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Independent Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage. September 20, 1952.

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Title:	Independent Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage [11 of 18]
Creator:	Chase, Martha
Contributor:	Hershey, Alfred Day, 1908
Publisher:	Journal of General Physiology
Date:	1952-09-20
Subject:	Molecular biology Molecular genetics
Description:	From the Journal of General Physiology Vol. 36, No. 1.
Type:	Text
Format:	text/plain
Language:	en
Identifier:	hersheychase-pg11.jpg
Source:	Master scanned with Epson GT-10000+ flatbed scanner at 600 dpi.
Rights:	http://osulibrary.orst.edu/specialcollections/coll/pauling/dna/copyright.html
Full Text:	A. D. HERSHEY AND MARTHA CHASE 49 cent of the phage phosphorus, half of which is given up without any mechan- ical agitation. 3. The treatment does not cause any appreciable inactivation of intracellu- lar phage. 4. These facts show that the bulk of the phage sulfur remains at the cell surface during infection, and takes no part in the multiplication of intracellu- lar phage. The bulk of the phage DNA, on the other hand, enters the cell soon after adsorption of phage to bacteria. Transfer of Sulfur and Phosphorus from Parental Phage to Progeny.---We have concluded above that the bulk of the sulfur-containing protein of the resting phage particle takes no part in the multiplication of phage, and in fact does not enter the cell. It follows that little or no sulfur should be trans- ferred from parental phage to progeny. The experiments described below show that this expectation is correct, and that the maximal transfer is of the order 1 per cent Bacteria were grown in glycerol-lactate medium: overnight and subcultured in the same medium for 2 hours at 37°C. with aeration, the size of seeding being adjusted nephelometrically to yield 2 X 101 cells per ml. in the sub- culture. These bacteria were sedimented, resuspended in adsorption medium at a concentration of 109 cells per ml., and infected with S11-labeled phage T2. After 5 minutes at 37°C., the suspension was diluted with 2 volumes of water and resedimented to remove unadsorbed phage (5 to 10 per cent by titer) and S°a (about 15 per cent). The cells were next suspended in glycerol- lactate medium at a concentration of 2 X 105 per ml. and aerated at 37°C. Growth of phage was terminated at the desired time by adding in rapid suc- cession 0.02 miT. HCN and 2 X 1011 UN'-killed phage per ml. of culture. The cyanide stops the maturation of intracellular phage (Doermann, 1948), and the UV-killed phage minimizes losses of phage progeny by adsorption to bac- terial debris, and promotes the lysis of bacteria ('_~taaloe and Watson, 1951). As mentioned in another connection, and also noted in these experiments, the lysing phage must be closely- related to the phage undergoing multiplica- tion (e.g., T2H, its la mutant, or T2L, but not T4 or T6, in this instance) in order to prevent inactivation of progeny by adsorption to bacterial debris. To obtain what we shall call the maximal yield of phage, the lysing phage was added 25 minutes after placing the infected cells in the culture medium, and the cyanide was added at the end of the 2nd hour. Under these condi- tions, lysis of infected cells occurs rather slowly. Aeration was interrupted when the cyanide was added, and the cultures were left overnight at 37°C. The lysates were then fractionated by centrifuga- tion into an initial low speed sediment (2500 G for 20 minutes), a high speed supernatant (12,000 G for 30 minutes), a second low speed sediment obtained by recentrifuging in adsorption medium the resuspended high speed sediment, and the clarified high speed sediment.