30 MMRICE .T. BKSSMAX 



phosphate are incubated separately, and the incorporation of label into 

 the acid-insoliiblo product is measured in each. This method has the 

 advantage of beino; applicable to very small syntheses and has been 

 used to measure base ratios of products foi'med vciy early in the reac- 

 tion. In the second method, i)roducts obtained from large-scale syntheses 

 are hydrolyzed and chromatographed to determine base content directly. 

 It is important that the primer represent only a small percentage of the 

 total DNA analyzed so that routinely 10- to 20- fold net syntheses are 

 employed in this procedure. 



In Table XIII, the base compositions of several different DNA 

 preparations are shown. First, in all cases, the percentage compositions 

 of the bases in the products are very similar to these values for the 

 respective primers. Second, the content of adenine approximates the con- 

 tent of tliymine and the guanine and cytosine contents are about equal. 

 Thus, purines ecjual pyrimidincs. These relationships are in keeping with 

 the proposed structure for DNA. 



With the use of the radioactive assay it was possible to show that 

 the comi)osition of the product is established early in the reaction. In 

 Table XIV it can be seen that when only 2% increase in DNA synthesis 

 had occurred in the reaction primed with Mycohacterinm phlei DNA, the 

 ratio of thymine to cytosine incorporation was equal to that of the primer. 

 This ratio remained constant with u]) to 1637^ increase in DNA. The 

 same is true for the reactions primed with calf thymus and Aerobacter 

 aerogenes DNA. The increase in ratios during the latter part of the 

 reaction can be explained by the formation of a polymer of deoxyadeny- 

 late and deoxythymidylate fomied late in the incubation. This polymer 

 will be discussed in Section IV,G. 



An important consideration in establishing what factor (s) is respon- 

 sible in determining the ultimate composition of the product is whether 

 the relative concentrations of the individual deoxj^-ibonucleoside triphos- 

 phates in the incubation mixture influence their relative concentrations 

 in the product. That this is not the case is indicated in Table XV, where 

 it can be seen that although the relative concentration of dTTP and 

 dATP were varied singly and together over a 5-fold range, the composi- 

 tion of the product remained constant. Thus, the determinant of product 

 composition seems to lie exclusively in the structure of the primer DNA 

 and the concentrations of the individual deoxyribonucleoside triphos- 

 phates do not play an important role here. It is clear from experiments 

 cited earlier that elimination of one of the triphosphates entirely from 

 the incubation mixture effectively stops DNA synthesis except under spe- 

 cial circumstances. Therefore, although the sfnictvre of the product is 

 not influenced by the concentration of the substrates, the rate of syn- 



