300 BACTERIAL VIRUSES. II 



4.5 cc. of T2r+ at 2.2 X 10^ ^ per cc. and 1.5 mg. of P in inorganic phosphate 

 containing about 0.05 milhcurie of P^\ The infected culture was aerated 

 vigorously at 37° for 1 hour. At zero time and at 1 hour, aliquots were 

 analyzed for protein-bound P, DNA, and radioactivity. 



The counts per minute of the medium were 93 per microgram of P. In 

 1 hour, the bacterial protein-bound P increased from 0.0109 mg. per cc. to 

 0.0163 or 5.4 y per cc. Newly assimilated P had a radioactivity of 98 

 counts per microgram. The DNA had increased from 24 to 72 y per cc, 

 or the ratio of newly formed DNA to original DNA was 2:1. 



Following the removal of aliquots after 1 hour, the entire culture was pre- 

 cipitated with 5 per cent trichloroacetic acid (TCA) and the sediment was 

 washed twice in the centrifuge with TCA. The supernatant fluids were 

 pooled. The sediment was washed twice with 95 per cent alcohol and twice 

 with ether. These supernatant fluids were combined with lipide extracts 

 prepared by three extractions of this sediment with boiling alcohol-ether 

 (1:1). The pooled lipide extracts were taken to dryness in vacuo and dis- 

 solved in 10 cc. of CHCI3. 



The dried lipide-free sediment was fractionated according to Schmidt and 

 Thannhauser (8). It was incubated with 2 cc. of n KOH at 37° for 20 hours 

 and chilled. To the solution containing a very slight flocculent precipitate 

 were added 0.4 cc. of 6 n HCl and 2 cc. of 5 per cent TCA. After 30 minutes 

 at 0°, the sediment, containing DNA, was removed by centrifugation and 

 washed with several cc. of water. The DNA fraction was dissolved in 0.1 

 N NaOH. The combined supernatant fluids were precipitated according 

 to Delory (9) ; essentially no P was found in this sediment and this fraction 

 was discarded. The supernatant fluid contained ribose-3-phosphate nu- 

 cleotides and comprised the RNA fraction. 



Both the DNA and RNA fractions prepared above were analyzed for 

 DNA. The former contained 6.9 mg. and the latter 0.6 mg. of DNA, or a 

 total of 7.5 mg. of DNA was recovered. The total DNA content of the 

 culture was 7.4 mg. Radioactivity measurements of the RNA fraction 

 were corrected for the radioactivity of the DNA it contained. This com- 

 prised 10 per cent of the P of this fraction. The radioactivities of the 

 nucleic acid fractions are summarized in Table IV. 



One-half of the P in the RNA fraction consisted of ribose-3-phosphate 

 nucleotides reactive in the Bial reaction (10). Pyrimidine nucleotides de- 

 rived from RNA and comprising one-half of the total P would not be 

 expected to react. Thus the RNA fraction with the exception of a smaU 

 amount of DNA contained ribose nucleotides of the expected character- 

 istics. 



It may be seen from Table IV that the newly synthesized DNA contained 

 P of essentially similar radioactivity to that in the medium, while the RNA 



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