324 



CHAPTER 35 



can be seen in the diagram at the left of the 

 Figure, P* should not occur in inorganic 

 phosphates but should sometimes appear in 

 other deoxyriboside 3'-monophosphates be- 

 sides the one containing C. When the experi- 

 ment was performed, the latter result was 

 obtained. Not only was P* absent from in- 

 organic phosphate, but it was found fre- 

 quently in all four kinds of deoxyriboside 

 3'-monophosphates. 



An additional test of the view that the DNA 

 chain grows at its 3' position is furnished by 

 treatment of the limited product by a dif- 

 ferent enzyme, snake venom diesterase. This 

 enzyme digests DNA by breaking the bond 

 between the phosphate and sugar at the 3' 

 position, starting at the nucleoside end of the 

 chain and proceeding toward the nucleotide 

 end. In this way, the DNA is gradually 

 digested into deoxyriboside 5'-phosphates as 

 indicated by the arrows in Figure 35-4. When 

 the limited product was treated this way, it 

 was found, as expected, that almost all the 

 radioactivity had been removed from the 

 chain, even though only a very small portion 

 of the DNA had been digested. Other results 

 show clearly that the product of a limited re- 

 action is DNA, which has one or very few 

 deoxyribotides added to the nucleoside end 

 of the chain. Still other evidence supports the 

 view that the 3' point of lengthwise linkage is 

 the same when net DNA is greatly increased 

 as it is when the limited reaction occurs. 



The fact that lengthening of a DNA chain 

 can occur in vitro, when any of the four com- 

 mon deoxyribosides happen to be at the 

 nucleoside terminus, is consistent with our 

 knowledge of the nondependence of DNA pri- 

 mary structure upon base sequence. Is there 

 any other evidence that the DNA synthe- 

 sized in vitro has the characteristics of DNA 

 synthesized in vivol Let us summarize some 

 of the physical properties of samples of DNA 

 composed of 90% or more of the product 

 synthesized in vitro. Such samples have 

 physical characteristics that are similar to 



>DNA 



p.p. 



FIGURE 35-4. Degradation of DNA {arrows) by 

 snake venom diesterase. 



that of DNA isolated from calf thymus, in- 

 sofar as sedimentation rate and viscosity are 

 concerned. From such characteristics a 

 molecular weight of about 6 million was cal- 

 culated, and it was inferred that the product 

 was not usually single-stranded. In support 

 of the latter inference was the finding that the 

 macromolecular structure of the in vitro prod- 

 uct was destroyed when heated for 10 minutes 

 at 100°C, as expected if this treatment pro- 

 duced single chains which collapsed to form 

 compact, randomly coiled structures. Like 

 thymus DNA, the enzymatic product shows 



