96 ALEXANDER L. DOUNCE 



One further point which should be mentioned concerning nuclei prepared 

 in aqueous media is that, in the absence of nuclear autolysis, the nuclei 

 should be capable of forming a peculiar type of structural gel upon the 

 addition of alkali or strong sodium chloride. The writer considers gel forma- 

 tion as an indication that little or no autolytic degradation of nuclear 

 structure has occurred.^* If nuclei are isolated from mammalian tissues 

 at pH values ranging from 6 to 7, they often will not form gels. Gels are 

 formed however by nuclei isolated in 70% ethylene glycol or glycerol; in 

 this case the high concentration of organic solvent appears to block auto- 

 lytic action. 



Cell nuclei isolated at a pH of about 6.8 in isotonic saline also can form 

 gels. Here it may be that the use of isotonic saline renders the nucleo- 

 protein complex so insoluble that it is not subject to rapid degradation by 

 autolytic enzymes. It has very recently been found in the writer's laboratory 

 that the use of low concentrations of calcium chloride also can prevent auto- 

 lytic degradation; more will be said about this later. The loss of ability to 

 form gels, resulting from intranuclear autolysis, may not be of any great 

 significance from the standpoint of the enzyme chemistry of the cell nuclei, 

 but it is interesting in that it demonstrates a type of chemical damage that 

 can occur without corresponding microscopically observable morphological 

 damage. 



In spite of the limitations just mentioned, the use of aqueous media for 

 isolating cell nuclei cannot be abandoned. The most important reason for 

 making this statement is that methods involving nonaqueous media can 

 cause damage to or destruction of a number of enzyme systems and there- 

 fore are not universally applicable. Such damage or destruction is partic- 

 ularly important in the cases of some of the insoluble mitochondrial enzymes 

 such as cytochrome oxidase and succinic dehydrogenase. In addition, 

 methods making use of aqueous solutions are much less time-consuming 

 than methods involving nonaqueous solvents and can be perfectly reliable 

 for investigations of materials such as lipid and DNA. 



b. Methods for Removing Fiber and for Homogenizing Tissue 



The first steps in isolating nuclei from tissues on a chemical scale consist 

 in breaking the cells and, where necessary, in removing fiber by special 

 procedures. When nuclei are to be isolated from a relatively nonfibrous 

 organ such as the liver of a young animal, special methods for removing 

 fiber may be dispensed with. It is sufficient in the case of liver to filter the 

 homogenized material through progressively finer grades of cheesecloth, 



5 A. L. Dounce, Science 110, 442 (1949). 



s A. L. Dounce, G. H. Tishkoff, S. R. Barnett, and R. M. Freer, J. Gen. Phijsiol. 33, 

 629 (1950). 



