DNA SYNTHESIS IN BONE MARROW STUDIED BY AUTORADIOGRAPHY 



Radiation delivered at the beginning of the cukure period in isotope 

 (Ox-6 : Ox-24) also markedly inhibited DNA synthesis. Since the grain 

 counts indicated that subsequent to irradiation no cells, even with long 

 culture times, synthesized more DNA than a 2-3 hours' non-irradiated 

 culture wovdd, it was suspected that the cells in the G^ period suffered a 

 latent damage. If the isotope was added to the cultures three or more 

 hours after irradiation (x-3-24) hardly any DNA synthesis was observed, 

 as opposed to those cultures in which the isotope was added immedi- 

 ately following the irradiation (Ox 24). These observations suggest that 

 cells in the G^ period, immediately before the S period receive a latent 

 damage from a dose of 5,000 r. During the first 3 hours following radiation 

 these cells may enter the S period ; after about 3 hours, however, even these 

 cells will be incapable of DNA synthesis. 



All G^ cells are damaged by a dose of 5,000 r. The grain counts indicate 

 that the difference between 6 and 24 hour cultures after radiation is pro- 

 duced by some G^ cells entering the S period for a limited time. The 

 radiation damage in these cells becomes apparent in that although they 

 may enter the S period, they cannot synthesize more than 2-3 hours' worth 

 of DNA as compared with the normal 12-15 hours' worth. The effect of 

 irradiation on the G^ period was indicated by the fact that no mitoses were 

 observed in any of the irradiated cultures. 



Formate ^'*C uptake — The incorporation of adenine or phosphorus is not 

 necessarily the true picture of DNA synthesis, therefore in a series of experi- 

 ments the uptake of formate ^*C as a purine and pyrimidine precursor was 

 investigated. Formate ^^C as a one-carbon compound takes part in many 

 of the reactions in the cytoplasm of the cell, and therefore it is surprising 

 that most of the labelled carbon taken up by the cells was localized in the 

 nucleus, even on the non-hydrolysed autoradiographs. This suggests a 

 preferential viptake of formate into DNA and RNA and also the central 

 role of the nucleus (? nucleolus) in RNA synthesis. 



On the acid hydrolysed preparations a quantitative assessment of the 

 formate ^*C into DNA was possible. The maximum uptake per nucleus 

 was of the order of 1 -2 X 10'' atoms DNA ^^C (50 grains in 10 days' expos- 

 ure, 2 grains/electron) which is only slightly greater than that found by 

 previous adenine ^*C experiments. (The concentration of adenine ^*C in 

 the cultures was 0-5[j.c/ml (specific activity 1 •56mc/mM) i.e. 0-3 milli- 

 molar. The concentration of the formate ^'*C : 2-0[jLc/ml (specific activity 

 l-04mc/ml) i.e. 2-0 millimolar.) The adenine stock was labelled in the 

 ratio of 1 : 45 and the formate stock in the ratio of 1 : 75. Therefore, if a 

 cell took up 1 ,000 molecules of adenine or formate they would include 22 

 molecules adenine ^^C or 13 molecules formate ^*C. Further, since corre- 

 sponding cultures contained 1,000 adenine molecules or 6,000 formate 

 molecules in equal volumes of the medium, if the uptake was parallel to the 

 concentration, then for each 22 labelled adenine molecules 78 labelled formate 

 molecules (i.e. over 3x) should have been taken up. This in fact was not the 

 case. It was considered that the concentrations used were in excess of those 

 permitting maximum uptake. Subsequent experiments have confirmed this. 



Effect of Aminopterin — As can be seen from Table I, aminopterin, in a con- 

 centration of 0-5[j.g/ml medium markedly inhibited the incorporation of 



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