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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 [5 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-pg05.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 43 heated to 800C. for 10 minutes, at which temperature unadsorbed phage is not sensitized to DNase. 3. The DNA of phage adsorbed to unheated bacteria is resistant to DNase, presumably because it is protected by cell structures impervious to the en- zyme. Graham and collaborators (personal communication) were the first to dis- cover the sensitization of phage DNA to DNase by adsorption to heat-killed bacteria. The DIVA in infected cells is also made accessible to DNase by alternate freezing and thawing (followed by formaldehyde fixation to inactivate cellu- lar enzymes), and to some extent by formaldehyde fixation alone, as illus- trated by the following experiment. Bacteria were grown in broth to 5 X 10' cells per ml., centrifuged, resuspended in adsorption medium, and infected with about two P'=-labeled phage per bacterium. After 5 minutes for adsorption, the suspension was diluted with water containing per liter 1.0 mm MgSO,, 0.1 m3c CaCI:, and 10 mg. gelatin, and recentrifuged. The cells were resuspended in the fluid last mentioned at a concentration of 5 X 108 per ml. This suspension was frozen at -15°C. and thawed with a minimum of warming, three times in succession. Immediately after the third thawing, the cells were fixed by the addition of 0.5 per cent (v/v) of formality (35 per cent HCHO). After .30 minutes at room temperature, the suspension was dialyzed free from formaldehyde and cen- trifuged at 2200 G for 15 minutes. Samples of P32-labeled phage, frozen-thawed, fixed, and dialyzed, and of infected cells fixed only and dialyzed, were carried along as controls. The analysis of these materials, given in Table III, shows that the effect of freezing and thawing is to make the intracellular DNA labile to DNase, without, however, causing much of it to leach out of the cells. Freezing and thawing and formaldehyde fixation have a negligible effect on unadsorbed phage, and formaldehyde fixation alone has only a mild effect on infected cells. Both sensitization of the intracellular P3- to DNase, and its failure to leach out of the cells, are constant features of experiments of this type, independently of visible lysis. In the experiment just described, the frozen suspension cleared during the period of dialysis. Please-contrast microscopy showed that the cells consisted largely of empty membranes, many apparently broken. In another experiment, samples of infected bacteria from a culture in salt-poor broth were repeatedly frozen and thawed at various times during the latent period of phage growth, fixed with formaldehyde, and then washed in the centrifuge. Clearing and microscopic lysis occurred only in suspensions frozen during the second half of the latent period, and occurred during the first or second thaw- ing. In this case the lysed cells consisted wholly of intact cell membranes, appearing empty except for a few small, rather characteristic refractile bodies apparently attached to the cell walls. The behavior of intracellular P" toward DNase, in either the lysed or unlysed cells, was not significantly different from