38 S. S. COHEN 



Most recently, it has been discovered (Allfrey et at., 1957a) that thymus 

 nuclei isolated in sucrose are equivalent in DNA, protein, and enzyme 

 content to thymus nuclei isolated by the nonaqueous Behrens method. These 

 workers considered such "sucrose" nuclei to be about 90 % pure and suitable 

 for use in studies of protein and nucleic acid synthesis, whose results will be 

 described below. 



Parenthetically, it may be noted that virologists have not yet reported an 

 attempt to study the reproduction of intranuclear viruses in isolated infected 

 nuclei. 



4. Some Data on the Composition of Nuclei^ 



a. Constituents of Normal Nuclei. Although the procedure described by 

 Hogeboom and Schneider (1952) is recommended to give high yields of nuclei, 

 it appears that Ca++ aggregates submicroscopic particles, causing their 

 incorporation into the mitochondrial fraction. At the present time, the use 

 of sucrose solutions in homogenization and in centrifugal fractionation seems 

 to permit the best balance in the separation of tissue components. Such an 

 analysis for Hver has been given by Muntwyler et al. (1950), comparing the 

 distribution of nitrogen, phosphorus, and the nucleic acids in normal liver 

 and in the liver of animals held under conditions of restriction of dietary 

 protein. Following homogenization, substances are separated accordmg to 

 their mass by the controlled use of defined gravitational force for limited time 

 intervals." Thus, nuclei are deposited at 200<7 for 10 minutes, mitochondria 

 at 8500^ for 10 minutes, and microsomes at 20000^ for 1 hour. 



As given in Table I, the DNA of the cell is entirely in the nuclear fraction, 

 whereas only 10-15 % of the total RNA was found in this material. This 

 fraction contained 15-20 % of the nitrogen (N) and phosphorus (P) of the 

 cell. Under conditions of dietary protein depletion a smaU loss of N and P 

 occurred per gram of hver. The major site of such loss was, however, in the 

 microsomal fraction, which had lost 35 % of its nitrogen. 



Although RNA decreases markedly in liver, kidney, and muscle under 

 conditions of a prolonged protem fast, the DNA content of these organs 

 remains constant. However, it has been reported that similar conditions 

 markedly reduce the DNA content of pancreas, adrenal, and spleen, as well 

 as their contents of RNA (Mandel et at., 1954). 



^ Spiegelman (195G) has briefly recorded the isolation of bacterial nuclei from B. 

 megatherium, after disrupting protoplasts with lipase. Also, see Spiegelman et al. (1958). 



^ Instead of relyhig on differences in sedimentation rate in a liquid of constant density, 

 recent procedures have successfully explored the separation of particulates within 

 homogenates in media of controlled density gradients (Holter et al., 1953; Kuff and 

 Schneider, 1954; Anderson, 1955; Ottesen and Weber, 1955). It can be noted that the 

 technique was introduced in the separation of plant virus particles (Braklie, 1951, 1953) 

 prior to its development for cytological study. 



