The Temporal Expression of T2r+ Bacteriophage Genes In Vivo and In Vitro

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RESUMO

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 × 105 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.

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