Organization and Replication of DNA in Vivo 



273 



pect to happen if these bacteria are quickly 

 washed, placed in a culture medium con- 

 taining only light nitrogen, and permitted to 

 continue their synchronous multiplication? 

 The DNA should replicate each time the 

 bacteria undergo cell division. During the 

 first replication of DNA, the two strands 

 containing heavy nitrogen should separate, 

 and each should synthesize a complementary 

 strand containing only light nitrogen. Thus, 

 after one DNA replication, the density of 

 the DNA molecules should be exactly mid- 

 way between completely light and com- 

 pletely heavy DNA. 



To test whether or not this expectation 

 is actually observed, DNA is extracted from 

 "all-heavy" bacteria and also from "all- 

 light" bacteria. These extracts, serving as 

 controls, first are ultracentrifuged separately 

 and then together in a fluid medium con- 

 taining cesium chloride. When a solution 

 of cesium chloride is ultracentrifuged for 

 about twenty hours, a gradient of densities 

 is established because the concentration of 

 cesium chloride is greatest at the bottom of 

 the ultracentrifuge tube and least at the top. 

 In the ultracentrifuge tube DNA assumes 

 the position corresponding to its own density. 

 In the density gradient the position of the 

 DNA can be detected by its absorption of 

 ultraviolet light at 2600 A. Two separate 

 bands of DNA are found in the medium, one 

 containing the all-heavy and the other the 

 all-light DNA. When DNA is extracted at 

 various time intervals after the originally all- 

 heavy bacteria have been placed in the all- 

 light nitrogen culture medium, the DNA 

 band in the ultracentrifuge tube is observed 

 to move from the all-heavy DNA position to 

 a position exactly intermediate between the 

 all-heavy and all-light positions (Figure 

 20-8). This result is exactly what is ex- 

 pected if after one replication the DNA is 

 "hybrid'' in density. 



What would one expect to find after an 

 additional DNA replication? In this case, 



the two strands of the hybrid DNA should 

 separate, and light complementary strands 

 should be made by both the light and heavy 

 single strands. So, after a second replica- 

 tion, half of the double-stranded DNA mole- 

 cules should be all-light, and half should be 

 intermediate between all-light and all-heavy 

 (that is, they should be hybrid). In fact, 

 the samples of DNA taken at later intervals 

 show the single band at the intermediate 

 position in the ultracentrifuge tube has be- 

 come two bands, one at the hybrid position 

 and one at the all-light 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 approximately the interval occupied by a 

 bacterial generation. 



Although these results are consistent with 

 the hypothesis of replication of double- 

 stranded DNA following chain separation, 

 they do not automatically exclude other pos- 

 sible explanations. It might be claimed, for 

 instance, that the double helix grows not 

 by separation of strands followed by the 

 synthesis of complementary ones, but by the 

 addition of new double strand material to 

 the ends of the original double strand. This 

 alternative explanation can be tested in two 

 ways. 



If the all-heavy molecules present initially 

 grew by adding light material to their ends, 

 they should be composed linearly of double 

 strands that are successively heavy and light. 

 It should then be possible for sonic vibra- 

 tions to fragment the macromolecules into 

 smaller segments, some all-heavy and others 

 all-light. This result should be detectable 

 in the ultracentrifuge tube by some of the 

 sonicated hybrid DNA assuming the all- 

 light and some the all-heavy positions. 

 However, nothing happens; the DNA re- 

 mains in essentially the same hybrid position 

 whether or not it is sonically fragmented. 



A second test of the view that DNA syn- 



