorporated into nucleic acids. Here, I argue that there are theoretical, experimental and computational reasons to believe that having four base types is a frozen relic from the RNA world, when RNA was genetic as well as enzymatic material.     

Purine metabolism in trypanosomatids.

Purine nucleotide biosynthesis was studied in culture forms of Trypanosoma cruzi strain Y, Crithidia deanei (a reduviid trypanosomatid with an endosymbiote) and an aposymbiotic strain of C. deanei (obtained by curing C. deanei with chloramphenicol). Trypanosoma cruzi was found to synthesize purine nucleotides only fring incorporated into both adenine and guanine nucleotides. Similar results were obtained with guanine, indicating that this flagellate has a system for the interconversion of purine nucleotides. Crithidia deanei was able to synthesize purine and pyrimidine nucleotides from glycine ("de novo" pathway) and purine nucleotides from adenine and guanine ("salvage" pathway). Adenine was incorporated into both adenine and guanine nucleotides, while guanine was incorporated into guanine nucleotides only, indicating the presence of a metabolic block at the level of GMP reductase. The aposymbiotic C. deanei strain was unable to utilize glycine for the synthesis of purine nucleotides, although glycine was utilized for synthesizing pyrimidine nucleotides. These results suggest that the endosymbiote is implicated in the de novo purine nucleotide pathway of the C. deanei-endosymbiote complex. The incorporation of adenine and guanine by aposymbiotic C. deanei strain followed a pattern similar to that observed for C. deanei.     

Studies on the Pathway of Incorporation of 2-Aminopurine into the Deoxyribonucleic Acid of Escherichia coli

A pathway for the incorporation of 2-aminopurine into deoxyribonucleic acid (DNA) was studied in cell-free extracts of Escherichia coli. It was demonstrated that the free base can be converted to the deoxynucleoside, and that the deoxynucleotide can be phosphorylated to the di- and triphosphates and then incorporated into the DNA. From a consideration of the individual reactions in crude extracts, it is likely that the rate-limiting step in this pathway is the formation of the deoxynucleotide. Of especial interest is the observation that 2-aminopurine may be viewed as an analogue of either guanine or adenine, depending on which enzymatic step is being considered. On the one hand, it resembles guanine in that it is specifically converted from the mono- to the diphosphate by guanylate kinase and not by adenylate kinase. On the other hand, it replaces adenine rather than guanine in the DNA synthesized with purified DNA polymerases. E. coli DNA polymerase utilizes aminopurine deoxynucleoside triphosphate as a substrate for DNA synthesis much better than does purified phage T5-induced DNA polymerase and is also much less inhibited by this analogue than the T5 enzyme. These experiments in vitro correlate with known differential effects of 2-aminopurine on E. coli and phage in vivo.     

Purine salvage in Entamoeba histolytica

Pulse-labeling of the nucleotide pool in Entamoeba histolytica with radioactive precursors, and subsequent high performance liquid chromatographic (HPLC) analysis of the radiolabeled nucleotides, indicate that E. histolytica is incapable of de novo synthesis of purine nucleotides. Hypoxanthine, inosine and xanthine could not be converted to nucleotides in E. histolytica, which suggests the absence of interconversion between adenine nucleotides and guanine nucleotides through formation of IMP. Adenosine was actively incorporated into nucleotides at an initial rate of 130 pmoles per minute per 10(6) trophozoites. Adenine, guanosine and guanine were also incorporated at much lower rates. The rate of adenine incorporation was enhanced by the presence of guanosine; the rate of guanine incorporation was significantly increased by adenosine. These stimulatory effects suggest that the ribose moiety of adenosine or guanosine can be transferred to another purine base to form a new nucleoside, and that the purine nucleosides are the immediate precursors of E. histolytica nucleotides. HPLC results showed that the radiolabel in adenine was exclusively incorporated into adenine nucleotides and that guanine was found only among guanine nucleotides, whereas the radioactivity associated with the ribose moiety of adenosine or guanosine was distributed among both adenine and guanine nucleotides.