HISTONES IN RELATION TO CONTROL IN 

 LIVING SYSTEMS 



Roger Chalkley 



Division ot Biology, California Institute of Technology, 

 Pasadena, California 



As this is a workshop, what I plan to do is 

 provide a broad outline of some of the things 

 which are being studied in Professor James 

 Bonner's laboratory at the California Institute 

 of Technology. We are concerned with the 

 molecular aspects of control mechanisms in 

 differentiated tissues. The strategy of attack 

 is first to isolate the chromosomal material 

 in a pure form. 



In Fig. 1 is shown a general scheme for 

 the isolation of chromatin. This scheme is appli- 

 cable to mammalian tissues and slight modifi- 

 cations are necessary for plant tissue, but the 

 general principle is the same. The tissue is 

 disrupted in a Waring blendor in increasing 

 volumes of the grinding medium and at increas- 

 ing speeds. The grinding medium consists of: 

 0.25 M sucrose, 0.003 M calcium chloride and 

 0.005 A/tris, pH 7.3. Grinding at increasing 

 volumes and increasing speeds removes peri- 

 nuclear contamination and gives rise to what 

 we think are reasonably pure nuclei. These 

 nuclei can be used for amino acid incorpora- 

 tion studies in vitro. The nuclei are washed 

 once with grinding medium and then with saline 

 EDTA. This inhibits the action of degrading en- 

 zymes and also removes the calcium that is 

 stabilizing the nuclear membranes. This makes 

 the next step, lysis in 0.01 M tris, more con- 

 venient. The lysed material is centrifuged 

 through a rough sucrose gradient at 22,000 

 rpm for two hours. This gives rise to a gel-like 

 pellet which, after dialysis against low con- 

 centrations of tris at pH 7.3, is known as "puri- 

 fied chromatin". Chromatin so prepared has a 

 high Svedberg constant and for the purpose of a 

 number of experiments it has proved advan- 

 tageous to shear the material and remove larger 

 aggregates by low speed centrifugation. The 



nucleoprotein remaining in solution (90%) is 

 commonly referred to as nucleohistone. 



The chemical compositions of some of the 

 chromatins that have been isolated are shown 

 in Table I. The histone:DNA ratio is roughly 

 1:1. In addition there is a very small amount 

 of RNA which is difficult to remove. This RNA 

 is partially resistant to RNase (1, 2). In the 

 case of pea cotyledon there is a more than 

 normal quota of RNA, but one has to recognize 

 that it is a rapidly developing system. It has 

 also been impossible to remove all of the non- 

 histone protein and this may have an important 

 contribution to make toward the chromosomal 

 apparatus. The histones themselves are acid- 

 soluble and this frequently provides a method 

 for their isolation. The molecular weight of the 

 acid- extracted material appears to be less than 

 10^. The molecular weight of lysine-rich his- 

 tones is usually estimated to be about 10,000 



CHRQMATIM ISOLATION! 



Washed Tissue 



Grindinq procedures in Wanna Blendor 



I 



Washed in qrindinq medium (2 X) 



Wa5hed in 0.15 M saline-EDTA 



I 



Lysed into Tris pH/.s 



Purified chromatin isolated after 

 centrifuqation through a sucrose qradient 



Fig. 1. 



131 



