The protamine to histone replacement in fertilized mouse eggs was studied by using antibodies to these proteins. Its course was followed with respect to DNA replication by autoradiography of 3H-thymidine-labeled fertilized eggs. It was found that protamines were replaced by histones before the onset of DNA replication.

No abstract

A procedure has been developed which allows the separation of chromatin into two fractions highly enriched in preexisting and in newly synthesized DNA, respectively. The procedure includes the following steps. 3]), the histone arrangements should be reproduced during DNA replication by means of specific interactions between DNA-bound (preexisting) and free (newly synthesized) histones. Such interactions could take place if the following conditions are fulfilled.a) The histone molecule should have positively charged sites for unspecific binding t u DNA and nonbasic loops for specific interactions with proteins.b) The strong interaction between the positively charged groups of histones and the negatively charged phosphate groups of DNA should be avoided.c) The preexisting histones should remain bound to the old DNA strand, while the newly synthesized ones should bind to the new DNA strand.Point (a) is well supported by studies on the primary structure of the five histone species, showing in all cases an uneven distribution of the positively charged groups and long (up to 29 residues) nonbasic, mostly hydrophobic sequences on the side opposite to the DNA binding site [4-71.Definition. A,,, unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260nm, when measured in a 1-cm pathlength cell. Point (b) is supported by a recent study on histone IV (F2al) which shows that its basic groups are acetylated before the histone molecule becomes incorporated into chromatin [S]. This finding indicates that one possible role of acetylation might be to allow the histones to find their places on the basis of specific (probably hydrophobic) interactions between chromatin proteins. Point (c) which is most important for the model proposed has not been experimentally tested. It has been shown only that the new histones, once incorporated into DNA, are conserved in the chromatin during the following cell generations [9]. There are no data as to whether the histones remain bound to DNA during replication and how they are distributed among the two DNA strands. There were several technical difficulties in testing these points. Separation of preexisting from newly synthesized chromatin components on the basis of iododeoxyuridinelabelling of DNA was practically unsuccessful due to the small difference in the buoyant density of the two nucleoproteins [lo]. Separation of the two DNA strands with the original histones still bound to them seemed to be impossible in view of some data indicating that at pH values causing denaturation of DNA the histone -DNA complex is dissociated [11, 121.However, our detailed studies on the alkaline denaturation of chromatin have shown that with increasing pH a stepwise release of histones takes Eur. J. Biochem. 43 (1974)