PROBLEMS IN POLYNUCLEOTIDE BIOSYNTHESIS 97 



TABLE I 

 The Incorporation of [^H]-TdR into DNA by Soluble Enzymes Prepared 



FROM SONICALLY DISRUPTED RaBBIT TISSUES [8] 



The reaction mixture contained o-i M phosphate buffer pH 8-i, 50 /imoles 

 glucose /ml., 2-5 /xmoles ATP/ml., 2-5 /imoles DPN/ml., 4/x moles MgCU/ml., 

 500 /xg.DXA/ml., 20 /Ltmoles NaCl/ml., and 2 /iC [^H]-TdR/ml. Incubation 

 time : 2 hr. 



„. DiS'A specific activity 



^^^"^ (c.p.m.//xmole DiXA-P) 



Rabbit bone marrow 24 750 



Rabbit thymus 9 275 



Rabbit appendix i 485 



Rabbit liver 131 



Rabbit kidney 71 



expected, the lowest values are found for the non-proliferating tissues such 

 as liver and kidney and the highest values in tissues such as bone marrow 

 in which cell division is very active. Extracts of such rapidly prolifering 

 tissues may be used to illustrate the net synthesis of DNA. Table II shows 



TABLE II 



S-ynthesis of Polynucleotide by Polymerase Present in Extracts of Ehrlich 

 Ascites Tumour Cells [io] 



The reaction mixture contained 100 /^imoles tris buffer pH 7-9, 0-25 /tmole 

 DPN, 5 ^moles MgCI.j, 25 /tg. ascites DNA and 07 ml. ascites cell extract in a 

 total vol. of I ml. Incubation time: 4 hr. Figures in parenthesis represent the 

 number of determinations. 



Additions 



Total DNA/tube 



Nil 26-2 (4) 



0-3 /^imole each of dATP, dCjTP, 



dCTP and TTP 47-7 (4) 



net synthesis by the polymerase present in a cell-free extract of Ehrlich 

 ascites carcinoma cells incubated with the four deoxyribonucleoside tri- 

 phosphates in the presence of primer DNA, magnesium ions and DPN. 



The activities of the kinases responsible for the formation of TTP are 

 shown in Fig. 2. Again the highest activity is found in extracts of those 

 tissues showing the most active cell proliferation. The activity in liver 

 tissue is very low indeed but when cell proliferation is induced by partial 

 hepatectomy a rise in the ability of extracts of the resulting regenerating 

 liver tissue to form TTP from TdR is apparent within 24 hr, and is very 



