PRECURSORS OF BACTERIOPHAGE NITROGEN AND CARBON* 



L. KozLOFF, F. W. Putnam, and E. A. Evans, Jr. 

 Department of Biochemistry, University of Chicago 



It has been known for some time that bacteriophage-infected cells 

 require an exogenous source of nitrogen and oxidizable carbon for 

 growth of virus (Spizizen, 1943). These early results imply that 

 nitrogen and carbon of the medium are assimilated for synthesis of 

 bacteriophage but do not demonstrate the incorporation of elements 

 of the medium into the phage nor reveal the intermediate pathways or 

 precursors. Isotopic investigations of workers in this laboratory (Koz- 

 loff, Knowlton, Putnam and Evans, 1950; Barry, Gollub-Banks and 

 Koch, 1950), which are reported herein, disclose the sources of bacterio- 

 phage nitrogen and demonstrate a substantial transfer of purines from 

 the host to the virus. While the medium is the ultimate source of most 

 of the virus N, P, and C, the investigation has been chiefly concerned 

 with the nature of the bacterial contribution. The results indicate that 

 the protein and nucleic acid of the virus are in part derived from 

 bacterial precursors and that a substantial portion of the bacteriophage 

 DNA arises from bacterial DNA. 



The principles and conduct of the experiments have already been 

 described (Kozloff and Putnam, 1950). Bacteriophage was grown by 

 multiple infection on labeled host cells or labeled medium (synthetic 

 lactate), purified by differential centrifugation, and characterized in 

 the analytical ultracentrifuge and by infectivity measurements. In 

 most instances the purified phage was partitioned into nucleic acid and 

 protein and the nucleic acid was hydrolyzed to give purines and pyrim- 

 idines. The purines were separated chemically, and the adenine or 

 guanine crystallized to constant radioactivity with added carrier or 

 were isolated by column chromatography without added carrier. 

 Thymine was purified by sublimation. In several instances the protein 

 was hydrolyzed and the acidic, basic, and neutral amino acid fractions 

 were separated by ion exchange columns. In a few experiments the 

 medium was labeled with P^^Of, or with N^^^H^Cl, but in most cases 

 the host was labeled. The bacteria were labeled as follows: a) fully 

 labeled with P^^ or N^^ (by growth on isotopic media so that all P or 

 N fractions were equally labeled), b) differentially labeled with P^^ 

 or N^^ (grown in isotopic medium as in (a) but the washed bacteria 

 then allowed to metabolize in growth limiting medium (for P^") or to 



'Aided by a Grant from The National Foundation for Infantile Paralysis. 



