HCN: A plausible source of purines,pyrimidines and amino acids on the primitive earth

Dilute (0.1 M) solutions of HCN condense to oligomers at pH 9.2. Hydrolysis of these oligomers yields 4,5-dihydroxypyrimidine, orotic acid, 5-hydroxyuracil, adenine, 4-aminoimidazole-5-carboxamide and amino acids. These results, together with the earlier data, demonstrate that the three main classes of nitrogen-containing biomolecules, purines, pyrimidines and amino acids may have originated from HCN on the primitive earth. The observation of orotic acid and 4-aminoimidazole-5-carboxyamide suggests that the contemporary biosynthetic pathways for nucleotides may have evolved from the compounds released on hydrolysis of HCN oligomers.     

Uracil synthesis via HCN oligomerization

Uracil is released from HCN oligomers upon acid hydrolysis in concentrations of 0.001% for 1 M HCN solutions to 0.005% for 0.1 M solutions. This yield is comparable with earlier reported, minor or nonbiological pyrimidines such as 5-hydroxyuracil and orotic acid. This is the first report of uracil itself via HCN oligomerization. Data are presented which establish that the observed uracil is not formed by decarboxylation of previously formed orotic acid, but via acid hydrolysis of at least two other precursors.     

Uracil synthesisvia HCN oligomerization

Uracil is released from HCN oligomers upon acid hydrolysis in concentrations of 0.001% for 1M HCN solutions to 0.005% for 0.1M solutions. This yield is comparable with earlier reported, minor or nonbiological pyrimidines such as 5-hydroxyuracil and orotic acid. This is the first report of uracil itselfvia HCN oligomerization. Data are presented which establish that the observed uracil is not formed by decarboxylation of previously formed orotic acid, butvia acid hydrolysis of at least two other precursors.     

Pyrimidine biosynthesis and its regulation in the estrogen-stimulated chick oviduct.

Studies on the incorporation of radio-labeled precursors into orotic acid and the pyrimidine nucleotides of RNA have established the occurrence of the orotate pathway for the de novo biosynthesis of pyrimidines in the chick oviduct. Measurements of the rate of incorporation of precursors into orotic acid in minces of oviduct revealed the activity of the orotate pathway to be accelerated in response to estrogen-stimulated nucleic acid synthesis and tissue growth. These data indicate that extrahepatic tissues of avian species meet their requirements for pyrimidine nucleotides through de novo synthesis rather than depend upon the liver or other exogenous sources for a supply of preformed pyrimidines. An examination of the influence of pyrimidine and purine nucleosides on the incorporation of radio-labeled precursors into orotic acid yielded evidence that pyrimidine biosynthesis in the chick is quite sensitive to inhibition by both purines and pyrimidines; the data indicate the reaction catalyzed by carbamoylphosphate synthetase to be the site of inhibition in both cases.     

The Cold Origin of Life: B. Implications Based on Pyrimidines and Purines Produced From Frozen Ammonium Cyanide Solutions

A wide variety of pyrimidines and purineswere identified as products of a dilute frozen ammoniumcyanide solution that had been held at –78°C for 27 years.This demonstrates that both pyrimidines and purines couldhave been produced on the primitive earth in a short time byeutectic concentration of HCN, even though the concentrationof HCN in the primitive ocean may have been low. We suggestthat eutectic freezing is the most plausible demonstratedmechanism by which HCN polymerizations could have producedbiologically important prebiotic compounds.     

Radiation-induced conversion of 5,6-dihydropyrimidines to the corresponding pyrimidines in N2O-saturated aqueous solutions

The effect of transition metal salts on the radiation-induced conversion of 5,6-dihydropyrimidines to the corresponding parent pyrimidines was studied in N2O-saturated aqueous solution at pH 7.0. The yield of the pyrimidines increased in sigmoidal forms with the increased one-electron reduction potential of the transition metal salts. The radiolysis of 5,6-dihydroorotic acid suggested that the 6-yl radical of the acid undergoes oxidation by transition metal salts to give orotic acid, whereas the corresponding 5-yl radical readily liberates CO2 to give uracil radical anion.     

Biomolecules from HCN

The mechanism of the condensation of dilute aqueous solutions of HCN and the products formed by these reactions have been investigated. The initial HCN condensation reactions yield3, a compound which is readily oxidized to4. A similar oxidation of5 to6 was also observed. Urea is formed on hydrolysis of4. The oxidation-reduction products formed from HCN may be in part a consequence of the oxidation of3. It has been established by combination GC/MS that the amino acids glycine, diaminosuccinic acid, α-amino-isobutyric acid, aspartic acid, alanine and isoleucine are released on acid hydrolysis of the ‘HCN polymer’. Hydantoin (7), 5,5-dimethylhydantoin (8) and 5-carboxymethyldenehydantoin (10) are also released on acid hydrolysis of the HCN condensation products. The direct conversion of the dicarbonyl derivative, of diaminosuccinic acid to orotic acid via10 at pH 8 has been observed. This conversion suggests a direct route to pyrimidines from HCN.     

Oligonucleotide-directed triple helix formation at adjacent oligopurine and oligopyrimidine DNA tracts by alternate strand recognition.

A significant limitation to the practical application of triplex DNA is its requirement for oligopurine tracts in target DNA sequences. The repertoire of triplex-forming sequences can potentially be expanded to adjacent blocks of purines and pyrimidines by allowing the third strand to pair with purines on alternate strands, while maintaining the required strand polarities by combining the two major classes of base triplets, Py.PuPy and Pu.PuPy. The formation of triplex DNA in this fashion requires no unusual bases or backbone linkages on the third strand. This approach has previously been demonstrated for target sequences of the type 5'-(Pu)n(Py)n-3' in intramolecular complexes. Using affinity cleaving and DNase I footprinting, we show here that intermolecular triplexes can also be formed at both 5'-(Pu)n(Py)n-3' and 5'-(Py)n(Pu)n-3' target sequences. However, triplex formation at a 5'-(Py)n(Pu)n-3' sequence occurs with lower yield. Triplex formation is disfavored, even at acid pH, when a number of contiguous C+.GC base triplets are required. These results suggest that triplex formation via alternate strand recognition at sequences made up of blocks of purines and pyrimidines may be generally feasible.     

Structural studies on HCN oligomers

Summary NMR spectral studies on the HCN oligomers suggest the presence of carboxamide and urea groupings. The release of CO₂, H₂O, HCN, CH₃CN, HCONH₂ and pyridine on pyrolysis is consistent with the presence of these groupings as well as carboxylic acid groups. No basic primary amine groupings could be detected with fluorescamine. Hydrazinolysis of the HCN oligomers releases 10% of the amino acids normally released by acid hydrolysis. The oligomers give a positive biuret test but this is not due to the presence of peptide bonds. There is no conclusive evidence for the presence of peptide bonds in the HCN oligomers. No diglycine was detected on partial hydrolysis of the HCN oligomers at pH 8.5 suggesting that HCN oligomers were not a source of prebiotic peptides.Chemical Evolution 38. For the previous papers see Ferris JP, Rao RV, Newton TA (1979). J Org Chem 44:4378–4381, 4381–4385; Ferris JP, Edelson EH, Mount NM, Sullivan AE (1979) J Mol Evol 13:317–330     

Influence of Purines and Pyrimidines on Cold Hardiness of Plants. III. Associated Changes in Soluble Protein and Nucleic Acid Content and Tissue pH

When applications of certain purines and pyrimidines enhanced the development or maintenance of cold hardiness, the content of water-soluble, trichloroacetic acid-precipitable protein and nucleic acids and tissue pH were higher in treated plants than in controls. The reverse was generally true when the treated plants were less cold hardy than the controls. In some instances, the purines and pyrimidines increased the content of these nitrogenous constituents in a nonhardy variety to a level equal to that found in untreated plants of a hardy variety.     

Purine and pyrimidine inhibitors of arginase.

1. Adenosine, inosine, adenine and uric acid are competitive inhibitors and cytidine and cytosine noncompetitive inhibitors of bovine liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1). 2. The affinity of the enzyme for these inhibitors was 10--100 times as great as for substrate in terms of Ki versus Km. 3. These nucleic acid metabolites may thus function in vivo to regulate the urea cycle. 4. Several naturally occuring competitive and noncompetitive inhibitors of arginase of unknown structure have been isolated from plant and animal tissue. From their properties and methods of isolation, they may be the purines and pyrimidines herein described. 5. These purines and pyrimidines have no effect on tryptic hydrolysis.     

Purine and pyrimidine contents of some desoxypentose nucleic acids

The distribution of purines and pyrimidines in desoxypentose nucleic acids prepared from a variety of animal and plant sources has been studied. 1. The nucleic acids were prepared from calf thymus, calf kidney, sheep spleen, horse spleen, chicken erythrocyte, turtle erythrocyte, trout sperm, shad testes, sea urchin sperm, wheat germ, and Pneumococcus Type III. 2. Separate hydrolyses were carried out for the determination of purines and pyrimidines. These procedures permitted nearly quantitative recovery of nucleic acid phosphorus in many of the preparations examined. 3. In the case of those preparations where a quantitative recovery was obtained it can be concluded that no bases other than adenine, guanine, thymine, and cytosine were present in appreciable amounts. 4. The distribution of purines and pyrimidines in all the nucleic acids studied renders the tetranucleotide hypothesis untenable. 5. The results of the analyses have indicated no great differences in the composition of these nucleic acids with respect to purines and pyrimidines.     

The Ultraviolet Light Inactivation of ΦX174 Bacteriophage at Different wave Lengths and pH's

The bacterial virus, ΦX174, which contains a single strand of DNA has been inactivated by different wave lengths of monochromatic ultraviolet light at pH 7, 2, and 12. The action spectra for inactivation at these three pH's all showed minima at 2400 A rather than at 2300 A, which is the characteristic absorption minimum of DNA. The shapes of the action spectra have been analyzed in terms of the effects of absorbed light on the pyrimidines and purines rather than the effect on nucleoprotein. In this interpretation the pyrimidines are at least 2 to 3 times more sensitive than the purines. The quantum yield for inactivation of the virus at 2650 A and pH 7 is 0.006. The quantum efficiency for quanta absorbed in the pyrimidines is 0.0085 and for the purines 0.0035. It is pointed out that action spectra for single- and double-stranded polynucleotides should have minima at different wave lengths, and that this difference may be used to distinguish between these two configurations in vivo.     

Phosphorylation and nucleotidylation of proteins in Rhodocyclus gelatinosus

Abstract Cells of Rhodocyclus gelatinosus were radioactively labeled by addition of [³²P]orthophosphate, [¹⁴C]inosine or [¹⁴C]orotic acid during anaerobic growth on citrate in the light. Protein analysis by two-dimensional gel electrophoresis and autoradiography of the gels revealed the presence of several radioactively labeled protein species in this organism. The molecular mass and the isoelectric point of all these proteins were determined. Treatment of the ³²P-labeled protein fractions with acid and alkaline phosphatase clearly showed that at least 8 protein species were modified by phosphorylation. The experiments conducted with the ¹⁴C-labeled precursors of purines and pyrimidines indicated the presence of 4 protein species which were modified by a compound containing a purine and phosphate, and a single protein simultaneously being labeled with pyrimidine and phosphate.     

Sensitivity of mitomycin C and nitrogen mustard crosslinks to extreme alkaline conditions

DNA-DNA crosslinks in cells treated with mitomycin C, nitrogen mustard, or decarbamoyl mitomycin C were measured in alkaline isopycnic gradients as a function of pH. Crosslinks from cells treated with mitomycin C and nitrogen mustard, which react with DNA purines, could be detected at pH 12.5 but not at pH 14. No crosslinks from cells treated with decarbamoyl mitomycin C were detected at either pH. Previous studies with cells exposed to psoralen derivatives plus 360 nm light, which produce DNA-DNA crosslinks with pyrimidines, demonstrated stable crosslinks at pH 14. These studies indicate that DNA-DNA crosslinks involving DNA purines are much less stable at high pH than those involving pyrimidines, and that methods involving exposure to extreme alkaline conditions may give inaccurate information for some agents.     

Pyrimidine biosynthesis and its regulation in the developing rat brain.

—Measurements of the incorporation of [¹⁴C]NaHCO₃ into orotic acid, uridine nucleotides and RNA in tissue minces establish the occurrence of the complete orotate pathway for the de novo biosynthesis of pyrimidines in rat brain. Selective inhibition of the incorporation of various radiolabelled precursors into orotic acid by uridine demonstrates the operation of a feedback control mechanism in brain minces and indicates carbamoylphosphate synthetase to be the site of inhibition; purine nucleosides were similarly found to inhibit the de novo biosynthesis of pyrimidines. The activity of the orotate pathway, as assessed by the rate of incorporation of [¹⁴C]NaHCO₃ into orotic acid, was found to be very high in fetal brain and to decline rapidly with neurological development; the mature rat brain exhibits less than 1% of the activity of the fetal brain at 18 days of gestation. Comparative studies on the ability of minces of the brain and several extraneural tissues to utilize [¹⁴C]NaHCO₃ and [¹⁴C]aspartate as precursors of orotic acid lead us to speculate that variations in the ability of tissues to synthesize orotic acid de novo are determined by similar variations in their ability to synthesize carbamoylphosphate.     

Triplex-forming oligonucleotides trigger conformation changes of a target hairpin sequence.

We used a DNA duplex formed between the 5' end of a 69mer (69T) and an 11mer (OL7) as a substrate for BamHI. The former oligonucleotide folds into a hairpin structure, the stem of which contains a stretch of pyrimidines in one strand and consequently a stretch of purines in the other strand. The oligomer 69T was used as a target for complementary oligodeoxypyrimidines made of 10 nt (OL1), 16 nt (OL5) or 26 nt (OL2) which can engage the same 10 pyrimidine-purine-pyrimidine triplets with the 69T hairpin stem. Although the binding site of OL7 did not overlap that of OL1, OL2 or OL5, the BamHI activity on 69T-OL7 complexes was drastically modified in the presence of these triplex-forming oligomers: OL1 abolished the cleavage by BamHI whereas OL5 and OL2 strongly increased it. Using footprinting assays and point-mutated oligonucleotides we demonstrated that these variations were due to different conformations of the 69T-OL7 complex induced by the binding of oligomers OL1, OL2 or OL5. Therefore, oligonucleotides can act as structural switchers, offering one additional mode for modulating gene expression.     

Nutrition of a Hemoflagellate (Leishmania tarentolae) Having an Interchangeable Requirement for Choline or Pyridoxal.

SUMMARY. Leptomonads of Leishmania tarentolae were grown continuously in a defined medium containing: inorganic salts', glucose, hemin, 17 amino acids, purines and pyrimidines, and a mixture of vitamins of the B group. In this medium the population of organisms reached about 20 to 50 million per ml. alter 1 week at 27°C. Only slightly better growth occurred in a partially defined medium containing bovine plasma fraction V. In earlier experiments, however, omission of the plasma fraction resulted in decreased growth, and under these circumstances cholesterol or lecithin had growth-stimulating effects. In later experiments in the fully-defined medium no effect of these lipids could be found. The leptomonads were shown to require at least the following substances: inorganic salts; a source of purines and pyrimidines; tryptophan and the nine other amino acids essential for the growth of rats, glutamic acid, tyrosine, proline, serine, one or more of the group alanine, glycine and aspartic acid; folic acid, biotin, pantothenic acid, nicotinamide, riboflavm, thiamine, and either pyridoxine plus choline or pyridoxal or pyridoxamine. Choline at 2 × 10⁻⁵ m gave optimal growth in the presence of pyridoxine at 1 × 10⁻⁵ m. In a medium with a suboptimal concentration of choline (0.4 × 10⁻⁵ m) the leptomonads grew through nine transfers but they were mostly somewhat rounded and aflagellate.     

Orotic acid biosynthesis in rat liver: studies on the source of carbamoylphosphate.

Measurements of the incorporation of radiolabeled precursors into orotic acid in tissue slices and minces provided evidence of the participation of the intramitochondrial carbamoylphosphate synthetase (CPSase-I) in the de novo biosynthesis of pyrimidines in rat liver. Ammonia, the only nitrogen source utilized by CPSase-I, markedly stimulated the incorporation of NaH¹⁴CO₃ into orotic acid in liver slices, and ornithine, which enhances the intramitochondrial consumption of carbamoylphosphate (CP) in citrulline synthesis, antagonized the stimulation by ammonia. Sensitivity of the incorporation of NaH¹⁴CO₃ into orotic acid to stimulation by ammonia was found to increase with age in concert with the emergence of CPSase-I in the liver during late fetal and neonatal development. Tissues lacking in CPSase-I activity did not exhibit the responses to ammonia and ornithine observed with the adult rat liver. While the occurrence of CPSase-I in the liver contributes extensively toward the exceptionally high capacity of that tissue for the de novo biosynthesis of orotic acid, our results also indicate that the physiological rate of orotic acid biosynthesis in rat liver is approximately one-third of capacity; the incorporation of NaH¹⁴CO₃ into orotic acid averaged 488 nmol/g of tissue in 3 h in the presence of toxic levels of ammonia, but declined to 160 nmol/g of tissue in 3 h when physiological levels of both ammonia and ornithine were provided. However, the rate of orotic acid biosynthesis observed with physiological concentrations of ammonia and ornithine could be reduced further, to about one-quarter of the physiological rate, by providing additional ornithine; thus, physiological levels of ornithine do not prevent the escape of intramitochondrial CP into the cytoplasm. Finally, over 80% of the incorporation of NaH¹⁴CO₃ into orotic acid at physiological levels of ammonia and ornithine was found to be ammonia dependent, and all but a small fraction of the ammonia-dependent incorporation could be blocked by providing ornithine in amounts in excess of physiological. These results indicate that CPSase-I is the major source of CP in the biosynthesis of hepatic pyrimidines under normal (physiological) conditions as well as in ammonia toxicity.     

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.