28 MOLECULES, VIRUSES, AND BACTERIA 



not think that this is hkely, we are currently attempting to disprove 

 this by demonstrating a de novo synthesis of the enzyme from metabo- 

 lites supplied exogenously after infection. 



Biosynthesis of thymidylate synthetase. Our studies with this 

 enzyme originated in part as a result of the similarities of the early 

 methods of assay for synthetase and hydroxymethylase (Flaks and 

 Cohen, 1957), Measuring the fixation of acid-stable C^'*H20, it was 

 shown that: (1) this enzyme was normally present in E. coli capable of 

 synthesizing thymine; (2) the enzyme rapidly increased six- to seven- 

 fold during infection of strain B with T2; (3) the increase did not occur 

 on infection with ghosts; (4) the increase of enzyme required protein 

 synthesis ( Flaks and Cohen, 1959b ) . 



Extending this work, we explored thymine-requiring mutants, 

 which had been observed to synthesize thymine after infection with T2 

 (Earner and Cohen, 1954) and T5 (Crawford, 1959). Using a sensi- 

 tive carrier assay, it was shown that although the extracts of thymine- 

 requiring strains lacked the synthetase, infection with T2 or T5 re- 

 sulted in the extensive production of the synthetase (Earner and 

 Cohen, 1959). It was found that in infections with T5, a virus which 

 does not contain HMC, production of the synthetase occurs without a 

 concomitant production of the dCMP hydroxymethylase. Indeed, pro- 

 duction of the synthetase in T5-infected cells exceeds that in T2- 

 infected cells. 



We have obtained two very active inhibitors for this enzyme, 

 which is crucial in the economy of most cells. In the absence of thy- 

 midylate synthesis and exogenous thymine, a normal synthesis of DNA 

 cannot occur. The unbalanced cell growth which then ensues results in 

 the irreversible loss of the ability to multiply, or "thymineless death" 

 (Earner and Cohen, 1954). Such a situation can be provoked by 

 5-fluorouracil deoxyriboside in many cells, including tumors, since the 

 nucleotide, fluorodeoxyuridine-5'-phosphate (F-dUMP) is a potent in- 

 hibitor for thymidylate synthetase ( Cohen et al., 1958 ) . Fluorouridine- 

 5'-phosphate is essentially inactive. 



In the search for a fluorouracil nucleoside which is less readily de- 

 graded by the mammal. Dr. J. Fox of the Sloan-Kettering Institute has 

 synthesized D-arabinofuranosyl 5-fluorouracil, and we have prepared 

 the 5'-phosphate of this nucleoside by an enzymatic method. This new 

 nucleotide (F-aUMP) is also an inhibitor of thymidylate synthetase, 

 although 1/100 as potent as F-dUMP. In the study of compounds 

 which can provoke thymineless death by this mechanism, T2 or T5 

 virus-infected bacteria are easily the best source of the thymidylate 

 synthetase. 



The production of thymidylate synthetase can be controlled by 

 three chemically diflFerent DNAs. These are the cytosine-containing 

 DNA of strain B, the HMC-containing DNA of T-even phages used to 



