Radioactive Precursors into Nucleic Acids

Incorporation of Radioactive Precursors into Nucleic Acids A. Precursors of Purines and Pyrimidines 

Formate serves as a precursor of carbons 2 and 8 of the purine ring and of the 5-methyl group of thymine. Thus one may obtain information on the dynamic state of nucleic acids in tissues by measuring the rate of incorporation of injected formate-C into nucleic acids. It must be emphasized that the specific activities of isolated nucleic acids taken at early time intervals after injection of formate-C may reflect changes in pool size and specific activities of the precursors, either formate or the nucleotides, which are the immediate precursors of the nucleic acids, in addition to reflecting changes in the rate of nucleic acid synthesis. 

It was found that vitamin E deficiency in the rat resulted in an increase in the incorporation of formate-C into nucleic acids of liver and skeletal muscle (Dinning, 1955). The rats used in these experiments were only mildly deficient in vitamin E, and subsequent experiments were conducted on rabbits in which it is possible to produce severe vitamin E deficiency with relatively short feeding periods. Vitamin E deficiency in the rabbit led to an increased specific activity of nucleic acids in several tissues following formate-C injection (Dinning et al., 1955). Measurements of the specific activity of CO2 expired by these animals indicated that the overall formate pool size was not affected by vitamin E deficiency. The increased specific activity of the tissue nucleic acids then could represent an altered specific activity of the nucleotide precursors or an altered synthetic rate of the tissue nucleic acids. 

The nucleic acids from bone marrow and skeletal muscle of these animals were fractionated into RNA and DNA. It may be seen from the results summarized in Table II that vitamin E deficiency led to an increase in the incorporation of formate into both RNA and DNA of bone marrow and skeletal muscle. However, while the increase due to the deficiency was approximately the same in both RNA and DNA of bone marrow, vitamin E deprivation appeared to increase rather selectively the incorporation of formate into DNA of skeletal muscle. An increase in turnover rate of 

Similar experiments were conducted in monkeys, and it was found that vitamin P] deficiency resulted in increased incorporation of formate into the nucleic acids of skeletal muscle and bone marrow (Dinning and Day 

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More direct evidence that polymyxin synthesis proceeds by a mechanism that differs from that of protein synthesis comes from experiments with growing cultures of B. polymyxa in which the effects of inhibitors of protein and nucleic acid synthesis on the incorporation of radioactive precursors into protein and polymyxin B were studied (Paulus and Gray, 1964). As shown in Table 7, chloramphenicol, actinomycin D, and to a lesser extent puromycin inhibit the incorporation of L-threonine- C into protein but stimulate its incorporation into polymyxin B. This differential effect of inhibitors on protein and polymyxin synthesis strongly supports the hypothesis that polymyxin synthesis does not proceed by the kind of template mechanism that operates in protein synthesis. 

The use of radiolabeled nucleosides as markers for anticancer activity has become a popular method due to the commercial availability of such compounds. The technique is based upon the knowledge that cells rendered unable to replicate or killed by the anticancer agent are unable to effectively incorporate nucleic acid precursors into their DNA or RNA structure. Therefore, a decrease in cell viability correlates with a decrease in radioactivity relative to a control cell population. Although specific procedures differ, the basic technique involves the incubation of tumor cells in the presence of the radiolabeled compound with or without anticancer agent followed by scintillation counting to determine the radioacti vity of the samples. 

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