Phosphorus cell labeling

This experiment is usually run using [3H]ethanol and locating the radioactive product by thin-layer chromatography. An alternative method would be to label the cells with [3H]oleic acid and [32P]phosphate and then monitor the ratio of the tritium label to phosphorus-32 label in any detectable phosphatidic acid and compare to the parent phosphoglycerides. The ratio would not change if the phosphatidic acid were derived by action of phospholipase D in the stimulated cells. However, the assay of choice at the present time is that associated with the formation of phosphatidylethanol. 

Tagging DNA. (a) Suppose that you want to radioactively label DNA but not RNA in dividing and growing bacterial cells. Which radioactive molecule would you add to the culture medium (b) Suppose that you want to prepare DNA in which the backbone phosphorus atoms are uniformly labeled with 32p Which precursors should be added to a solution containing DNA polymerase I and primed template DNA Specify the position of radioactive atoms in these precursors. 

In nucleotides the positions of (spin = 1/2) resonances in NMR spectra depend on the charges on the phosphates and therefore change with pH. The intracellular pH in vivo can therefore be measured. Furthermore, atoms bound to phosphorus yield upfield shifts of the P signals, and the enzymatic reaction between O-labeled ADP and 0-labeled orf/io-phosphate in cells can be followed by P-NMR. A useful property of the nucleus (spin = 5/2) is the drastic shortening of P relaxation times leading to line broadening to the point where the P signal may virtually disappear. In this context, it is also of importance that... 

After penetrating into bacterial cells the DNA of this phage undergoes the typical fate which we saw for RNA in cases of virus reproduction considered above it is replicated by complementary synthesis with the preliminary formation of double-stranded DNA. This process was discovered in later experiments (Sinsheimer, 1961 Sinsheimer et al., 1962) by fractionation of nitrogen- and phosphorus-labeled DNA of infected cells in a density gradient of cesium chloride at various times after infection. This double-stranded form of phage DNA was called the replicative form (RF). 

The effect of Roentgen rays on turnover rate of phosphatides present both in tissue and in nuclei was investigated as well. Two groups of twelve rats, after irradiation with 1000 r, are given labeled phosphate, while nonirradiated, control groups are treated in a similar way. After the lapse of two hours the animals are sacrificed and the sarcoma and livers are pooled separately. An aliquot is used in the determination of specific activities of inoi anic and phosphatide phosphorus of the tissue, while from the bulk of the material cell nuclei are isolated by the method of Bounce (42). The specific activities of the corresponding phosphorus fractions of the nuclei are also determined, and furthermore the activity of the inorganic phosphorus of the pooled blood plasma is measured. As seen in Tables XXXI and XXXII, the rate of turnover of phosphatides in liver... 

Despite the fact that the introduction of one unit of a virus within a living cell of a susceptible host is followed by the production of millions of virus units, almost nothing is known about the reproductive process. It seemed possible that preparation and isolation of tobacco mosaic virus containing radioactive phosphorus and inoculation of this virus into the diseased plants with the subsequent extensive multiplication of the virus should provide some information concerning the process of virus reproduction. This line of thought induced Stanley (169) to investigate the radioactivity of Turkish tobacco plants inoculated with labeled tobacco virus. Similar investigations were also carried out by Born and associates (25). 

---