24 MOLECULES, VIRUSES, AND BACTERL\ 



sec"^ Analysis of the specific activities in the peak of enzyme elution 

 indicates that the enzyme would probably be a major protein com- 

 ponent. From the S2o,w for this component, assuming a frictional ratio 

 of 1, a molecular weight of 49,000 and a turnover number of the order 

 of 125 moles dCMP per mole of enzyme per minute have been 

 calculated. 



With these numbers we may estimate the number of enzyme mole- 

 cules per cell. In the Biogen experiment, 10^" infected cells gave rise to 

 about a milligram of extract protein capable of converting 0.3 micro- 

 mole of dCMP in 20 minutes. It can be calculated that an infected cell 

 contains about 8,300 molecules of hydroxymethylase in 15 minutes or 

 produces about nine molecules of enzyme per second over a 15-minute 

 interval. Since the appearance of enzyme occurs at almost a constant 

 rate, it can be calculated that at the time DNA synthesis begins ( about 

 seven minutes), the hydroxymethylase content of the cells can main- 

 tain a rate of dHMP synthesis sufficient to produce 120 T2 virus par- 

 ticles per cell if continued for 14 minutes. This value is quite close to 

 the usual yield of a T-even phage per cell in a one-step growth experi- 

 ment in the media used. 



We can now ask whether any active enzyme is to be found in un- 

 infected cells. In order to test this, it has been necessary to run assays 

 with approximately 2 x 10'^ cpm per assay tube or 2.4 micromoles of C^^- 

 H2O at 7 ixc per micromole. Extracts of uninfected cells were prepared 

 and amounts of protein (1.6 to 4.7 mg. ) were used derived from 8.2 x 

 10^ to 2.6 X 10^*^ cells. Among the blanks used were the reaction mixture 

 minus protein, and the reaction mixture with an extract lacking added 

 dCMP. In another control, tlie reaction mixture was run with an extract 

 of infected cells. The expected activity of enzyme was obtained in 

 spite of unusual radioactivity of HCHO used. After 20 minutes, 0.55 

 jnicromole dHMP was added as carrier, and this substance was purified 

 initially on Dowex 50 - H + and subsequently by paper chromatography 

 in isobutyrate-NH40H. The nucleotide was hydrolyzed, and the free 

 HMC was purified by paper chromatography in butanol-NH40H and 

 isopropanol-HCl. The purification steps for carrier HMC are summa- 

 rized in Table I. It can be seen that after the final chromatography, the 

 activity present in carrier HMC in all assays was far less than could be 

 expected on the hypothesis that the cells contained at least one mole- 

 cule of dCMP hydroxymethylase per cell. Furthermore, there was no 

 suggestion of a trend in fixation as the amount of cell protein was in- 

 creased. It may be remarked that the exclusion of activity for one mole- 

 cule of enzyme in any auxotroph or otherwise "deficient" bacterium 

 does not seem to have been reported previously. 



Biosynthesis of dCMP hydroxymethtjlase . Enzyme cannot be de- 

 tected in uninfected cells, but it is found in large amount in all infected 

 cells (8,300 molecules per cell, or 0.7 per cent of the protein extracted 



