LEONARD J. COLE AND MARIE E. ELLIS 



action of DNA-ase under these conditions was, therefore, 2 per cent of the 

 total celkilar DNA. 



In the next experiment, 5 ml of fresh spleen cell suspension (containing 

 5-2 X 10'' cells per ml) was centrifuged at 2,500 rpm for 10 minutes, the 

 supernatant was discarded, and the packed centrifugal residue resuspended, 

 by shaking, in 5 ml of saline. The suspension was centrifuged once again, 

 the supernatant discarded, and the residue again resuspended by shaking. 

 The suspension was then incubated with DNA-ase at 25° C for 30 minutes. 

 After incubation, the tubes were chilled in ice, and centrifuged at 2,500 rpm 

 for 10 minutes in a refrigerated centrifuge. The ultraviolet absorption 

 spectra of the resultant supernatants are shown in Figure 2. The specific 

 and large absorption at 260 mij. as a consequence of the action of DNA-ase 

 on the "disrupted {e.g. twice centrifuged and resuspended by shaking) cell 



Univashed sp/een 

 cell suspension after 

 incubalion at 25 "C 

 for 30 min. I 



Suspension a/one 

 ° Suspension \ DNA-ase 

 n DNA-ase alone 

 \^ Net lvalues 

 (calculated) 



I 



^ 0-6 



% 



I 



> 0-2 



o Disrupted cell suspension 



incubated pvitl? DNA -ase 

 Disrupted cell suspension 

 incubated- no enzyme 



^ Cell suspension-untvastied- 

 ^ incubated ivitfi DNA 



I 



i\ 



ase 25% for 30 mn. 



^ 



220 



260 300 



IVai^elength 



S'^O 



220 



Ta[L 



260 300 



Wa'/elength 



SfO 



Figure 1. Effect of deoxyribonuclease treatment 

 on unwashed mouse spleen cell suspension 



Figure 2. Effect of deoxyribonuclease treat- 

 ment on disrupted spleen cell suspension 



suspension, is in striking contrast to that of the unwashed cell suspension 

 incubated with DNA-ase. The optical density at 260 m;^ {i.e. 0-758) 

 corresponds to a Hberation of 39 per cent of the total cellular DNA in the 

 disrupted cell suspension by the DNA-ase. 



A study was next made of the effect of DNA-ase on a washed spleen 

 homogenate preparation, the cell count of which was approximately equal 

 to that of the cell suspension used above. The spleens obtained from one- 

 week-old mice and weighing 280 mg were homogenized in a Potter ground- 

 glass homogenizer, washed twice with cold 0-14M NaCl, rehomogenized, 

 and resuspended in 10ml 0-14 M NaCl. Two homogenates were prepared 

 concurrently ; one contained 6-9 X 10*5 cells and/or cell bodies per ml : 

 the other 7-9 X 10*= per ml. Both homogenates were incubated at 25°C for 

 30 minutes, one (A) with added DNA-ase, the other {B) without enzyme. 

 After incubation, the flasks were chilled, the homogenates centrifuged, and 

 the clear supernatants withdrawn for analysis. The supernatant from B 



143 



