Regulation of Gene Synthesis 



451 



This formula completes a summary of the 

 pathways involved in producing the deoxy- 

 riboside triphosphates required for replica- 

 tion of E. coli DNA. 



In the preceding discussion we noted that 

 an infecting virulent phage carries spec- 

 ifications for the manufacture of a specific 

 thymidylate synthetase and probably for 

 specific nucleoside monophosphate kinases. 

 This conclusion suggests that virulent phages 

 carry instructions for making a number of 

 specific proteins. Within two minutes after 

 injection of T phage DNA, phage specific 

 RNA appears; within four minutes phage 

 specific proteins appear; and within six min- 

 utes phage DNA is synthesized — five times 

 faster than is DNA in uninfected cells. Host 

 DNA is destroyed soon after infection by 

 T-even phages. Although the mechanism 

 is not completely clear, it is thought to in- 

 volve a new DNase that appears after phage 

 infection. Roughly half an hour after in- 

 fection 100 to 200 new phages are produced 

 and liberated by lysis. These activities lead 

 us to hypothesize that, after virulent phage 

 infection, all DNA and messenger RNA syn- 

 thesis in the bacterial cell is directed by the 

 phage DNA. That E. coli DNA contains 

 C, whereas the T-even phages contain 5- 

 hydroxymethyl cytosine {HMC) to which 

 glucose is attached in different ratios for dif- 

 ferent T-even phages, suggests another test 

 of this hypothesis. 



Within several minutes after infection 

 with T-even phage, dCP is converted to 

 rfHMCP by a hydroxymethylase. This en- 

 zyme is newly produced, since uninfected 

 cells or cells infected with T5 (which has 



no HMC in its DNA) have no hydroxy- 

 methylase activity. Through the action of 

 kinase — also produced only in T-even in- 

 fected cells — dHMCP is phosphorylated to 

 dHMCPPP. All the nucleoside monophos- 

 phate kinase activity for G, T, and HMC 

 in phage-infected cells may be due to a sin- 



gle new enzyme. In T2-infected cells an- 

 other new enzyme appears which splits 

 pyrophosphate away from c/CPPP and ortho- 

 phosphate away from dCPP converting these 

 to dCP which, as described, is the substrate 

 for making t/HMCP. The dephosphorylat- 

 ing activity of this enzyme, dCPPP-dCPPase, 

 is 60 times greater than the kinase phos- 

 phorylating activity and has no effect upon 

 dHMCPPP. Such a mechanism seems to 

 be adequate in excluding C from T-even 

 phage DNA. 



The biochemical pathways described in 

 uninfected and T-even infected cells result 

 in the synthesis of dHMCPPP, dAPPP, 

 dGPPP, and dTPPP— the raw materials re- 

 quired for DNA polymerase action in the 

 synthesis of T-even phage DNA. As is true 

 for other phage-induced enzymes, DNA 

 polymerase shows a high level of activity in 

 phage-infected cells. Is a new DNA poly- 

 merase developed in response to T2 phage 

 infection, a different one from the E. coli 

 DNA polymerase formed in uninfected 

 cells? The DNA polymerases in samples 

 from uninfected and T2-infected cells are 

 found to differ in antigenic properties, mi- 

 gration during chromatography, and inac- 

 tivation sensitivity to specific chemicals. 

 Moreover, E. coli DNA polymerase can use 

 native, double-stranded DNA as primer- 

 template, whereas the polymerase from the 

 infected cells is virtually inert. Given single- 

 stranded primer-template, E. coli polymer- 

 ase can increase the amount of DNA ten- 

 to twentyfold, whereas the polymerase from 

 infected cells produces less than 100% in- 

 crease. We may conclude, therefore, that 

 a new DNA polymerase, T2 DNA polymer- 

 ase, is formed in T2-infected E. coli. 



As mentioned, T2, T4, and T6 have dis- 

 tinctly different glucose distributions in the 

 HMC of their DNAs. The glucose residues 

 are added to HMC in the DNA polymer 

 through the action of enzymes called glu- 



