THE USE OF CENTRIFUGATION IN BIOLOGY 87 



The purpose of the sugar (or salt) is to stabilize the contents so that 

 minor shaking or convection will not remix the separated components. 

 The viscosity of the sugar solution contributes greatly to the stabiliza- 

 tion. Ordinary buffer solutions are not stable enough. This density 

 gradient separation method gives no absolute quantitative data, but does 

 give quantitative separation of components. 



AN EXAMPLE OF THE USE OF CENTRIFUGATION IN BIOLOGY 



One of the fundamental problems of genetics is whether the nuclear 

 material, especially the nucleic acid, stays in one piece during replication. 

 In principle three different fates are possible for the nucleic acid. First, 

 it could always remain intact. This would imply that the new nucleic 

 acid which goes to one of the daughter cells after mitosis is built by 

 copying the parental nucleic acid without disrupting it at all. Since such 

 a mechanism entirely conserves the parental molecules, it is called the 

 conservative method of replication. 



Second, the Watson-Crick model of DNA affords the possibility that 

 the nucleic acid comes apart into just two pieces, which are separately 

 replicated by pairing with the proper components. Thus each daughter 

 cell would receive exactly half of the parental nucleic acid, in one single 

 piece. If this model is correct, then in a second generation, resulting in 

 four daughter cells, two would contain one each of the parental half- 

 strands; the other two cells would contain none of the parental nucleic 

 acid atoms. This scheme conserves half of the parental molecules of 

 nucleic acid and is therefore called semiconservative. 



Third, the nucleic acid might come apart into a number of pieces which 

 are incorporated at random into various maturing DNA strands by some 

 unknown mechanism. In this scheme the parental nucleic acid molecules 

 are dispersed among the daughter cells and the method of replication is 

 therefore called dispersive. 



A combination of isotope utilization and density gradient centrifuga- 

 tion has permitted an unequivocal decision about the mechanism for 

 bacteria. The bacteria in question were grown in a nutrient medium con- 

 taining much heavy nitrogen (N 15 ) instead of normal nitrogen (N 14 ) 

 and heavy carbon (C 13 ) rather than normal carbon (C 12 ). Thus the 

 nucleic acids of such cells would be more dense than the nucleic acids of 

 cells grown in a normal medium. 



In this experiment, cells were grown for many generations in the dense 

 medium and then transferred to a normal medium. Samples of cells 

 were taken immediately upon transfer to the normal medium and at 

 various times thereafter; the DNA of the sample cells was extracted and 

 run in a CsCl density gradient. The results are shown in Fig. 42. 



