Organization, Replication, and Types of DNA in Vivo 



315 



in the ullracentrifuge tube is found to become 

 two bands, one at the hybrid position and one 

 at the ail-hght position. It should be noted, 

 moreover, that the time required for the 

 change from all-heavy to all-hybrid molecules, 

 or for the change from all-hybrid to half all- 

 light and half hybrid molecules, is approxi- 

 mately the interval occupied by a bacterial 

 generation. 



Although these results are consistent with 

 the hypothesis of replication of double-strand 

 DNA by chain separation, they do not auto- 

 matically exclude other possible explanations. 

 It might be claimed, for instance, that the 

 double helix grows, not by separation of 

 chains followed by the synthesis of comple- 

 mentary ones, but by the addition of new 

 double chain material to the ends of the 

 original double chain. One can test this al- 

 ternative explanation in two ways. 



If the all-heavy molecules grew by adding 

 light material to their ends, they should be 

 composed linearly of double chains that are 

 successively light, heavy, and light. Then it 

 should be possible to fragment the macro- 

 molecules by sonic vibrations into smaller 

 segments, some of which should be all-heavy 

 and others all-light. This should be de- 

 tectable in the ultracentrifuge tube by some 

 DNA assuming the all-light and all-heavy 

 positions. This does not happen. The DNA 

 remains in essentially the same hybrid posi- 

 tion whether or not it is fragmented sonically. 



A second test of the view, that synthesis is 

 at the ends of the double chains, is made pos- 

 sible by the following fact. If double- 

 stranded DNA is heated to an appropriate 

 temperature (near 98° C), the H bonds are 

 broken and the complementary strands sepa- 

 rate. (Double-stranded DNA's with high 

 A + T/G -f C ratios become single-stranded 

 at lower heats than do those with low ratios. 

 This is expected since high-ratio DNA is 

 richer than low-ratio DNA in A-T, each pair 

 of which has one less H bond than a C-G 

 pair, so that less energy is needed to break the 



smaller total of H bonds present in the 

 former than in the latter.) If the appropri- 

 ately heated mixture is cooled quickly, the 

 chains remain single. That this heat de- 

 naturation followed by quick cooling produces 

 single strands from double helices can be con- 

 firmed by the loss of that part of the DNA 

 X-ray difTraction pattern which denotes poly- 

 strandedness. The change to single-strand- 

 edness is also accompanied by an increase of 

 as much as 40% in the absorbence of ultra- 

 violet light of 2600 A. The second test of 

 endwise DNA synthesis involves converting 

 all-light and all-heavy DNA to the single- 

 stranded condition and locating the positions 

 of the two types of single strands in the ultra- 

 centrifuge tube. Then "hybrid" DNA is 

 made single-stranded and ultracentrifuged. 

 This preparation shows only two major com- 

 ponents, one located at the all-light single- 

 strand position and the other at the all-heavy 

 single-strand position. This result also is 

 inconsistent with the hypothesis under test. 

 Not only do the two tests eliminate the view 

 that appreciable endwise synthesis of DNA 

 occurs in bacterial DNA, but they offer ad- 

 ditional support for the hypothesis of replica- 

 tion after chain separation. 



Experiments like those done with bacteria 

 have been performed, with similar results, 

 using the unicellular plant, Chlamydomonas, 

 and other higher organisms, including man. 

 The general agreement in the results of all 

 these experiments furnishes conclusive, or 

 at least almost conclusive, evidence of the 

 correctness of the Watson-Crick hypotheses 

 for the double helix configuration of DNA in 

 the chromosomes, and for DNA replication 

 after chain separation. 



The work we have just discussed shows 

 clearly that the great proportion of chromo- 

 somal DNA exists in double helix configura- 

 tion, and replicates by chain separation once 

 a generation, as would be expected were this 

 substance genetic material. We can ask now 

 whether all the DNA found in chromosomes 



