The discovery that the desoxyribonucleic acid (DNA) of bacteriophage T2 lacks cytosine (Marshak, 1951), and contains instead 5-hydroxymethylcytosine (Wyatt and Cohen, 1952), permits one for the first time to study viral growth in terms of the synthesis of a distinctive chemical constituent. Other recent findings suggest, moreover, that DNA plays a dominant role in initiating the infection (Hershey and Chase, 1952). This paper describes the changes in cytosine and hydroxymethylcytosine content of the intrabacterial DNA after infection of Escherichia coli with bacteriophage T2. Materials and MethodsCultural Conditions.--The variety of T2 known as T2H was propagated on a strain of E. ~li, designated R2, chosen for its relative resistance to premature lysis following multiple infection. All cultures were grown in "peptone-broth" containing per liter 10 gin. bacto-peptone, 3 gm. NaC1, 1 gin. glucose, 1 m~ MgSO4, 0.1 m~ CaC12, and 5 mg. P added in the form of phosphate buffer of pH 7.0. Beef extract was omitted because it interferes with the diphenyiamine reaction for DNA.Bacteria were grown with aeration at 37°C. to a concentration of 2 × 10 s per ml. in 720 ml. peptone-broth, sedimented, and infected with 5 phage per bacterium in a non-nutrient adsorption medium (Hershey and Chase, 1952). The infected cells were then resedimented and transferred (at "time zero") to warm aerated broth at 2 X 10 s cells per ml. Each culture was analyzed to obtain the following data: colony counts of bacteria before infection; plaque counts of input phage; colony counts of bacteria escaping infection, and plaque counts of infected bacteria. In nearly all experiments 95 to 99 per cent of the bacteria were infected, and the total cell counts made before and after infection agreed within 15 per cent, showing that most of the bacteria were capable of yielding phage.Samples of the culture were taken for estimation of total DNA (diphenylamine reaction) at time zero and atone or more additional times; and for titration of infective intracellular phage at 30 minutes and at one or more additional times. The phage yields were measured from 106-fold dilutions in chilled broth containing 0.01 xf NaCN, which gives the yields at the time of dilution (Doermann, 1952). The diluted samples were titrated after warming 30 minutes at 37°C. and again after standing overnight in the refrigerator. The two assays usually agreed within 30 per cent.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.