6000 



CALF THYMUS HISTONES (COLUMN STANDARDIZATION] 



13700 

 CPM 

 300 1 3600 



13550 

 200 I 3500 



II 21 31 41 SI a 71 81 91 101 III 121 131 l<X 151 ISI 171 181 191 



Fig. 2. 



Elutlon of hlstones from the cation exchange resin GC-50, 

 using a gradient of guanidinium chloride (8-40%, meas- 

 ured as refractive index). The quenching of C ( a ) or 

 H3 (A ) during the increasing salt concentration in the 

 eluted fractions is shown. 



and the arginine-rich histones are somewhere 

 in the order of 25,000 (3). Elevated ionic 

 strengths dissociate histones from the chromo- 

 somal apparatus (4). They contain neither try- 

 ptophan nor cysteine (5). Within a given species 

 the electrophoretic pattern obtained on an acryl- 

 amide gel is comparable from organ to organ, 

 but between species there are sometimes small 

 but characteristic changes in patterns. Histones 

 can be identified further by elution from a cation 

 exchange resin, and this has proven to be a very 

 useful tool. 



A typical pattern is shown in Fig. 2. This 

 shows acid-extracted histones from calf thymus. 

 They were applied to the resin in 8% guani- 

 dinium chloride and eluted with the gradient 

 shown. One invariably finds a run-off peak 

 (R.O.), the nature of which is a matter for con- 

 siderable speculation right now. Histones la and 

 lb are very lysine-rich, while lib is moderately 

 lysine-rich and HI and IV are arginine-rich. 



One of the earlier studies done in the group 

 was to see if the isolated chromosomal material 

 could do some of the things that one would expect 

 of the in vivo material. One of these was to see 

 if it could act as a template for DNA-dependent 

 RNA synthesis and to compare the template 

 activity of the chromatin with that of DNA which 

 had not been isolated from an identical prepara- 

 tion of chromatin (Fig. 3). Here one sees the 



4000 



2000 



a, 

 i. 



Q 



< 



a: 



g 6C00 



a: 

 o 

 o 



a 



< 



NTP(x 10 



4000 - 



2000 



05 



NTP(xlO M)EACH 

 Fig. 3. 



DNA-dependent RNA synthesis - a comparison of the tem- 

 plate activities of liver chromatin and rat liver DNA. 

 (a) The effect of increasing nucleoside triphosphate 

 (NTP) concentration upon the template activity of DNA 

 and chromatin (present in equal amounts); (b) the effect 

 of Increasing NTP concentration upon the template activity 

 of different concentrations of DNA. (Fig. 7, Marushlga 

 and Bonner, j. Mol. Biol. 15, 160, 1966; reproduced with 

 permission of Academic Press.) 



incorporation of AMP into RNA using as tem- 

 plate either chromatin or DNA isolated from an 

 equivalent batch of chromatin. It appears that 

 the chromatin is unable to make RNA at the same 

 rate as an equal amount of DNA. These particu- 

 lar experiments were performed by Dr. 

 Marushiga. In Table n you see some more simi- 

 larities with in vivo experiments. The synthesis 



132 



