200 



1.0 2.0 



MOBILITY 



Fig. 12. 



Free boundary electrophoresis of the product of nucleo- 

 hlstone-primed DNA synthesis. The faster component has 

 the mobility of DNA (peak at 2.2), the slower has that of 

 nucleohistone (peak at 1.6). 



studies to this end have been initiated at Cal 

 Tech. In particular I wish to discuss a number 

 of experiments related to the release of dor- 

 mancy in the potato tuber, to the increase in 

 enzyme activity of the liver induced by hydro- 

 cortisone, and to the effects of estradiol in 

 preparing the endometrial layer of the uterus 

 for implantation following fertilization. The 

 first experiments were performed by Dorothy 

 Tuan (9). The system she has studied is the 

 dormant bud in the potato tuber, the dormancy 

 of which is relieved by ethylene chlorohydrin. 

 Dormant buds of potato tubers are treated with 

 ethylene chlorohydrin for three days and im- 

 mediately there is an increase in DNA and RNA 

 synthesis (Fig. 13). The RNA synthesis is 

 actinomycin-D sensitive. If RNA synthesis is 

 inhibited at this time in the development, DNA 

 synthesis is also stopped. Therefore, the 

 strategy of her next experiments with the sys- 

 tem was to isolate chromatin from the buds at 

 an early period where it is making very little 

 RNA, and to compare its in vitro template activ- 

 ity with that of chromatin isolated from the 

 buds at a later period in the development where 

 RNA synthesis was much increased in vivo. 

 Table IV shows the result of this type of in- 

 vestigation. Potato tuber chromatin was found 

 to have an exceedingly low template activity. 

 However, this is not necessarily significant since 

 isolation of the chromatin from the tuber pre- 



sents considerable technological difficulties due 

 to the almost infinite amount of starch present. 

 In the case of chromatin isolated from the 

 dormant bud, we see that chromatin can direct 

 the synthesis of RNA, but at a very low level. 

 At the end of three days' treatment with ethylene 

 chlorohydrin, the template activity of the bud 

 chromatin is seen to increase. A significant 

 increase in the amount of RNA synthesized is 

 observed. Thus the template activity of isolated 

 chromatin has mirrored its change in the pat- 

 tern of RNA synthesis in development. 



A similar approach has been applied to the 

 study of the effect of hydrocortisone upon RNA 

 synthesis in the liver of the rat. These studies 

 have recently been reported by M. Dahmus and 

 J. Bonner (10). The experimental design was to 

 compare the template activity of liver chromatin 

 adrenalectomized rats before and after hor- 

 mone administration. 



A characteristic result of this type of ex- 

 periment is shown in Fig. 14. The template ac- 

 tivity (rate of RNA synthesis) is plotted as a 

 function of the increase in concentration of the 

 DNA or purified chromatin in the incubation mix- 

 ture. In the in vivo experiments the increase in 

 RNA is some 300% (11). However, in this sort 

 of study it is not nearly so dramatic. This may 

 be due to a difficulty of getting some of the RNA 

 in this system to leave the template. Due to the 

 relatively small increase in chromatin template 

 activity, statistical studies were applied (10) to 

 this system and the difference shown to be sig- 

 nificant at the 95% level. The possibility that an 

 increase in RNA synthesis following hormone 

 administration might be due to less RNase or 

 ATPase in the in vitro system was checked and 

 found not to be the case. The size of the DNA in 

 the induced and noninduced chromatin was the 

 same, as deduced from analytical ultracentri- 

 fuge studies. The deproteinized DNA from both 

 types of chromatin were identical in their ability 

 to direct DNA-dependent RNA synthesis. 



In our experiments with estradiol we have 

 adopted a different approach. Estradiol, applied 

 in vitro to the endometrial cells of immature 

 calves, stimulates RNA and protein synthesis 

 followed by DNA synthesis and mitosis about 

 44 hours after the initial hormone applica- 

 tion (12). The fact that it is possible to demon- 

 strate such mitosis by microphotography (12) 

 shows that the tissue incubated in vitro appears 

 to be responding in the same way that the 

 endometrial tissue is in the calf. One thing which 

 interested us and which is pertinent to the 

 problem of histones and the relationship of 



140 



