L. G. LAJTHA, R. OLIV'ER AND F. ELLIS 



This h)drohsis was found to remove all non DNA phosphorus or adenine 

 from the cells while leaving DNA phosphorus and adenine behind (Lajtha^). 

 After hydrolysis autoradiographs were prepared using the stripping film 

 technique, and were exposed in light-tight boxes in a refrigerator. After 

 processing, the slides were stained without removing the photographic 

 emulsion (Lajtha^' *). The stained autoradiographs thus obtained allow 

 differential and grain counting (see Figure 1) . The number of cells showing 

 positive autoradiographs can be expressed as a percentage of the total 

 counted. Grain counting over individual nuclei is also carried out and the 

 maximum activity is expressed as 'upper average' grain count (the average 

 grain count of 10-15 cells judged visually to show maximum activity). 



RESULTS 



[a] The cell cycle — If the cells synthesized DNA throughout the entire 

 intermitotic period, then, after a few hours of culture in the presence of the 

 isotope all the cells should contain labelled DNA. This, however, was 

 found not to be the case. Experiments with ^^P or adenine ^'*C indicated 

 that when cells were cultured for progressively greater lengths of time, a 

 gradually increasing percentage of the cells showed DNA autoradiographs 

 (per cent positive nuclei). The grain counting (upper average grain count 

 per nucleus) demonstrated that the amount of labelled DNA per nucleus 

 also increased with time, until a maximum was reached in about 15-18 

 hours. These findings indicate that DNA synthesis takes place during a 

 limited period in the cell cycle. 



Lajtha, Oliver and Ellis^ showed that labelling of the DNA indicated 

 a length of period of DNA synthesis (S period) of the order of 12-15 hours, 

 and a total cell cycle time (intermitotic period) of about 40-45 hours. When 

 only the mitotic figures were counted on the smears (metaphase and ana- 

 phase) it appeared that, while in a 2-4 hours' culture with '^-P the majority 

 of the mitotic figures did not show autoradiographs, in a 6 hours' culture 

 most of the mitoses already contained labelled DNA. This observation 

 suggests that immediately prior to mitosis there is a 3-4 hours' stage of the 

 cell cycle (Gg period) during which no DNA synthesis takes place. Prior 

 to this second gap is the period of DNA synthesis (S), which is preceded by 

 the long first gap (G^) during which again no DNA synthesis takes place. 

 The cell cycle related to DNA synthesis measured bv the incorporation 

 of 32p or adenine i^C into DNA : ' 



25-30 h 12-1 5 h 3-4 h 



(,i S G. 



This timing, in principle, agrees well with that found in the bean root by 

 Howard and Pelc^. 



Ejfect of X-irradiation — Large doses of X-rays (5,000 r in 15 minutes, 

 140kV 1mm Al filter) exerted a marked inhibition on DNA synthesis. 

 When in a 0-6 hour culture in isotope, radiation was delivered in the 

 third hour (0-3x-6) then both the number of cells showing DNA auto- 

 radiographs and the grain count over the nuclei give similar counts to those 

 in a 3-hour culture (0-3). This suggests that cells in the period of DNA 

 synthesis are immediately and completely prevented from synthesizing DNA. 



217 



