The kinetic order of synthesis of
deoxycytidylate deaminase (EC 3.5.4.12), deoxycytidylate hydroxymethylase (EC 2.1.2.b),
dihydrofolate reductase (EC 1.5.1.3),
5-hydroxymethyldeoxycytidylate kinase (EC 2.7.4.4), and
thymidylate synthetase (EC 2.1.1.b) after
infection of Escherichia coli with T2r(+) bacteriophage was found not to correlate with their order of synthesis in an in vitro
protein-synthesizing preparation. The in vivo and in vitro synthesis of
enzyme-specific
messenger RNA measured in the
protein-synthesizing preparation preceded each
enzyme by about 1 min. Through the use of sheared
DNA, it was shown that the
thymidylate synthetase gene was most susceptible to a loss in template activity, which suggests that this gene is further removed from its promoter than the other genes are from theirs. With
a DNA segment of 2.5 x 10(5) daltons, the synthesis of
dihydrofolate reductase alone was obtained, but at a much reduced rate. Translation of the
RNA from phage-infected cells treated with
chloramphenicol yielded amounts of
dihydrofolate reductase and deoxycytidylate hydroxymethylase activities similar to those obtained with
RNA from untreated infected cells. These results suggest that the
chloramphenicol RNA, which consists primarily of immediate-early
RNA, may contain most, if not all, of the information required for the synthesis of phage
dihydrofolate reductase and deoxycytidylate hydroxymethylase.