CHALKLEY: Yes. 



Dr. Maurer and myself have been concerned 

 with the problems of hlstone metabolism. This 

 is an interesting field in which the results tend 

 to depend upon the methods employed for the 

 isolation of the different histone fractions. We 

 have been fortunate to have the advantage of 

 using the relatively standardized IRC- 50 micro- 

 separation techniques which have been developed 

 at Cal Tech. In order to keep the system as 

 simple as possible, we decided to investigate 

 systems in which no DNA was being made. In 

 the presence of DNA replication, as the DNA: 

 histone ratio appears to be efficiently main- 

 tained, all histones must be synthesized. One 

 promising system that we had available to us, 

 in connection with some hormone studies which 

 we were doing, was the endometrium tissue iso- 

 lated from immature calves. We can get a great 

 deal of this tissue which, in the absence of 

 estradiol treatment, is not involved in DNA 

 replication. The tissue was incubated in the 

 presence of CI-* -leucine and histones were iso- 

 lated directly from the nucleus by acid extrac- 

 tion. The results of the subsequent fractionation 

 of the histones are shown in Fig. 5. Again the 

 optical density pattern is similar to that de- 

 scribed previously. However, the most striking 



thing is that histones la, lb, and lib, so far as 

 we can see, do not incorporate radioactive 

 label to any significant degree. Peaks III and 

 IV and the material in the run-off peak (R) do 

 incorporate label. In order to demonstrate that 

 this was standard protein synthesis, we ran con- 

 trol experiments with puromycin present in the 

 incubation medium and the incorporation was 

 decreased by a large amount in both instances 

 (Fig. 6). 



We were interested in seeing if this labeled 

 histone was really attached to the chromatin 

 which we isolated following procedures de- 

 scribed earlier. We combined this with the 

 study of the incorporation of amino acids into 

 peptides in isolated nuclei. Figure 7 shows the 

 results of a nuclei incubation followed by chro- 

 matin isolation from the nuclei. Histones were 

 obtained by acid extraction of the chromatin. 

 Again, there is incorporation of the label into 

 III and IV and into the run-off peak. I should 

 add that if one measures the specific activity 

 in the peaks from the whole tissue incorpora- 

 tion, the specific activity approaches that of the 

 whole cytoplasmic protein. 



We wondered if this was a general effect or 

 whether it was just a rather unusual result 

 found in the one tissue. In Fig. 8 you see what 



O 04 

 t 



1400 

 1200 

 1000 

 800 



80 I OU 120 



fra:tion mumber 



Fig. 5. 



The biosynthesis of histones of incubated endometrium tissue In the absence of DNA repli- 

 cation. (Fig. 2, Chalkley and Maurer, Proc. Natl. Acad. Sci. U.S. 54, 498, 1965; repro- 

 duced with permission of the National Academy of Sciences.) 



135 



