Nucleic acids, fractionation

The 96-nucleotide primer can also be isolated by HPLC on RPC-5 at neutral pH (Eshaghpour and Crothers, 1978, Sanger et al., 1980). For a discussion on the application of this technique to nucleic acid fractionation see Wells et al. (1980). 

Recently we have thiolated nucleic acid fractions isolated from Ehrlich ascites (EA) cells. The activities of thiolated DNA, ribosomal RNA and transfer RNA were studied in a DNA-polymerase system from Friend leukemia virions. Of all the fractions tested transfer RNA, thiolated 1-3% showed a maximum inhibition of DNA polymerases of oncorna viruses. Table 23 shows some of the results obtained using thiolated tRNA. 

As follows from Table 23 the unmodified EA-tRNA does not inhibit the DNA polymerase activity of FLV at the concentrations used in the incubation mixture. However, at the same concentrations the thiolated EA-tRNA is a very strong inhibitor of DNA-polymerase activities, endogenous as well as template-dependent, of FLV. Experiments are in progress to modify nucleic acid fractions of viral origin. 

The importance of the photoreaction with fatty acids becomes more evident if we know the comparative and quantitative results of furanocoumarin photoreaction with different cell fractions. Caffieri, et al. [293] showed that the highest proportion of photobinding of 8-MOP was to lipid fraction (60%) followed by protein and nucleic acid fractions (20% each). Similar results have been reported for 4,6,3 -TMA [269,294]. 

Whereas sample preparation is a standard procedure, the most convenient procedures for sample recovery may be less well known to the reader. In SEC and RPC, nucleic acid fractions may be precipitated with ethanol after elution, with no added steps. In contrast, ethanol precipitation cannot be carried out with lEC and HIC because high salt concentrations would have to be used for elution and therefore, co-precipitation of the vast excess of salt would occur. The high sdt content could potentially be removed by dialysis, but such a procedure would be time consuming and possibly degradation of the nucleic acids could lead to the loss of sample. 

The demonstrated substrate activity of allopurinol and oxipurinol for nucleoside phosphorylases made this route plausible (Equation 1). However, conversion of the base to a ribonucleotide and subsequent dephosphorylation (Equation 2) could not be dismissed. A search for nucleotides in the acid soluble extracts of tissues and in nucleic acid fractions [1] gave negative results, but the methods permitted only the establishment of an upper limit of occurrence at about lO " M. Similarly, Kelley and Wyngaarden were unable to detect allopurinol derivatives among the cellular acid solubles [3]. Presently available, much more sensitive methods have now permitted the detection in tissues of ribonucleotides corresponding to all 3 of the ribonucleosides previously in hand. However, the precursors of the urinary ribonucleosides may not be predominantly the ribonucleotides, although the latter have been shown to be somewhat short-lived in tissues. 

Isopycnic separation depends solely on the buoyant densities of the particles to be separated and not on their shape or size. Thus, the method is not useful for separation of proteins many of which have similar buoyant densities Inspite of differences in molecular weights. It is however, very useful for separation of such intracellular organelles as mitochondria, lysosomes, and peroxisomes which do not differ in size but differ in their buoyant densities. The method is also useful for nucleic acid fractionation. 

All fractions are made up to measured volumes and aliquots are removed for analysis. RNA may be measured by the orcinol reaction and DNA by the di-phenylamine reaction, the values obtained being referred to those obtained upon standard solutions of pure RNA and DNA, respectively. (In the S method, these are determined in the one and only nucleic acid fraction.) In addition, total and inorganic phosphorus in each fraction may be measured. The assay procedures are described in detail in Section III. 

The addition of purines and pyrimidines accelerates the rate of protein synthesis in washed Staph, aureus as shown above. If penicillin in high concentrations is added to the incubation mixture, the additional protein synthesis due to the presence of purines and pyrimidines is abolished, although the basal protein synthesis is not significantly affected, as shown in Fig. 29. This effect is accompanied by a decrease in the purine-stimulated nucleic acid formation, but the concentration of penicillin which abolishes the additional protein synthesis does no more than halve (and frequently less than halve) the additional nucleic acid synthesis. Estimation of the proportions of the purine and pyrimidine bases in the nucleic acid fraction by the method of Smith and Markham (1950) modified by Wyatt (1951) shows that the changes lie in the ribonucleic acid fraction and that there is no significant alteration in the proportions of the various bases whether penicillin is present or not (Gale and Folkes, 1953b). 

The preparation of pure nucleic acids is as yet an unsolved problem. It is quite easy to prepare nucleic acid fractions that are free of proteins, polysaccharides, etc., but invariably they are mixtures of many very similar nucleic acids whose further separation becomes extraordinarily difficult. Most investigations on the chemical structure of nucleic acids have been carried out on such nucleic acid fractions, just as in the beginning of protein research all structural principles were derived from heterogeneous protein mixtures. The validity of the results need not be doubted on this account, however. 

Furanocoumarins can photoreact with unsaturated fatty acids. For example, angelicin can form an adduct with linolenic acid. The unsaturated fatty acids may have important role in the phosphaddylinositol system, and furano-coumarin adducts may change the regulatoiy function of this system. Also, these adducts may inhibit phospholipase and thus prevent the activation of protein kinase C. The greatest extent of photobinding of xanthotoxin and 4,6,3 -trimethoxy was found in the lipid fraction followed by the protein and nucleic acid fractions [98]. 

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