Fractionation methods nucleotides

Cambella and Antia [385] determined phosphonates in seawater by fractionation of the total phosphorus. The seawater sample was divided into two aliquots. The first was analysed for total phosphorus by the nitrate oxidation method capable of breaking down phosphonates, phosphate esters, nucleotides, and polyphosphates. The second aliquot was added to a suspension of bacterial (Escherichia coli) alkaline phosphatase enzyme, incubated for 2h at 37 °C and subjected to hot acid hydrolysis for 1 h. The resultant hot acid-enzyme sample was assayed for molybdate reactive phosphate which was estimated as the sum of enzyme hydrolysable phosphate and acid hydrolysable... 

Treatment with hot organic solvents was the next step in the tissue fractionation, to remove lipid-phosphorous and breakdown lipid-protein interactions. In the Schneider procedure, nucleic acids were then extracted in hot dilute trichloroacetic or perchloric acid, leaving a protein residue with any phosphoprotein links still intact. This method was to become particularly useful when 3H thymidine became the preferred label for DNA in the early 1960s. For investigations where both RNA and DNA were to be examined the Schmidt-Thannhauser process was often chosen. Here the lipid-extracted material was hydrolyzed with dilute sodium hydroxide releasing RNA nucleotides and any hydroxyamino acid bound phosphorus. DNA could be precipitated from the extract but the presence in the alkaline hydrolysate of the highly labeled phosphate released from phosphoprotein complicated... 

Other methods used to characterize and identify DOP involve bioassays with Chlorella to study the biological availability and biouptake of the HMW SRP fraction (4, 6). These bioassays indicate that the algal growth responds similarly to HMW SRP and to PO. A preference for PO 3- was detected, and not all of the reactive HMW fraction was used. Enzymatic assays used by Herbes et al. (13) tentatively identified inositol hexaphosphate as part of the DOP. Using an anion-exchange HPLC system with a phosphorus-specific post-column reactor, Minear and co-workers (15,16) possibly have detected inositol hexaphosphate, DNA, and nucleotide fragments in lake waters. 

Nucleotide fractions appear in the boxes on the right side of the GUI. They can be entered manually or calculated by clicking the Compute by GALO button at the bottom of the GUI. In the current version of the GALO method [13], nucleotide fractions are calculated that are multiples of 1% of the total number of synthons in the mixture. Nucleotide fractions can be edited at any time. Click the Compute Manually button to recalculate the fractions of amino acids. 

A variation of the use of standards is the method in which predetermined quantities of a standard radioactive compound are added to the solution. The fractions are collected, and, by plotting the counts of the fractions, the peak of interest can be identified. This method is especially useful in following cell metabolism of purine and pyrimidine analogs. A plot of the nucleotides in a cell extract of schistosomes containing 14C-labeled adenine and guanine nucleotides is shown in Figure 7.3.5... 

The infected system was shown to contain deoxymidine 5-phosphate this observation is interesting, since this nucleotide has not yet been shown to be present in normal cells. The nucleotide is presumably used for the synthesis of thymidine 5-triphosphoric acid. Detectable quantities of 5-(hydroxymethyl)cytosine or of 5-(hydroxymethyl)cytosine nucleotides were not present, despite the fact that this base is a normal constituent of the phage deoxyribonucleic acid. Explanations for this observation are that (a) the amount present in the acid-soluble fraction at any given moment is too small for detection by the methods of anal3rsis employed, or (b) the newly synthesized 5-(hydroxymethyl)cytosine is directly incorporated into deoxyribonucleic acid. 

A useful procedure for estimating adenylate cyclase in intact cells and tissues is to incubate the tissue with labelled adenine and then measure the rate of labelling of cyclic AMP [112]. Adenine readily penetrates cells and is partially converted to ATP. In heart slices, the ATP newly synthesised from radioactive adenine was found in equilibrium with the existing pool used for the production of cyclic AMP the specific activity of the newly-formed cyclic AMP was similar in the presence and in the absence of stimulatory hormone [113]. The prelabelling method has been compared with the protein-binding method in brain slices [114,115]. Increases in total levels of cyclic AMP and increases in levels of radioactive cyclic AMP derived from intracellular adenine nucleotides labelled by prior incubation with radioactive adenine occurred on similar time courses and to similar extents. Radioactive cyclic AMP represented a small (7-13%) but relatively constant fraction of the total amount of cyclic AMP. These results provided no evidence for the presence of more than one major compartment of adenine nucleotides in brain slices that serve as a source of nucleotide precursor for cyclic AMP. The nucleotides of this compartment were uniformly labelled by incubation with radioactive adenine [116]. 

For any given tissue or cell preparation, it is desirable to compare the amount of radioisotope which the cyclic AMP contains with the absolute levels of the cyclic nucleotide. The result of such a comparison for heart tissue slices demonstrates that the radioactive cyclic AMP formed reflects the changes of the absolute levels of the cyclic nucleotide [113], A similar result has been obtained in brain slices [114,115]. Thus, the prelabelling method provides a convenient means of studying dynamic changes in cyclic AMP levels in tissues and cells. It is essential that cyclic AMP be isolated free of other labelled adenine products, and this involves fractionation on Dowex 50 columns and/or paper chromatography of the tissue extract. 

A variety of powerful methods is available for fractionating short oligonucleotides. Work on nucleotide sequence analysis of small RNA molecules (n=80 or 120) and more recently viral RNA molecules (n= 3000) and even ribosomal precursor RNAs (up to 45 s) (Holley et al. 1965a Brownlee 1972 Maden et al. 1972) has stimulated the development of these procedures. They can also be used for synthetic oligonucleotides (e.g. Hachman and Khorana, 1969). The methods depend largely on chromatography and electrophoresis on filter papers, diethylaminoethyl(DEAE) paper, cellulose acetate, thin layers of cellulose or polyethyleneimine (PEI)-cellulose, or columns. 

Its main limitations are that it is not usually the best method for small oligonucleotides (n < 10), it does not distinguish nucleotide composition differences clearly, -and it is difficult to scale up for preparative use. It is widely used for analytical and small scale preparative separations because it is quick, sensitive and has high resolving power. Very little specialised apparatus is required and routine fractionation can be carried out by a technician. 

In general, thin layer methods are quicker and more sensitive than paper methods and in principle paper methods can be directly transferred to cellulose thin layers. In practice these methods have not been widely used for nucleotide fractionation (but see 1.2 and 3.4). The following manufacturers for example provide general information on the use of the products for thin layer chromatography H. Reeve Angel (Whatman) Camlab (Glass) Ltd Shandon Southern Ltd Eastman-Kodak. It is not proposed to go into more detail here. 

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