128 ALEXANDER L. DOUNCE 



plasmic PNA,^*'^^ independent synthesis in the cytoplasm of some of the 

 cytoplasmic PNA is to be suspected. Such a situation in which cytoplasmic 

 PNA would be partly dependent and partly independent of nuclear PNA 

 would fit well with the concept of plasmagenes expressed by Spiegelman 

 and Kamen.^* 



4. Composition of the Cell Nucleus with Respect to Proteins 



The principal known protein fractions of the cell nucleus are the histone 

 fraction, a globulin fraction, a newly discovered acid protein, ^^'^"^ and 

 residual insoluble protein. The acid protein, however, may be identical 

 with the protein of the residual chromosomes of Mirsky.^" It is not clear 

 whether cell nuclei contain appreciable quantities of albumins, but at 

 least small amounts may be present. It is very likely that albumins, if 

 present, will be lost, together with some of the globulin fraction, from nuclei 

 isolated in aqueous media. 



a. Scheme for Separating Nuclear Protein Fractions. The following scheme can be 

 used to fractionate the major proteins of cell nuclei : (a) Extract the nuclei, previously 

 isolated so as to keep the DNA firmly bound, twice with 0.9% NaCl solution at 

 pH 7.0, and wash twice with water. The globulin fraction together with any albumins 

 that may be present will pass into solution, (b) Extract the residue twice with 0.2 N 

 HCl and wash twice with water. (A quantity of HCl equivalent to five times the 

 volume of the packed nuclei suffices for each extraction.) The histones will be ex- 

 tracted, (c) The residue contains DNA, presumably PNA, and the lipoprotein of 

 Mayer et al.^"" and Laskowski and Engebring,''' '"^ which may be identical with the 

 residual chromosomal protein of Mirsky'** and with the "chromosomin" of the Sted- 

 mans.i"^" It is apparently possible to obtain the lipoprotein in solution by treatment 

 of the residue with deoxyribonuclease, followed by extractions with alkali accord- 

 ing to Laskowski.'^' '•" It should be noted that the residue forms the "unwinding" 

 type of gel typical of nuclei containing firmly bound DNA. The DNA thus is prob- 

 ably bound firmly to the residual protein or liproprotein of cell nuclei rather than 

 to the extractable histone. The method of Mayer et a/."*" for isolating the acid lipo- 

 proteins seems to be applicable only to nuclei that do not have the DNA firmly 

 bound. 



All operations should be carried out at a temperature as close to 0° as possible. 

 Globulins can be recovered from the NaCl extract by dialysis at pH 5 to 6, and his- 

 tones can be recovered from the HCl extract by the addition of ammonia which 

 causes them to precipitate. If insufficient ammonia is added, only part of the histone 

 will precipitate. One part of concentrated ammonia by volume to ten parts of his- 



95 D. Elson and E. Chargaff, Federation Proc. 10, 180 (1951). 



9«A. Marshak, J. Biol. Chem. 189, 607 (1951). 



" G. W. Crosbie, R. M. S. Smellie, and J. N. Davidson, Biochem. J. 54, 287 (1953). 



«8K. Spiegelman and M. D. Kamen, Science 104, 581 (1946). 



99 M. Laskowski and V. K. Engebring, Federation Proc. 11, 246 (1952). 

 ">» T. Y. Wang, D. T. Mayer, and L. Thomas, Exptl. Cell Research 4, 102 (1953). 

 "' V. K. Engebring and M. Laskowski, Biochim. et Biophys. Acta 11, 244 (1953). 

 '"'* E. Stedman and E. Stedman, Cold Spring Harbor Srjmposia Quant. Biol. 12, 224 

 (1947). 



