114 ALEXANDER L. BOUNCE 



and to show the presence of one enzyme, arginase, in the liver cell nuclei. Important 

 work on the localization of DNA in the nuclei was carried out. 



(2) Subsequent Modifications. In subsequent modifications of the Behrens tech- 

 nique, one very marked improvement has been the use of modern lyophilizing tech- 

 niques. Another improvement has been the substitution of cj'clohexane for benzene 

 in Mirsky's laboratory.^* A different modification, sometimes employed, which 

 may not be so sound on theoretical grounds, is the use of excess acetone in the cold to 

 prepare an acetone powder from which nuclei can then be isolated by the specific 

 gravity flotation procedure. This modification was introduced by Mayer and Gulick''^ 

 and later by Behrens and Taubert." An objection to such a procedure is that con- 

 siderable translocation of material, especially that of low molecular weight, might 

 occur before the concentration of acetone has become high enough to prevent it. 



The most complete descriptions of the isolation of various types of cell nuclei by 

 the Behrens procedure have been given by Mirsky et al.^^ Word for word reproductions 

 of these descriptions would require quite considerable space, while condensations 

 would not be of much value, and, since the material is published in a readily available 

 journal, the reader is referred to the original article for details. Something further 

 will be said, however, about the general principles of the method and the processing 

 of the tissue prior to isolation of the nuclei. 



Before lyophilization, the tissue, which has been frozen by means of acetone-dry 

 ice or liquid nitrogen, is placed in a towel or other piece of cloth and is hammered into 

 small pieces. This should, if possible, be done in the deep-freeze room to avoid local 

 areas of wetting. A more elegant procedure for freezing, in which the tissue is ground 

 with dry ice in a mixer, has been described by Behrens.''* A very finely divided frozen 

 powder is obtained by this procedure. The frozen tissue is next placed in the flasks 

 to be attached to the lyophilizing apparatus. Centrifuge bottles are used in the appa- 

 ratus shown in Fig. 4. This apparatus is of sufficient capacity to dry a relatively large 

 amount of tissue without having to empty the traps, but care should be taken that 

 the traps do not become plugged up. Two hundred grams of tissue, which will contain 

 about 65 to 70% water on the average, forms a convenient amount to lyophilize. 



After lyophilization, the tissue can be shredded in a Waring Blendor before being 

 ground in the ball mill, according to Mirsky et al.*^ When liver tissue is used, the 

 pieces of dry tissue may simply be crushed with a mortar and pestle and, for grinding, 

 a commercial porcelain ball mill of 1 -liter capacity with 160 irregularly shaped pebbles 

 of 15 to 20 mm. average diameter may be used.* Fifty grams of the lyophilized tissue 

 is a convenient amount to grind, although Mirsky et al.'^^ ground 100-g. portions 

 in a similar mill. For a 50-g. portion of tissue, 200 ml. of petroleum ether, boiling 

 point 50 to 60°, may be added while Mirsky et al. use 450 ml. of the same solvent for a 

 100-g. portion. For a 50-g. portion of dry liver, 24 hr. is the proper length of time for 

 grinding, while Mirsky et al. recommend 44 to 48 hr. for a 100-g. portion. 



After the grinding operation, it is necessary to remove fiber by filtration through 

 some sort of cloth or screen. Fine cheesecloth is very satisfactory for this purpose. 

 The fiber remaining in the cheesecloth and the pebbles are washed with several por- 

 tions of petroleum ether to avoid undue losses of nuclei. The washings must also be 

 filtered through cheesecloth. 



In principle the isolation of the nuclei from the ground, filtered tissue is simple, 

 but in practice the procedure is laborious and time-consuming, and according to Kirk- 



« V. G. Allf rey, H. Stern, A. E. Mirsky, and H. Saetren, J. Gen. Physiol. 35, 529 (1952) . 



^« D. T. Mayer and A. Gulick, /. Biol. Chem. 146, 433 (1942) . 



" M. Behrens and M. Taubert, Z. physiol. Chem. 291, 213 (1953). 



«M. Behrens, Z. physiol. Chem. 291, 245 (1953). 



