Uracil incorporation into nucleic acids

The penicillin-inhibited incorporation of uracil-C into nucleic acids resulted in a corresponding accumulation in the nucleotides. The separation... 

Pyrimidine 1,3-diazine, a heterocyclic compound, consisting of a six-membered ring with 2 nitrogen atoms (Fig.l), M, 80.1, m.p. 20-22°C, b.p. 124°C. The P. ring system is present in many natural compounds, e. g. antibiotics (nucleoside antibiotics), pterins, purines and vitamins, it is especially important in the pyrimidine bases. Cytosine (see). Uracil (see) and Thymine (see), which are constituents of nucleic acids. Pyrimidine itself does not occur naturally. Pyrimidine analogs (see) can also be incorporated into nucleic acids. 

Rat liver catabolizes uracil at a high rate, and, as has been mentioned in Chapter 12, this base is consequently not readily incorporated into nucleic acids. However, as shown by the data in Table 13-1 (S), by increasing the concentration of uracil the catabolic enzyme system of rat liver slices can be saturated, and significant amounts are then incorporated into RNA. 

Incorporation. The structural analogs can be incorporated into nucleic acids or proteins instead of the natural building blocks. In this way 2-thiouracil and 5-fluoruracil are incorporated into RNA instead of uracil, 5-bromouracil into DNA instead of thymine, and ethionine into protein instead of methionine. These substitutions lead to altered nucleic acids or proteins which cannot carry out their functions or do so inadequately. Thus, one speaks sometimes of fraudulent nucleic acids or proteins. 

Uracil mustard is not a uracil antagonist for E. coli Bu, which requires this nutrilite [479]. However, in rats with Walker 256 carcinosarcoma, incorporation of uracil-2- C into the RNA of intracellular tumour and spleen fractions is markedly inhibited [488]. UracU mustard also inhibits the incorporation of nucleic acid precursors into RNA and markedly inhibits DNA synthesis in tissues of A/J mice [489]. Inhibition of arginine incorporation into nuclear proteins was likewise noted [490]. 

A study of the mechanism of uracil incorporation into uridine phosphates was carried out in the Ehrlich ascites tumor, a tissue which utilized uracil as well as small molecule precimsors for nucleic acid formation (312). Uridine 5 -phosphate (UMP) was formed from uracil, ATP, and ribose 1-phosphate (R-l-P). Uridine was an intermediate in the formation of the nucleotide and was formed by the reaction of uracil and R-l-P with pyrimidine nucleoside phosphorylase (313, 314). Nucleoside kinase reacted the nucleoside with ATP to form UMP. The sequence is ... 

Hotchkiss (1949) found that suspensions of Staph, aureus incubated with mixtures of amino acids, purines, and pyrimidines in the presence of penicillin took up more uracil than in the absence of penicillin. It is possible that there is a connection between these various investigations. Since there is no obvious alteration in the uracil content of the staphylococcal nucleic acid formed in the presence of penicillin (Gale and Folkes, 1953a Park, private communication) it seems that the formation of the uridine-5 -pyrophosphate derivatives may represent a stimulation of a side reaction rather than a side-tracking of the incorporation of uracil into nucleic acid. On the other hand, should the incorporation of an essential base into nucleic acid be inhibited, it is probable that the whole nucleic acid synthesis would cease rather than that the relative proportions of the bases within the nucleic acid should change. The true answer must probably await the development of a method for the study of nucleic acid synthesis in detail. George and Pandalai (1948) have claimed that the action of penicillin can be reversed by the addition of nucleic acid to cultures. 

Azauracil [1,2,4-triazine-3,5(2,4)-dione] inhibits the growth of various micro-organisms. When grown in the presence of 6-azauracil- -C, Streptococcus jaecalis accumulates radioactive metabolites in the acid-soluble fraction of the cells. A major metabolite is D-ribofuranosyl-6-aza-uracil. This material is identical with material prepared by condensing tri-O-benzoyl-D-ribofuranosyl chloride with the mercuric derivative of 6-azauracil, followed by debenzoylation. A second major metaboUte was tentatively shown to be D-ribosyl-6-azauracil 5-phosphate. Bacteria develop resistance against 6-azauracil and its D-ribosyl derivative. Resistant Streptococcus faecalis will not convert 6-azauracil to its D-ribosyl derivative or to other bound forms, and the bacterium has also lost the ability to incorporate uracil into the nucleic acids of its cells. 

The binding properties of 2 -0,-4 -C-ethylene bridged nucleic acids (ENA) (65) have been examined and compared with LNA. NMR demonstrated that ENA adopts the iV-conformation, as does LNA. In hybridisation studies, ENA showed an enhanced affinity towards RNA, but was slightly less stable than the corresponding LNA analogues. However, it was reported that ENA exhibits superior nuclease resistance to LNA. The bicyclic uridine nucleoside (15,5S,6S)-6-hydroxy-5-hydroxymethyl-l-(uracil-l-yl)-3,8-dioxabicyclo[3.2.1]octane (66) has been incorporated into ODNs where it was expected to restrict the sugar moiety into an OA -endo conformation. It was found that the presence of the analogue caused some destabilisation in duplexes with either DNA or RNA, thus a locked nucleic acid does not always lead to duplex stability. 

In 1968, my graduate student Charles Walsh and I addressed the following question What are the pyrimidine sources for nucleic acid synthesis by Plasmodium lophurae We found the parasite synthesized pyrimidines de novo (Walsh and Sherman, 1968b). The evidence for a de novo synthesis was the presence of the key enzymes, thymidylate synthetase and oroti-dine-5-monophosphate pyrophosphorylase, as well as the demonstration of the incorporation of 14C-bicarbonate into cytosine, uracil and thymine. Finding a de novo pathway for the synthesis of pyrimidines by the malaria parasite would, in the next three decades, provide a biochemical basis for the mechanism of action of anti-folate anti-malarials as well as contributing to an understanding of the unique properties of the malaria parasite mitochondrion. 

Eimeria tenella. It appears that the sporulated oocysts of E. tenella are capable of de novo pyrimidine synthesis since sporozoites incorporate " C-labeled aspartate and orotate into pyrimidine nucleotides (103). Besides de novo synthesis, E. tenella is also capable of pyrimidine salvage. E. tenella will incorporate uracil, cytidine and uridine but not thymidine into its nucleic acids (104). This parasite is dependent on UMP for the synthesis of TMP and thymidylate synthase activity is present in extracts of E. tenella (105). As with both Plasmodium and the kinetoplastids, the thymidylate synthase of E. tenella exists as a bifunctional protein (91). 

Both 5-hydroxyuracil (isobarbituric acid, I) and 5-hydroxyuridine (II) can be prepared from the corresponding 5-bromopyrimidines by mild basic hydrolysis [5-8]. 5-Hydroxyuracil inhibits enzymatic degradation of uracil [9] and is one of three pyrimidine analogues (the other two are 5-bromouracil and 6-azauracil) that inhibit the rate of incorporation of uracil into nucleic... 

A great many fraudulent bases related to cytosine and its nucleosides have been synthesised, either with the chemotherapeutic object of inhibiting one or more enzymatically controlled anabolic processes involved in nucleic acid syntheses, or of attempting incorporation into the DNA or RNA, with the object of producing mutations of the normal replication. Chemical reactions have also been carried out on the intact nucleic acids, for this purpose. Among the latter, one which is highly specific to cytosine is the reaction with hydroxylamine. This apparently adds first across the 5,6-double bond, followed by conversion of the 4-amino group to —NffOH, elimination of the first hydroxylamine, and hydrolysis to uracil [280, 281]. The G-C pair is... 

They demonstrated that N -labeled ammonium salts were rapidly incorporated into the nucleic acids of the internal organs of the rat, and that the extent of labeling of the pyrimidines was approximately equal to that of the purines. Since the pyrimidine ring is structurally part of the completed purine molecule, the question naturally arose at the time as to whether pyrimidines might be the precursors of the purine molecule. The later studies of Plentl and Schoenheimer showed that the N -labeled pyrimidines, uracil and thymine (Fig. 8), when fed to rats, did not appear in either the pyrimidine or purine bases of the nucleic acids. The other naturally occurring pyrimidine base, cytosine (Fig. 8), was investigated... 

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