5l6 MOLECULAR MECHANISMS OF DIFFERENTIATION 5 



nucleus in situ has been frequently demonstrated by the incorporation of amino 

 acids into nuclear proteins. In these investigations it remained undecided to which 

 extent this uptake depended on the presence of the cytoplasm. However, in a recent 

 investigation protein formation in isolated nuclei could be demonstrated (AU- 

 frey, 1954; Allfrey, Mirsky and Osawa, 1955). Using an isolation procedure which 

 precludes, loss of nuclear proteins, the observed uptake of alanine-'^'C into the 

 nuclear protein fractions was found to be much higher than in previously studied 

 nuclear preparations. It became evident that the energy svipplied by metabolic 

 reactions occurring in the nucleus itself was utilized for protein formation since 

 metabolic inhibitors prevented incorporation. Fractionation of the nuclear pro- 

 teins showed highest incorporation in a non-histone protein which was bound to 

 deoxyribonucleic acid. A soluble non-histone protein fraction also showed con- 

 siderable activity while the histone fraction contributed only a small portion of 

 the total activity. The observed differences between the activities of different 

 protein fractions were essentially in line with activities obtained in protein frac- 

 tions of nuclei obtained after injection of labelled glycine-^^N into the intact 

 animal and subsequent separation of the nuclear protein fractions from liver, 

 pancreas and kidney cells (Allfrey, Daly and Mirsky, 1955).' The significance of 

 the differences in the incorporation rates of these proteins is obscure at the present 

 time. Rapid formation of all fractions is certainly necessary during marked pro- 

 liferative activity of cells. Whether the incorporation into one of the fractions, e.g. 

 histones, follows more directly the proliferative rate while the other nuclear protein 

 fractions may retain a high rate of incorporation when cytoplasmic proteins are 

 being formed, is again a question which awaits a systematic study in a tissue during 

 its transition from the predominately proliferative to a predominately differentiat- 

 ing and eventually to a mature adult state. The embryonic development of the 

 lens, of muscle, or of connective tissue may yield valuable information on this point. 

 The importance of the undisturbed maintenance of nucleo-cytoplasmic inter- 

 actions in embryonic development has become apparent from studies of lethal 

 hybrids (Briggs and King, 1955, p. 219-224; P.S. Chen, 1954; Gregg and Lovtrup, 

 1955). By an original analysis of careful analytical work on merogonic hybrids of 

 Triturus cristatus (sperm) x Triturus palmatus (egg cytoplasm) Zeller (1956) could 

 demonstrate the occurrence of an interesting deviation of the RNA content in 

 these embryos. In following the RNA content in eggs and embryos of either 

 Triturus palmatus or cristatus it was observed that the scattering of the values 

 changed in a characteristic fashion during development. In the early part of 

 development the range between the RNA values of different clutches contributes 

 more than one half of the total scattering, the rest of the scattering being due to 

 variations between eggs from the same clutch. As development continues the 



^ These incorporation rates cannot necessarily be regarded as a direct quantitative index 

 of the rate of formation of the respective proteins. If the investigated proteins differ greatly 

 in their composition, for example, in their alanine content and if alanine is used as the 

 labelled tracer, the protein with lowest alanine content would show the lowest activity per 

 protein nitrogen. Only expression of the data as labelled alanine per total protein alanine, 

 would give values which would make incorporation data of different proteins directly 

 comparable. 



