t2 and other bacterial viruses 467 



deproteinized by stirring with chloroform-octanol (9 to 1), and tlie protein 

 centrifuged off. The nucleic acid can then be precipitated from the super- 

 natant by the addition of 2 volumes of cold 95 % ethanol. After subsequent 

 dialysis and lyophilization, 80 to 90 % of the original nucleic acid can be 

 recovered in this mamier. 



It is also possible to separate viral DNA in the T-even series by osmotic 

 shock and removal of the ghost particles by centrifugation. The nucleic acid 

 is subsequently precijDitated with ethanol. With those phages in which tlie 

 distal protein can be removed by the Cd++-cyanide complex, complete libera- 

 tion of the viral nucleic acid into the supernatant can be effected by the 

 addition of the amnio acid lysine. After centrifuging down the protein frag- 

 ments, the DNA in the supernatant can be further purified by the procedures 

 listed above. 



Since the DNA content of T2 is about 2 X 10~^^ gm. of nucleic acid per 

 particle, the molecular weight of the DNA would be around 120,000,000 if it 

 existed as a single molecule. Measurement of the DNA liberated by the urea 

 treatment (Cohen, 1947) gives values of around 25,000,000. 



A. Analysis of Components of Viral Nucleic Acid 



The further analysis of viral DNA involves acid or enzymatic hydrolysis, 

 and separation and determination of the liberated nucleic acid components, 

 either by cliromatographic procedures or specific reagents. Recognition of 

 the fact that perchloric acid hydrolysis led to destruction of the character- 

 istic pyrimidine, 5-hydroxymethyl cytosine (5-HMC) has induced the use of 

 either 6 N HCl for 3 hours at 100°C. under CO 2 in sealed tubes (the latter 

 precaution is omitted by some workers), or 88 % formic acid (0.5 ml. per 1.5 

 mg. of virus) in a sealed tube for 30 minutes at 75°C. The further separation 

 and determination of the purine and pyrimidine bases is made usually by 

 paper chromatography with elution and determination of the characteristic 

 ultraviolet (UV) absorption of the individual bases. One- and two-dimensional 

 chromatographs are used, propanol-HCl and propanol-NH40H being the 

 most common solvents (Wyatt and Cohen, 1953; Hershey et at., 1953). The 

 bases can also be separated and determined by ion exchange chromatography 

 (Jesaites, 1957). Table II is a summary of values from the literature for the 

 purine, j^yrimidine, and glucose content of the T series of phages, the tem- 

 perate phage A which lysogenizes E. coli strain, and the coliphage CI 6 

 (Burnet, 1933). This represents the extent of our quantitative knowledge of 

 the nucleic acid components of the bacterial viruses. 



The most striking feature of the DNA of the even-numbered coliphages 

 and phage CI 6 is the presence of the pyrimidme, 5-hydroxymethyl cytosine, 

 (Fig. 2) w^liich apparently replaces cytosine (since the latter is not found in 



