140 ( HAHI.KS A. THOMAS, JR. 



This view was supported by the findings of both Stent et al., and 

 I.evintJKd that these rehitively hirp;e pieces were not further dispersed 

 on transfer to a second <j;('n('ratioii of jji^o^cny phage. 



With the a(lv(>nt of density hd)eHng techni(iues, two groups })egan 

 to reinvestigate these pi-oblenis: Kozinski, using o-bromodeoxyuridine 

 (BU), and Roller and Meselson employing N^^ and C^-^ (Kozinski. 1961 ; 

 Roller, 1961). In the case of Kozinski's ex])eriinents, normal P'*--labeled 

 phage were allowed to singly infect "heavy" bacteria growing in a 

 medium containing 5-bromodeoxyuridine. Progeny phage were collected 

 and DNA extracted by the phenol procc^lure. This DNA was banded in 

 CsCl and the P'*- (which came from the parental DNA) was now found 

 to be in a band whicii overlapped the band for totally new or "heavy" 

 DNA. This experiment can be done in reverse as a result of perfecting 

 methods to grow highly infective, uniformly BU-labeled phage (Kozin- 

 ski, 1962). In Roller's experiment "heavy" phage are allowed to infect 

 "light" bacteria and the progeny DNA molecules are examined by 

 centrifugation in CsCl. Again the conclusion is the same, namely, that 

 all of the parental label is now found in molecules having a density 

 which is essentially the same as that of the newly synthesized "light" 

 DNA. Although it is difficult to estimate the upper limit to the size of 

 the transferred pieces, it seems clear that only a small fraction of the 

 parental DNA now resides in progeny molecules where it makes up as 

 much as 10% of the total molecule. Following breakage by shear 

 (Roller, 1961) or sonication (Kozinski, 1961) the parental label now 

 assumes a position in the band corresponding to molecules made up of 

 half-hea\y and half-light DNA. When the majority of the DNA has 

 been sheared to a molecular weight of about 10 X 10® (as estimated by 

 sedimentation coefficient), the majority of the parental DNA is found 

 at the half-heavy position. One would like to conclude directly that these 

 fragments are hybrid duplexes, in view of the fact that the majority of 

 the phage molecule is duplex. However, the possibility still exists that 

 the parental subunits now preferentially reside in some special regions 

 of the molecule, the structure of which is not known, and the existence 

 of which cannot completely be ruled out at this time. 



Unfortunately, there are no published experiments in which the 

 density labeling techniques have been employed to examine a second 

 generation of phage. There is at present very little information available 

 from these experiments on the distribution of the sizes of the transferred 

 pieces, and even less on the perpetuation of this distribution during 

 another cycle of infection. 



There is a conflict between the earlier results and those derived from 

 the density labeling experiments just mentioned. It could be one of 



