Guanine incorporation into nucleic acids

Adenosine was shown to be incorporated into nucleic acid purines, but to only about half the extent of adenine itself (L2). The incorporation of guanosine was also studied and here again the guanine derivative was a poorer precursor than the adenine derivative, but the utilization of guanosine was considerably greater than that of the aglycone (L22). The administration of the adenine nucleotides revealed that these compounds, whether as the 3 isomer or the 5 isomer, were poorer precursors than the free base adenine (Wl). With all nucleotides studied, the incorporation of the 5 isomer was less extensive than of the 3 isomer (R9). Roll et al. have shown that guanine nucleotides are considerably... 

From the species names, one may recognize some of the various participants in purine metabolism. The important purines guanine (Gua) and adenine (Ade) are both synthesized from inosine monophosphate (IMP). They may then be derived to (d)GTP and (d)ATP, respectively, prior to incorporation into nucleic acids like DNA and RNA. This simple explanation betrays the true complexity of purine metabolism, and elucidating the properties of such complex systems depends on sophisticated computational tools, which we describe presently. 

The findings that inosinate was the end product of the pathway of de novo synthesis and that adenylate and guanylate were the first metabolites formed in the course of the incorporation into nucleic acids of adenine and guanine, were most consistent with interconversion at the ribonucleo-... 

There is little evidence regarding the cyclic operation of these reactions. One cycle may function in muscle where the extensive deamination of adenylate accompanying muscle function must be followed by its rapid resynthesis. McFall and Magasanik S3) have suggested that in cultured L cells, guanine is converted to adenine nucleotides for storage and then converted back to guanine nucleotides for incorporation into nucleic acids. 

In the above-mentioned experiment, labeled inosine, adenine, adenosine, and adenylic acid were incorporated into nucleic acid adenine to a greater extent than into nucleic acid guanine. This suggests that all the compounds, including inosine, may be related in their utilization by conversion to a common metabolite. 

Adenine is inert in the nucleoside phosphorylase systems of both mammalian tissues and microorganisms, but isotopically labeled adenine is effectively incorporated into nucleic acid purines, both in rats " and in yeast.This poses a question as to the possible role of nucleoside phosphorylase in polynucleotide synthesis. It has been suggested that hypoxanthine or guanine nucleosides (or nucleotides) are synthesized first. Then an exchange reaction with free adenine (or a derivative) might occur, For example, adenine might react with inosine to form adenosine and hypoxanthine. Some known exchange reactions are discussed below. 

The fate of other purine-ribose compounds was studied in the rat and it was found that C Mabeled adenosine (211) and adenylic acid (212) were utilized for the s3Tithesis of RNA adenine and guanine, but to a much smaller extent than adenine (191). Similarly, growing yeast utilized the purine base, adenine, far more readily than the corresponding nucleoside or nucleotide (195). It was believed that the ribose derivatives were poorly utilized because they were first cleaved to free adenine, which was incorporated subsequently into polynucleotides. It is curious that the attachment of ribose or a ribose pho hate moiety to adenine or guanine did not facilitate their incorporation into nucleic acids. In contrast, inosine, the ribonucleoside of hypoxantbine, was utilized considerably by the rat as a nucleic acid precursor (211) the corresponding deoxyriboside, deoxyinosine, was not (213). 

S-Adenosyl ethionine carries out ethylation reactions or ethyl transfer, and this is presumably involved in the carcinogenesis. Administration of ethionine to animals leads to the production of ethylated bases such as ethyl guanine. This may account for the observed inhibition of RNA polymerase and consequently of RNA synthesis. Incorporation of abnormal bases into nucleic acids and the production of impaired RNA may also lead to the inhibition of protein synthesis and misreading of the genetic code. 

M5. Mandel, H. G., and Carlo, P., The incorporation of guanine into nucleic acids of tumor-bearing mice. J. Biol. Chem. 201, 335-341 (1953). 

Hypoxanthine, on the other hand, which accounts for only a fifth or so of the urinary uric acid is an active intermediate. It is degraded to xanthine and then to uric add by xanthine oxidase. This enzyme is found mainly in liver, kidney, and bowel, while guanase is widely distributed and would quickly deaminate any guanine formed. The product xanthine is a poor substrate for hypoxanthine phosphoribosyltrans-ferase (HPRT). Most of the hypoxanthine formed is reutiliiced by conversion to inosinic acid. Similar conclusions were reached by Ayvazian and Skupp in 1965 when they administered C-labeled purines to patients (A2). Furthermore, these studies and those earlier studies show that the xanthine is converted to hypoxanthine, presumably at the nucleotide level, and on the basis of what we know about microorganisms, we would assume it to be via guanine nucleotides (M2). Since label was found in urinary 7-methylguanine as early as 4 hours after administration of C-labeled purines, and since methylation of RNA occurs at the macromolecular level (B13), interconversion must be rapid and incorporation of some of these products into nucleic acids must also occur quickly. 

The fact that purine bases could be used directly for nucleotide and nucleic acid i thesis was first established by the use of labeled compounds. Plentl and Schoenheimer (5) were not able to demonstrate incorporation of N- anine into nucleic acids of rat viscera in 1944, but Brown later was able to show that it was utilized by mice. (It is now known that guanine is degraded more rapidly in rats than in mice.) Brown and his colleagues (4, 5) also demonstrated the incorporation of N-adenine into nucleic acids, and between 1948 and 1954 numerous studies were made of the incorporation of N- or >KJ-labeled purines into nucleotides and nucleic acids (see reference g). 

Abrams and co-workers first showed that the precursors of nucleic acid purines were the same as those of uric acid by demonstrating the incorporation of N -glycine into adenine and guanine of the nucleic acids of growing yeast. Heinrich and Wilson, using the rat, also explored the precursors of purines in nucleic acids, and their results confirmed the earlier observations of Buchanan and collaborators for uric acid. It has now been well substantiated that formate, glycine, and CO2 aU con-... 

Autoradiography with labeled substrates on cultured fibroblasts has been used to visualize HG-PRT deficiency cells of deficient individuals lack the ability to incorporate hypoxanthine or guanine into nucleic acids, because these purine bases can not be converted to their corresponding mononucleotides (1,2). The alternative pathway to form IMP or GMP via inosine or guanosine is not likely, for, although nucleoside phosphorylase is present, there is no definite evidence for the existence of inosine- or guanosine kinase in human cells (3,4). 

The effect of 6-mercaptopurine on the incorporation of a number of C-labelled compounds into soluble purine nucleotides and into RNA and DNA has been studied in leukemia L1210, Ehrlich ascites carcinoma, and solid sarcoma 180. At a level of 6-mercaptopurine that markedly inhibited the incorporation of formate and glycine, the utilization of adenine or 2-aminoadenine was not affected. There was no inhibition of the incorporation of 5(or 4)-aminoimidazole-4(5)-carboxamide (AIC) into adenine derivatives and no marked or consistent inhibition of its incorporation into guanine derivatives. The conversion of AIC to purines in ascites cells was not inhibited at levels of 6-mercaptopurine 8-20 times those that produced 50 per cent or greater inhibition of de novo synthesis [292]. Furthermore, AIC reverses the inhibition of growth of S180 cells (AH/5) in culture by 6-mercaptopurine [293]. These results suggest that in all these systems, in vitro and in vivo, the principal site at which 6-mercaptopurine inhibits nucleic acid biosynthesis is prior to the formation of AIC, and that the interconversion of purine ribonucleotides (see below) is not the primary site of action [292]. Presumably, this early step is the conversion of PRPP to 5-phosphoribosylamine inhibited allosterically by 6-mercaptopurine ribonucleotide (feedback inhibition is not observed in cells that cannot convert 6-mercaptopurine to its ribonucleotide [244]. 

Once the fusion has taken place, it is necessary to eliminate any unfused myeloma cells and to select only hybrid cells secreting antibody. This is primarily achieved by the use of hypoxanthine aminopterin thymidine (HAT) media and cells that are deficient in the enzyme responsible for incorporation of hypoxanthine into DNA. Figure 3 illustrates this process. The unfused splenocytes are not immortal and naturally die off in culture. The elimination of the unfused myeloma cells is carried out by the initial use of mutant myeloma cells selected for a deficiency in the enzymes hypoxanthine guanine phosphoribosyl transferase (HGPRT) and thymidine kinase (TK), rendering them unable to use the salvage pathway for nucleic acid synthesis. The myelomas will die off... 

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